**Selected Nanomaterials' Application Enhanced with the Use of Stem Cells in Acceleration of Alveolar Bone Regeneration during Augmentation Process**

### **Wojciech Zakrzewski 1, Maciej Dobrzynski 2, Zbigniew Rybak 1, Maria Szymonowicz <sup>1</sup> and Rafal J. Wiglusz 3,\***


Received: 31 May 2020; Accepted: 16 June 2020; Published: 22 June 2020

**Abstract:** Regenerative properties are different in every human tissue. Nowadays, with the increasing popularity of dental implants, bone regenerative procedures called augmentations are sometimes crucial in order to perform a successful dental procedure. Tissue engineering allows for controlled growth of alveolar and periodontal tissues, with use of scaffolds, cells, and signalling molecules. By modulating the patient's tissues, it can positively influence poor integration and healing, resulting in repeated implant surgeries. Application of nanomaterials and stem cells in tissue regeneration is a newly developing field, with great potential for maxillofacial bony defects. Nanostructured scaffolds provide a closer structural support with natural bone, while stem cells allow bony tissue regeneration in places when a certain volume of bone is crucial to perform a successful implantation. Several types of selected nanomaterials and stem cells were discussed in this study. Their use has a high impact on the efficacy of the current and future procedures, which are still challenging for medicine. There are many factors that can influence the regenerative process, while its general complexity makes the whole process even harder to control. The aim of this study was to evaluate the effectiveness and advantage of both stem cells and nanomaterials in order to better understand their function in regeneration of bone tissue in oral cavity.

**Keywords:** stem cells; nanomaterials; bone augmentation; nanohydroxyapatite

#### **1. Introduction**

Nowadays, the progress that has been made in dental surgery allows for far developed tissue regeneration in oral cavity and is expected to expand in the nearest future. Because implant dentistry has become a desirable option for replacement of missing teeth, the effectiveness of this technique is mostly dependent on the proper quality and quantity of alveolar bone [1]. The excessive bone loss forbids the placement of dental implants in the ideal prosthetic position [2]. Among many undesirable conditions, bone may be compromised owing to tumour, trauma, periodontal disease, and so on. It was confirmed by Neophytos D. et al. [3] that alveolar bone with a width of 5 mm requires augmentation procedure before successful implant placement.

Bone is biologically privileged tissue, because it has the capacity to undergo regeneration as a part of repair process [4].

There are several bone manipulation techniques that are used to achieve a predictable long-term success for dental implants, and as is later on explained in this study, autologous bone graft still remains the "gold standard" for the process of bone augmentation, as it is characterised by the most effective osteogenic, osteoconductive, osteoinductive, and immunogenic properties [5].

Lack of dental tissue in the alveolar ridge is eventually destructive for either maxilla or mandible, which will be discussed later in this study. When it comes to optimal implant and periodontal aesthetics, preservation of the labial appearance of the alveolar process in frontal region of maxilla and mandible is crucial [6].

It is important to underline that, when there is an insufficient amount of bone to sustain primary and/or secondary implant stability, the alveolar ridge needs to be augmented before placement of the implant. Inadequate alveolar bone height and width often require bone manipulation before, at the time of, or even after the implant surgery. In the case the bone is reduced, but there is enough of it for primary stability of implant, it is possible to directly cover the parts of implant that are still exposed after implant placement with bone graft [7]. In the case of implants, primary stability is crucial, and inability to acquire such a status is one of the most important contraindications for patient implantation.

Among grafts, it is possible to distinguish among the following: autologous grafts, allografts, and xenografts, which will be thoroughly explained in the study.

Alveolar process is a bony ridge that is present on both maxillary and mandibular bone.

The aim of this study is to summarise current research on bone tissue engineering for the clinician with a focus on stem cells, and to review the success of bone augmentation with their help. This work also attempts to confirm the utility of nanomaterials and stem cell-based therapies in acceleration of bone augmentation processes.

#### **2. Changes of the Alveolar Process Following Extraction**

#### *2.1. Degradation Period*

Generally, in order to avoid the degradation processes of bone after extraction, and preserve a proper extraction socket architecture, it is recommended to apply the technique of immediate implant placement at the time of extraction. It is commonly known that the bone modelling process occurring in alveolar process after tooth extraction should be avoided. The bone develops together with teeth, which influence its volume and shape. The alveolar bone supporting the teeth is characterised by distinctive features like rapid and continuous remodeling in response to stimuli by force [8–10]. According to Bodic F. et al. [11], the alveolar bone is subjected to mechanical loads for only 15–20 min per day. Because both maxilla and mandible are tooth-dependent tissues, after loss of tooth, it reacts with a reduction of alveolar ridge in both apicocoronal and buccolingual dimensions [12,13] owing to compromised blood supply and resorption of thin bundles of bone during healing [14]. The process is called atrophy. Changes in the alveolar ridge quickly result in alterations of soft tissue (gingiva), which is attached directly to the former structure. Major changes in the extraction site tend to occur within the first 12 months after the extraction [12,15], while the loss of height of the alveolar bone occurs during the first 3 months. These processes occur because of the fact that tissue must adapt its mass and structure to changing mechanical demands. In the absence of stimuli, such as forces derived from swallowing and mastication, the alveolar bone undergoes resorption [16]. After a tooth extraction, there is a cascade of inflammatory reactions that are activated, while the extraction socket is temporarily closed by the blood clot. Although tissue integrity is quickly restored, the residual ridge is being formed, which has life-long catabolic remodeling effects on either maxilla or mandible. There is no major difference in bony tissue degradation development when it comes to different regions of extraction sockets in oral cavity.

#### *2.2. Healing Period*

The alveolar bone healing process is complex, and its effectiveness depends on the efficacy of hosts' inflammatory response [16]. The bone microarchitecture analysis allows to evolve healing with trabecular thickness, and progressively increase the number over time [10]. The healing process starts within 12 months after tooth extraction [12], while reorganisation of lamina dura takes place throughout its duration. Because oral tissues are located in an environment rich in microorganisms, the healing process is always impaired by the lack of sterility [17]. The alveolar bone healing process usually occurs without histological cartilage formation, while long bone healing is a process of endochondral ossification [18].

Fracture healing is a process in which the restored bone shows a lack of scar tissue and formation of blood clot is a crucial step in order to begin such a healing process [19]. Cascade of reactions following blood clot formation allows effective tissue healing in alveolar bone, because platelets in the clot carry specific growth factors (GFs). Both osteoblasts and osteoclasts have direct contact with lymphocytes, which suggests a regulatory role of the immune system, especially in later stages of the healing process [19]. Non-alveolar and alveolar bones differ in nature of the cells surrounding them. For example, the latter lacks muscle stem cells which play essential role in fracture healing [20,21].

In the later stages of the healing process, the blood clot is being replaced with granulation tissue [22], which then leads to the development of newly formed vessels. When modelling processes commence, at first, apical and lateral walls of the alveolus are restored. Later, the healing process goes toward the centre and the coronal region of the alveolus. According to Scala A et al. [23], it takes around one month to close the extraction socket with a newly formed bone. The process eventually ends with corticalization of the socket and formation of bone marrow.

Residual ridge is an alveolar process that is formed after healing of soft tissues and bone, followed by extractions. Although it is a life-long process, the reduction of bone is most aggressive during the first 6 months. Residual ridge resorption (RRR) depends mostly on the site of the ridge and occurs differently among individuals. The basic structural change in RRR is about reduction of the size of the ridge under mucoperiosteum.

When a structure, for instance, maxillary bone, undergoes stress, it becomes deformed. The mechanical aspect of bone remodelling is mostly associated with Wolffs's law [24] of bone transformation, which simply says that bone remodels in response to the forces applied, although this explanation describes very briefly such a complex physiological process like bone remodelling.

The biggest amount of bone loss occurs in the horizontal dimension and happens mainly on the facial aspect of the ridge. On the other hand, vertical ridge height occurs most intensely on the buccal aspect [14]. Long-term lack of tooth in the bone results in increased narrowing and shortening of the ridge, which generally relocates palatally/lingually. Stages of alveolar ridge reduction after tooth loss are presented in Figure 1.

**Figure 1.** Stages of alveolar ridge reduction after tooth loss, order 1—pre extraction, order 2—post extraction, order 3—high, well-rounded, order 4—knife edge, order 5—low, well-rounded, order 6—depressed.

#### **3. Factors Influencing Alveolar Bone Loss and Regeneration**

#### *3.1. Bone Loss Factors*

After tooth extraction, the alveolar ridge undergoes uneven atrophy processes [22,25]. Although loss of teeth results in naturally irreversible alveolar bone resorption [26], the destructive process of the bone may start even before extraction of the tooth. It may be complicated with gingivitis present, which leads to periodontopathy, endodontic lesions, or trauma injury. After such a situation, further loss of bony tissue owing to extraction may result in severe complications that occur more quickly than in a case in which the bone stays intact before tooth removal.

Bone degradation can be also caused by several metabolic bone diseases, like vitamin D-resistant rickets (VDRR), focal infections, hyperparathyroidism, age-related parietal bone atrophy, or Paget's disease. VDRR for instance, is a disease affecting mainly dentin, while enamel remains unchanged [27]. Spontaneous pulpal abscesses, which are formed without carious lesions, are detectable. Big tubular clefts in the region of pulpal horns are visible, with submicroscopic defects in the enamel layer, leading to facilitated invasion of bacterial toxins [28]. Increased bacterial invasion of teeth results in accelerated teeth loss, eventually causing accelerated bone resorption.

#### *3.2. Bone Regeneration Factors*

Despite bone having a mineral nature, it is a vital and dynamic organ. The histogenesis of bone is directly from mesenchymal connective tissue in the intramembranous bone formation process, and from pre-existing cartilage in endochondral bone formation. Following tooth removal, the normal healing process takes approximately 40 days, starting with clot formation and culminating in a socket filled with bone covered by connective tissue and epithelium [29,30]. The biological principles of bone regeneration comprise the following: osteoinduction, osteogenesis, and osteoconduction. Optimization of these processes has been the goal of new materials used in hard tissue engineering. Osteoinduction process allows migration, followed by proliferation of unspecialised connective tissue cells into bone-forming cell lineage [31]. It induces osteogenesis [32] and GFs determine its action [33].

During osteogenesis, a formation of new bone from both Haversian systems and osteoblastic cells of the grafted bone takes place [34,35]. A direct transfer of vita cells to the area that will regenerate new bone occurs.

Osteoconduction, which is the last of the aforementioned principles, focuses on bone growth on a surface. It implies recruitment of non-adult cells and their stimulation to preosteoblasts [36]. The osteoconduction process means bone growth on a surface. It is a phenomenon often seen in the case of bone implants. Because its function is to provide space and substrates for the biochemical and cellular event progressing the bone formation process, it eventually results in osteogenesis [32,37]. After successful implant insertion, proper biologic width and aesthetics should allow for remodelling of the soft tissue and bone, occurring between 6 months and 1 year [38].

#### *3.3. Osteoinductive Factors*

Bone repair is a multistep process that involves migration, differentiation, and activation of considerable amount of cell types [39,40]. Taking into consideration that bone tissue is highly vascularised, it requires both bone tissues and blood vessels to be formed in a tight integrity [41]. Current bone regenerative strategies pursue mimicking natural bone regeneration. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factors (VEGFs) are two key regulators of osteogenesis and angiogenesis, acting by promoting osteogenic and endothelial differentiation of stem cells, respectively [42,43]. Both factors act synergistically during bone regeneration.

BMPs are one of the most researched and crucial morphogenetic signals coordinating tissue architecture in the whole organism. Having the appropriate concentration and being placed on specific scaffold, they are capable of inducing new bone formation by turning mesenchymal stem cells into chondroblasts and osteoblasts [44]. BMPs are being increasingly used in surgeries.

They belong to the transforming growth factor (TGF) beta superfamily [45]. There are currently at least 20 members of the aforementioned family and, among them, BMP-2 is one of the most common factors in use.

Among the most useful functions of BMPs, one can distinguish among the following: induction of cell replication, chemotaxis, induction of differentiation, anchorage-dependent cell attachment, osteocalcin synthesis/mineralisation [46], and alkaline phosphatase activity [47]. Recent studies confirmed that using recombinant BMP in order to correct bony defects, furcations, and fenestration leads to periodontal regeneration with ankylosis [48]. On the contrary, when BMP-7 was used in the augmentation process, it resulted in serious periodontal increase without ankylosis. BMPs can also be used to alleviate implant wound healing. Rutherford et al. [49] have shown that application of Osteogenic protein-1 (OP-1) around the extraction socket escalated bone growth measured histologically at 3 weeks.

VEGF is the signal protein produced by cells, having the ability of vasculogenesis and angiogenesis. It additionally mediates osteogenesis [50]. Street et al. [40] prove, that localised VEGF delivery is beneficial even for osteoblasts migration and bone turnover. This means that, delivered to a bone defect, it is an effective strategy to accelerate bone healing [51]. Additionally, VEGF has been successfully used to improve maturation of newly formed bone. VEGF belongs to a sub-family of GFs, that is, the platelet-derived growth factor family of cystine-knot GFs. The serum concentration of VEGF increases in chronic hypoxic conditions like diabetes mellitus [52], because it is a part of the system responsible for restoring oxygen supply in the case of inadequate oxygenation of tissues.

#### **4. Augmentation Techniques**

The augmentation procedure in dentistry is aimed to increase the volume of alveolar bone, particularly when placement of intrabony implant would otherwise be considered problematic [53]. In order to regenerate a sufficient amount of bone to allow successful implant placement, a ridge augmentation technique is recommended. The intramembranous bone formation pathway is used when intraoral bone augmentation techniques are applied by the dental surgeon. The bone augmentation technique, which is used in order to reconstruct different alveolar ridge defects, depends on the horizontal and vertical extent of the defect. Predictability of corrective procedures is influenced by the span of the edentulous ridge and amount of attachment on neighbouring teeth [54].

#### *4.1. Guided Bone Regeneration (GBR)*

GBR is similar to guided tissue regeneration, but focuses on development of hard tissues. It is a surgical procedure based on using barrier membranes, with or without bone graft/bone substitutes. Bony regeneration by GBR depends on the migration of osteogenic and pluripotential cells to the defect site in bone and exclusion of cells impeding bone formation [55,56]. It is important to underline that, in order to accomplish successful regeneration of a bone defect, the rate of osteogenesis must exceed the rate of fibrogenesis from the surrounding soft tissue [30,57]. The GBR technique requires four principles in order to successfully fill osseous defect-space maintenance for bone in-growth, stability of the fibrin clot to make the uneventful healing possible, exclusion of epithelium and connective tissue to allow space to be filled with bony tissue, and primary wound closure to promote undisturbed healing [58].

The mechanism of GBR is focused on selective in-growth of bone-forming cells into a bone defect region, which is enhanced when adjacent tissue is kept away with a membrane [59]. This additionally allows to protect the wound from both salivary contamination and mechanical disruption.

There are several techniques used in GBR regarding tri-dimensional bony tissue reconstruction. They are all based on packing bone substitutes into the bony defect and covering it with resorbable or non-resorbable membranes.

#### *4.2. Bone Augmentation Methods and Precise Implant Placement*

The goal of successful augmentation following tooth loss is to allow performing effective prosthetic replacement that is in harmony with the rest of the adjacent natural dentition.

Resorption of alveolar bone is a natural consequence of tooth loss. This process causes clinical problems, especially in terms of aesthetics. In order to overcome the changes in the oral cavity, it is required to carry out treatment that makes it possible to preserve the natural tissue shape, in order to prepare for prosthetic appliance like an implant [60]. The clinical outcome of implant treatment is challenged especially in compromised bones of elderly patients [61].If more alveolar ridge is preserved, it will guarantee optimal implant placement and proper functioning of prosthetic appliance. Nevertheless, nowadays, clinicians are usually faced with the necessity to place implants in the alveolar bone of smaller volume. Such a situation requires the clinician to carry out a proper pre-treatment with augmentation techniques that will promote a more predictable regenerative outcome [54]. As Figures 2 and 3 show, the properties of alveolar bone can be assisted by several methods.

**Figure 2.** Lekholm and Zarb classification: Type I, whole bone is built of very thick cortical bone; Type II, thick layer of cortical bone surrounds the core of dense trabecular bone; Type III, thin layer of cortical bone surrounds the core of trabecular bone of good strength; Type IV, very thin layer of cortical bone with low density trabecular bone of poor strength.

**Figure 3.** Ridge defect classification of edentulous patients according to Seibert (1983).

The ideal alveolar ridge width and height make the placement of a natural appearing pontic possible, which provides maintenance of a plaque-free environment [62]. The structural loss of the residual alveolar ridge can occur as a result of tooth extraction, surgical procedures, periodontal disease, or congenital defects [63,64]. In a situation with a bone missing, the overlying soft tissue tends to collapse into the bone defect, making it difficult to recreate oral cavity aesthetics after application of functional prostheses. Alveolar deformities classification is based on quantity of volumetric horizontal and vertical tissue loss within the alveolar process. Such a classification was established to standardise communication between clinicians in the selection and sequencing of reconstructive procedures [65]. It is crucial to thoroughly evaluate the contour of the partially edentulous ridge before starting the process of fixed partial denture fabrication. As presented in Figure 3, according to Seibert, Class I

represents bucco-lingual loss of tissue with normal height of ridge. Class II defect is represented by the loss of alveolar height in apico-coronal axis. On the other hand, Class III has a combination of bucco-lingual and apico-coronal loss of tissue. According to this classification, the bone augmentation technique is dependent on the horizontal and vertical extent of the defect.


Immediate implant placement can be achieved without additional surgical treatment. However, slight hard tissue augmentation may be needed to add support to periimplant mucosa. There are situations that require soft tissue addition in order to aid maintenance [66,67]. There are several important factors that need to be taken into consideration during planning optimal placement of implants in the alveolar bone, that is, soft and hard tissue management, aesthetic factors, and proper quality of prosthetic restoration.

Conventionally, the placement of dental implants sacrifices much bone tissue during the drilling procedure. However, there are several implantation technique ideas that allow limited bone removal, especially in the case of patients with a limited amount of alveolar bone [68,69].

In order to perform a successful implantation, the dentist has to remember the periimplant values of hard and soft tissues. If the implants are placed too tightly, it will result in insufficient vertical blood supply to the papillae. Angulation of implants is crucial for a proper papilla development afterwards. There is no sufficient support for the papillae in two divergent crowns, while convergent crowns do not allow soft tissue to develop naturally. Tarnow et al. [70] demonstrated a proper relationship with regard to both implant to natural tooth and implant to implant. Regarding the former, in order to avoid horizontal bone loss, which will affect adjacent tooth, the distance should be about 2 mm. The latter requires a distance of at least 3 mm, which, when avoided, creates accelerated bone loss patterns in such areas [71]. It is important to underline that each implant loses periimplant bone within the first year and then stabilises [72].

The crown-to-implant ratio should be 1:1 or less, while the minimum height of the implant is 10–12 mm. The lower height of implant has already been proven to show a high failure rate [73]. In general, the actual height of bone is required to be 12 mm of bone actual height for a macroretentive screw-type implant to properly support occlusal forces [71].

#### *4.3. Membranes in GBR*

A GBR membrane acts as a barrier preventing fast-growing soft tissue from invading space required to be filled with a new bone [74]. Membrane materials for bone tissue engineering are usually divided into natural biomaterials [75], like chitosan, inorganic materials represented by nanohydroxyapatite [76], and synthetic polymer materials with polylactide-co-glycolide (PLGA) [77] as an example. Current approaches on graft materials exclusively face serious limitations [78]. Membranes themselves are not able to recover the defects in bone tissues completely, owing to their lack of satisfactory osteoinduction. The results change definitely with the incorporation of osteoinductive factors into semisolid or porous membranes-scaffolds [79,80].

Providing adequate space for bone regeneration is one of the fundamental principles of GBR. Various animal studies have proven that excluding the epithelium and connective tissue makes it possible to create that space, permitting slow migration of osteoblasts to the wound, which results in new bone formation [58,81]. Reinforced membranes allow the space maintenance by preventing membrane collapse that can occur owing to increased pressure from neighbouring tissues.

#### *4.4. Resorbable Membranes*

Degradable membranes can be made from collagen (natural), or poly (l-lactic acid) (PLLA) and PLGA [82] (polymeric). Many of these membranes are formulated with antibiotics, usually tetracyclines [83]. There are two important challenges in osteoinduction process that need to be overcome. The first one is to retain the osteogenic factor for a sufficient amount of time to generate the desired biological response, while the second concerns the biocompatibility of the material.

Achieving a desired tissue response is strictly dependent on both degradation components of the extracellular scaffold and concentration of inductive factors released from the matrix.

It is important to underline that bone can only grow when provided with space to do so. Thin, polymeric membranes allow to hold back soft tissues, but provide no significant mechanical support during bone healing. Polymeric adhesive or calcium phosphate cement are required for greater mechanical strength. In such a case, the bone is repaired alongside cement degradation, which can be a challenge.

One of the biggest advantages of resorbable membranes is the fact that they do not require a second surgical procedure over time. They undergo disintegration. On the other hand, it is important to underline, that such materials also have their limitations. Neiva R. et al. [84] have confirmed that using membranes to produce similar gains of keratinized tissue formation was failed in comparison with connective tissue grafts. Collagen material is resorbed by the host with the use of neutrophils and macrophages, causing no inflammation, while expanded polytetrafluoroethylene (ePTFE) material undergoes hydrolysis reaction, which, in some cases, may cause it.

#### *4.5. Autogenous Bone Grafts*

The "gold standard" bone graft material in traditional augmentation techniques is autologous bone graft. At the same time, allografts avoid donor site issues, but can cause a higher risk of infection and immune reaction of host tissue [85,86]. Autologous material is better than allograft, because it maintains bone structures, such as minerals, collagen, viable osteoblasts, and BMPs. Although autografts are a gold standard, they still present several significant limitations. Their harvesting is connected with the second concurrent surgical procedure, high donor site morbidity, and resorption [87,88].

Soft-tissue grafts are free gingival grafts that can be harvested from several sites in the patient oral cavity. In the reconstruction of minor alveolar defects, bone grafts from the retromoral region are one of the best intraoral sources possible [89–91]. Surgical operation concerning this region of oral cavity causes minimal discomfort for the patient, has relatively uncomplicated surgical access, and is in proximity of donor and recipient sites, which can lower the anesthesia doses required and cause only minor complications [92]. Its downside poses an unnecessary risk of complications owing to the involvement of the second surgical site. However, when the patient has inadequate thickness of palatal tissues, it is difficult to harvest a sufficient amount to place the graft properly.

Microvascular flap use is one of the states of the art and effective techniques for the repair of significant bone and soft tissue defects. While the vascularised pedicle allows proper perfusion to the harmed area, it also results in complete osseointegration of the bone graft [93]. On the other hand, even this technique represents several disadvantages—it requires a long intraoperative time for the patient, and may result in a permanent deficit, when a muscle or bone are included in the flap [94–96].

#### *4.6. Allografts*

Allograft is a tissue graft between individuals of the same specimen, but of nonidentical genetic composition. Generally, cadaver bone is a source for allografts, as it is available in large quantities [97]. One should realise and remember that the aforementioned bone has to undergo multiple treatment sequences in order to make it neutral for the host's immune system and to avoid cross-contamination of disease. Allografts can be used as an alternative, but they have very limited osteogenicity and resorb more rapidly than autogenous bone. It is a useful material in case of patients requiring a non-union

type grafting, who have inadequate autograft bone quantity, or when it is hard to obtain tissues from the donors' site.

The main disadvantage of allografts is related to the relatively poor capacity for osteoconduction and osteoconduction, when compared with autologous graft. Another disadvantage of allografts is connected with an improper rate of resorption. It has to be clearly emphasised that bone tissue tends to be resorbed quickly, even after augmentation support, unless loading is provided with dental implant. Implants can be placed at the time of surgical procedure or 6 months later, after the stabilisation of the graft, which minimizes the resorption process.

One of the possible alternatives for soft-tissue grafts is acellular dermal matrix allografts (Alloderm). Alloderm is a donated human dermis, composed of a structurally integrated complex that constitutes basal membrane and an extracellular matrix [98]. Their use reduces the likelihood of cross-infection. Wagshall et al. [99] claim that if a graft material could be used to replace the palatal grafts, then all the possible complications connected to donor site would be immediately eliminated. This would result in alveolar ridge augmentation, being more acceptable for the dental patients.

#### *4.7. Xenograft*

Xenografts are a graft specimen from the inorganic portion of animal bones. Bovine is one of the most common sources for their extraction. In order to remove their antigenicity, the removal of the organic component is processed, whereas the remaining inorganic components both provide a natural matrix and serve as an excellent source of calcium. The disadvantage of xenografts is that they are only osteoconductive, and the resorption rate of bovine cortical bone is slow [15].

#### *4.8. Bone Substitute Materials and Genetic Engineering*

Genetic engineering can be done in two different ways. The first idea focuses on direct in vivo delivery of genes. There are reported cases in which recombinant human BMP-2 was mixed with an absorbable collagen sponge to treat open long-bone fractures [100]. Govender et al. [101] explained in their study that there was 44% reduction in the risk for failure in healing with less secondary invasive interventions and reduced healing time. The factor used during this study was recombinant BMP-2 called rhBMP-2/ACS. The second idea focuses on using autologous bone alternatives of either animal, human, or synthetic origin [102,103]. Such a technique has its limitations, for example, the risk of bacterial contamination [104], or effectiveness limited mainly to reconstruction of small bony defects [105].

#### **5. Nanomaterials Application in Alveolar Bone Regeneration**

#### *5.1. Nanohydroxyapatite (n-HAp)*

Nanomaterials, when compared with bulk materials, possess features like macroscopic quantum tunnelling or quantum size, causing altered physiochemical properties [106,107]. In oral biology, nanotechnology applications are mainly focused on augmentation procedures for osseous tissue regeneration and implants osseointegration enhancement [106]. Even though supraphysiological doses are necessary to combat the poor pharmacokinetics of this compounds, the nanocarriers can overcome such limitations by stabilizing the bioactive molecules.

Although bone autografts are considered the "gold standard" in clinical bone repair, they still have several limitations owing to the amount of bone that can be used, as well as an increased risk of donor site morbidity. Because of that, a considerable amount of study has been undertaken in order to develop effective regenerative strategies, leading to bone augmentation with limited side effects [108,109]. The n-HAp has a hierarchical architecture at multiple levels, including macrostructure (cancellous and cortical bone), microstructure (trabeculae), sub-microstructure (lamellae), nanostructure (embedded minerals and fibrillary collagen) [110], and sub-nanostructure (proteins and minerals). The presence of nanotubes or nanocrystals in the composite materials allows for enhancing the

mechanical properties of the scaffolds. The nanosized materials present enhanced characteristics, like wettability, charge, roughness, and adsorption of proteins. Moreover, the nanotextured surfaces enhance in vitro osteogenesis and promote mineralization. Furthermore, in the case of the nanomaterials, aqueous contact angles become three times smaller, leading to increased adhesion of the osteoblasts in comparison with micro-sized materials.

Special composition and architecture allow them to have self-regenerative and self-remodelling ability in response to damaging signals and mechanical stimuli [111]. This makes nanomaterial an ideal candidate for bone graft development, as it is capable of recapitulating the organisation of the natural extracellular matrix, in order to regulate bone forming cells activity [112], as can be seen in Figure 4.

**Figure 4.** Schematic representation of how a synergistic combination of compositional, nanoelements, well-defined structure, and growth factor administration may endow nanomaterials with a "self-regenerative" capacity for the regeneration of large critical defect bone in a natural bone-healing way, especially at an in vivo level.

Synthetic biomaterials for bone repair should provide mechanical support and biological compatibility in order to promote bone tissue regeneration based on healing. Hydroxyapatite is an interesting inorganic mineral with potential dental [113], maxillofacial [114], and orthopaedic applications [115] that has a typical lattice structure as (A10(BO4)6C2) which defines A, B, and C by Ca, PO4 <sup>3</sup>−, and OH<sup>−</sup> [116]. Hydroxyapatite (Ca10(PO4)6(OH)2 is the principal inorganic mineral component of animal and human bones and teeth, and is difficult to dissolve in a solution where the ration of the calcium-to-phosphorus is 1:67 [117]. There are other forms of calcium phosphate present in nature, but HAp is the least soluble of them. The enamel is the hardest substance consisting of relatively large HAp and fluorapatite (FAp) crystals that are 25 nm thick, 40–120 nm wide, and 160 to 1000 nm long. In contrast to enamel, hydroxyapatite is present in bone as plates or needles, while its dimensions range from 40–60 nm long, 20 nm wide, and 1.5 to 5 nm thick.

Nano-HAp is a nanoform of hydroxyapatite with a range of unique properties and diameters ranging in size between 1 and 100 nm [118]. From these dimensions derives a distinct activity of the particles. It has been one of the most studied biomaterials in the medical fields, and has also proven to have strong biocompatibility [119], stability, and nontoxicity. Material possesses an ability of intense ion-exchange against various cations, causing HAp to have high bioactivity [120]. Diversity of n-HAp utility can be seen in Figure 5 and Table 1.

**Figure 5.** The application of nanohydroxyapatite (n-HAp) scaffolds in bone tissue engineering. PLGA, polylactide-co-glycolide.

#### *5.2. Examples of Bone Regeneration Using Nanohydroxyapatite and Stem Cells in Published Studies*

Use of stem cells for bone healing and regeneration still remains in its infancy [121,122]. Composite grafts are able to incorporate osteogenic, osteoconductive and osteoinductive properties onto a compound [123,124]. For instance, local autogenous bone marrow can be harvested in order to combine with a bioceramic material. Also, use of antibiotic-loaded or antimicrobial bone graft substitutes has advantages over nonresorbable antibiotic carriers due to its biodegradability [125]. Nowadays, Nowadays, bone is the second most common transplanted tissue in comparison with blood tissue [126]. Implant bone graft-carrier allows to release the incorporated growth factor at the desirable rate and concentration. Additionally, it can be formed to be structured for facilitate cellular infiltration and growth [127].

**Table 1.** The application of n-HAp scaffolds in bone tissue engineering. BMP, bone morphogenetic protein.


Dahabreh et al. [128] check in their studies influence of bone graft substitutes on osteoprogenitor cells in terms of proliferation, differentiation and adherence. Moreover, Bojar et al. [129] confirmed effectiveness of alloplastic materials as an alternative for autologous transplants and xenografts in oral surgery and dental implantology.

The chemical composition most of them is hydroxyapatite. However, there is still a doubt to be successfully used in regenerative medicine. Although, the treatment using the allograft tissue is preceded by tissue freezing and freeze-drying as well as sterilization, there is always a risk of disease transmission from a donor [130] or rejection [131]. Furthermore, Kattimani et al. [132] proved that the limitations of allograft's use with stem cells and nanohydroxyapatite has resulted in new alternatives.

Nanosized bioceramics highly active surfaces and size make them a promising platform for bone regeneration. Such ceramics present increased osteoblast adhesion when compared with regular sized ceramics [133]. Their nanometre grain size is responsible for the increased osteoblast functions, like adhesion, proliferation, and differentiation. One of the examples of nanophase ceramics is n-HAp. It has been successfully used, among other applications, as a coating of orthopaedic implants, filler of composites, and bone filler [134]. According to several authors, the aforementioned material, in its pure form, is limited owing to its brittleness [135,136]. Wide ranges of solutions have been proposed in order to compensate problems with nanohydroxyapatite use, like incorporation of

chitosan-biopolymer [137]. Wang et al. (2015) [138] proved, in their study, that scaffolds containing n-HAp, chitosan, and polylactide-co-glycolide (CS, PLGA) proved to have higher compression and tensile modulus, when compared with the same scaffolds that had no nanohydroxyapatite, which proves its important superior function. Bhyiyan et al. (2017) prepared a study in which they developed a multicomponent covalently-linked biodegradable biomaterial called n-HAp-PLGA collagen [139]. Its properties were similar to cancellous bone, and maintained high mechanical strength, even in an aqueous environment [139]. PLGA provides strong biodegradability, while hydroxyapatite bioceramic is responsible for osteoinduction/osteoconduction, while collagen allows biological stimulation for cell proliferation, similar to extracellular matrix in vivo [140]. PLGA is a synthetic biodegradable polymer. The main reaction used to create PLGA is ring opening polymerization and polycondensation of glycolic and lactic acids. On the basis of the studies of Tsai et al. (2010) [141], it can be confirmed that human mesenchymal stem cells' (hMSCs') growth on n-HAp-PLGA-collagen films is vast and comparable to collagen—a widely used substrate for hMSC attachment and proliferation [142]. Table 2 shows the versatile applications of n-HAp scaffolds in bone tissue regeneration.



#### *5.3. Nanohydroxyapatite Doped with Rare Ions*

In order to enhance n-HAp use in the regeneration field, it can be doped with specific materials. Evis et al. (2011) confirmed that the biodegradation rate of hydroxyapatite doped with metal ions was slower than that of pure mineral. The rate of resorption appeared to be minimal when the aforementioned material was doped with magnesium ions [143].

In implantology, absorption of the hydroxyapatite is crucial. It occurs simultaneously with replacement by bony tissue, and it can be achieved by matching implant resorption bone regeneration rates [144].

Qin et al. (2013) [145] demonstrated that osteogenic potential can increase as a result of the influence of Ag nanoparticles in human urine-derived stem cells, while also significantly increasing osteoblast lineage differentiation and mineralization in vivo [146]. On the other hand, Au particles also demonstrated promising application in both bone and cartilage repair [147].

Usually, one of the methods of n-HAp synthesis is the co-precipitation technique. In the case of Ag-HAp nanoparticles, it is similar, but with an addition of 1% Ag in the form of silver nitrate or 1% Au, respectively [148]. At the cellular level, n-HAp, Ag, and Au can be useful as possible promoters of osteogenic differentiation [149,150]. Hsu et al. (2007) [147] confirmed that Au nanoparticles enhance osteogenesis process through the mitogen-activated protein kinases signalling pathway, which can explain how Ag and Au can positively influence bone regeneration. n-HAp doped with such particles can be a representative of a powerful novel approach in the field of regenerative medicine.

#### *5.4. Carbon Nanotubes as a Bone Regeneration Sca*ff*old*

Nowadays, a number of studies investigating the scaffold use of carbon nanotubes (CNTs) has been increasing [151,152]. In atomic scale, CNTs are hexagonal sheets of graphite wrapped into single or multiple sheets. They can be metallic or semiconducting, depending on their chirality. CNTs have thermal conductivity that is twice that of diamond and are stable up to 2800 ◦C. Its high bone affinity to serve as a scaffold makes it a promising material to use in regenerative medicine. CNTs possess

outstanding mechanical properties, with their tensile strength in the range of 50–150 GPa, and a failure strain in excess of 5%. Several scientific works confirmed its effectiveness by checking osteoblasts adhesion to such complex [153], influence on proliferation of osteoblasts and osteocytes [151], and CNTs' promotion of osseous tissue formation in vivo [154,155]. Tanaka et al. (2017) [151] confirmed that multi-walled CNTs (MWCNTs) blocks can serve as filler materials, because they are solid, with nano-sized surface irregularities and non-porous interiors, causing surrounding cells to not be capable of entering the scaffold. By allowing osteoblasts to proliferate on the MWCNT block surface, such a scaffold can have osteoconductive abilities. Their previous studies about CNT [156] additionally proved that this material can be successfully used as a functional scaffold for bone formation and promote the process of bone tissue regeneration.

#### *5.5. 3D-Printed Sca*ff*old Nanomaterials for Bone Application*

Current surgical procedures for bone regeneration utilise transplantation using autografts, allografts, or xenografts, and have to deal with repair, renewal, and replacement of the bone tissue defect. Some of the main disadvantages of such operations are donor site morbidities, unavailability of large tissue volumes, additional risk of infections, communicable diseases, and severe pain [157,158].

Recently, 3D printing techniques appeared to be profitable as a tool for making scaffolds with controlled microarchitectures. They represent an attractive alternative for the synthesis of new scaffolds, allowing the modulation and control of the geometry with high precision over the pore size, outer shape of the scaffold, or porosity, all these together with cost-effectiveness and a rapid manufacturing process [159].

#### *5.6. Graphene-Based Nanomaterial in Bone Regeneration*

Graphene demonstrates the true sense of biomaterial by having two surfaces without bulk in between. Its single-atom-thick carbon-based honeycomb structure allows it to have uncharacteristically strong optical and mechanical electron properties. Currently, the methods such as the introduction of growth factors, genetic modifications, and cytokines have been used to help control stem cell differentiation [160]. Graphene is a 2D-nanostructure, which has similar mechanical, thermal, and electrical properties to carbon nanotubes and has potential for technological and scientific applications. The cytotoxic effects of graphene have been assessed, and it has been confirmed that layers made out of this material produce fewer toxic effects, which is crucial for bone regeneration. Low toxicity allows the final composite not to be rejected or induce an inflammatory reaction when placed inside the organism.

Biris et al. (2011) [161] have proven that nanocomposites can be synthesised in situ with a singular growth process, and they are characterised by high biocompatibility in osteoblastic bone cells' proliferation in vitro. Graphene implementation in tissue engineering has offered unique scaffold structures with exceptional electrical and mechanical properties [162]. Its potential functionalization in combination with carbon backbone, nanoscale size, antibacterial activity [163] has been used as an enhanced method of controlled cell proliferation [164]. The recent biocompatible graphene nanocomposites can be prepared with the use of radio-frequency chemical vapour deposition (rf-CVD), using methane and acetylene as the carbon sources. Nanoclusters of such particles are evenly dispersed over HA with 2~7 nm diameters, and act as a catalyst for graphene synthesis [165]. The unique properties of nano-scale materials matched with cell sensitivity can be exploited to help improve the regeneration process [166].

Graphene-based HA nanocomposites can be prepared in the form of scaffolds, bulks, coatings, or powders. Both bulk composites and powders can be successfully used to repair the bone defects or small non-unions, as well as in coating metallic implants to increase bone-binding abilities. Porous graphene-based HA nanocomposites can be successfully used for larger bone defects. Such materials possess enhanced osteogenic activity and are promising when scaffolds are made out of nanomaterials. They also provide better outcomes in providing guided cell differentiation than in a situation when the

cells are distributed directly into the defect. Nano-sized scaffolds are believed to better control the differentiation process, owing to their interaction with extracellular matrix (ECM) [162].

Loaded GFs, as well as adsorbed drugs on graphene and its derivatives, were able to increase osteogenic differentiation owing to increased local concentration. At the same time, the bone morphogenetic proteins (BMPs) are the most potent osteoinductive proteins for bone tissue regeneration [163].

#### *5.7. Structural E*ff*ects of Nanomaterials on Bone Regeneration*

At the time of bone regeneration, the porous architecture of scaffolds provides sufficient microenvironments for nutrient/waste exchange, cell proliferation, differentiation, and angiogenesis. The special structure of natural nanomaterials enhances bone with dynamic biological functions and mechanical durability. Nowadays, studies confirm that nanostructures at different dimensional levels play different roles in the regulation of bone regeneration [112]. For instance, during the initial period of implantation, biomaterials must provide structural support in the defect site for bone regeneration. Their nanofeatures act as a good enhancer to acquire proper mechanical properties and stability of osseous regeneration [165]. Nanoparticles can be incorporated into materials to form nanomaterials with adjustable mechanical strength, which can be used to induce stem cells' osteogenic differentiation [166]. Lastly, nanoparticles alone can have the ability to improve osteogenesis for bone regeneration, for instance, Laponite [112]. In such materials, there is a possibility to adjust the conformation of GFs to increase their bioactivity for bone regeneration.

Nanoscaffolds have considerable drug loading abilities, high mobility of drug loaded particles, and efficient in vivo reactivity toward nearby tissues [167]. They can be used for labelling cells, in order to enable monitoring and continuous cell tracking [168], as well as enhancing osteoinduction, osteoconduction, and osseointegration [169].

Tissue engineering and regenerative medicine (TERM) aims to create functional substitutes for diseased and damaged tissues. The strategy behind TERM combines three essential elements, namely, scaffolds, GFs, and stem cells, as can be seen in Figure 6. Scaffolds provide support for tissue formation and are seeded with stem cells. GFs are also included as they regulate the differentiation and proliferation processes [169].

**Figure 6.** Tissue engineering triad.

#### *5.8. Hydrogels*

Microengineering technologies can be successfully used in order to make hydrogel scaffolds mimic in vivo extracellular matrix (ECM). These techniques include lithography, micromolding, biopaterning, and microfluidics. According to Geckil et al. (2010) [170], hydrogels are three-dimensional, insoluble, cross-linked hydrophilic polymeric networks that are capable of providing scaffold to facilitate cell growth, infiltration, and differentiation [171], and can be used to deliver cells with regenerative

functions, as pictured in Figure 7. Polymers in hydrogel can absorb a large amount of biological fluid or water with the help of interconnected microscopic pores. In order to increase the biological (hydrophilicity, cell-adhesiveness), mechanical (stiffness, viscoelasticity), and biophysical properties like porosity, combinations of either synthetic or natural hydrogels can be utilized. Such biomaterial composition makes them amenable to surface modification and biomimetic coatings. It is a type of polymer scaffold that has several potential advantages in bone repair. Hydrogels are materials that are able to mimic natural ECM of the bone, allowing to encapsulate bioactive molecules or cells. Network structure of the aforementioned materials allows proteins that are entrapped inside to be confined in the meshes of gel and released as required [172]. Moreover, those materials are absorbable and demonstrate magnificent integration with surrounding tissues, which removes the necessity of its surgical removal and additional trauma [173].

**Figure 7.** Schematic illustration of hydrogel-assisted bone regeneration. MSC, mesenchymal stem cell; GF, growth factor.

There are still some challenges that require further investigation. A controlled release of encapsulated drugs is one of them. Both burst and delayed release of the drug can affect actual therapeutic effect, and the use of inappropriate polymers can also cause toxic reactions [174]. Mimicking such a 3D-cell microenvironment in vitro with the use of hydrogels is crucial for various applications like constructing tissues for repair.

#### *5.9. Nanostructured Sca*ff*olds for Bone Tissue Engineering*

Scaffolds can be utilised in bone tissue engineering in order to deliver biofactors including cells, genes, and proteins to generate bone and assessment of vascularity formation, together with overall tissue maturation [175]. There are three rules that a scaffold needs to comply with in order to be useful in tissue engineering. Firstly, it is required that the scaffold enhances the regenerative capability of the chosen biofactor; secondly, it must provide the correct anatomic geometry in order to maintain space for tissue regeneration; and thirdly, the scaffold needs to provide temporary mechanical load bearing within the specific tissue defect.

Many materials have been proposed as synthetic bone substitutes. Hydroxyapatite is regarded as one of the most bioactive bone substitute materials, mainly because of its superior osteoconductivity. On the other hand, synthetic octacalcium phosphate has been shown to be a good precursor of biological apatite in both teeth and bones, and it also presented better biodegradable and regenerative characteristics when compared with the other calcium phosphate bone substitute materials [176]. Hence, one of the disadvantages of such materials is their inability to achieve close apposition of the material to the neighbouring bony tissue, as well as brittleness of the ceramic materials. This can

be overcome by mixing ceramic with, for example, polyesters, in order to form a composite that has good biodegradability, a high affinity for cells of polyesters, as well as osteoconductivity together with mechanical strength of calcium phosphates.

Mechanical properties can be enhanced by cross-linking. Arvidson et al. (2011) [175] give examples of polypropylene fumarate and CaSO4/-TCP materials that are similar to those of cancellous bone substitutes, with compressive strength of 5 MPa and modulus of 50 MPa during degradation [177].

Within the stem cell niche, nanoscale interactions with ECM components form another source of passive mechanical forces that can influence stem cell behaviours [178]. The ECM is built of a broad spectrum of structural polysaccharides and proteins that span over different length scales. Such a connection between stem cells and their nano-environment enables long-term maintenance and control of stem cell behaviour. The possibility to fabricate such small-scale technologies and platforms makes it possible to gain valuable insights into stem cell biomechanics [179].

Currently, scaffolds manufactured from nanotubes, nanoparticles, and nanofibres have emerged as promising candidates for better mimicking the nanostructure of natural ECM. They resemble it, and can be efficiently used to replace damaged tissues [110].

The ideal bone tissue scaffold should be osteogenic, osteoinductive, and osteoconductive [180]. The aforementioned biomimetic efforts include choosing biomaterials that are naturally present in bone, like collagen or hydroxyapatite. Other factors include incorporating growth factors like BMPs and fabricating multiple scale architectures in the scaffold.

It was confirmed by Gong et al. (2015) [178] and Kim et al. (2013) [181] that using nanogrooved matrices mimicking the natural tissues made it possible for the body and nucleus of hMSCs with the sparser nanogrooved pattern to become elongated and orientated more along the direction of nanogrooves than those with the relatively denser nanogroove patterns. The formation of cytoskeleton is crucial for the shape effect on the stem cell differentiation, while a type of synthetic ECM comprised of hierarchically multiscale structures can provide native ECM-like topographical cues for controlling the adhesion and differentiation of hMSCs. Interestingly, the platform that integrates hMSCs into the PLGA scaffold showed potential to regenerate the osseous tissues without the need for further surgical treatments.

Synthetically nanofabricated topography is also a factor that can influence the cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organisation [182]. The conclusion is that there is an involvement of cytoskeleton into the stem cells' physiology, suggesting the importance of the force balance along the mechanical axis of the ECM–integrin–cytoskeleton linkage, and their regulation by the mechanical signals in the stem cell niche [183]. Nowadays, there are additional possibilities of printing biocompatible scaffolds using the selective laser melting (SLM) method [184].

#### **6. Tissue Engineering-Stem Cell Application in Bone Augmentation**

Stem cells play vital roles in the repair of every tissue of the organism. They are undifferentiated cells, capable of renewing themselves and, by differentiation, they can be induced to develop into many different cell lineages [185]. They have been proved to be promising in tissue regeneration, as well as in the augmentation process. During bone reconstruction procedures, surgeons harvest autologous bone from the patient in order to transplant the graft to the injured site [186]. Moreover, autologous bone grafts still have an unpredictable resorption rate [187]. Nowadays, regeneration of large bony defects is still difficult to manage, despite many advances in bone regeneration treatment [37]. A tissue-engineering model is being promoted as an efficient, "state-of-the-art" technology for major osteogenesis [188,189].

In the view of increasing demands for bone grafting and limitations of "gold standard" procedures, surgeons are looking for a better approach. Tissue engineering allows combining synthetic scaffolds and molecular signals together with mesenchymal or bone marrow stem cells [175] to form hybrid constructs. The classical approach of tissue engineering concerns harvesting stem cells from the bone

marrow; then isolating and expanding them; and, at the end, inserting the cells on a suitable synthetic or natural scaffold, before implantation into the same patient [190].

The goal of the modern approach is to reach stem cells present in more accessible sources in the human body, like periodontal ligament or deciduous and permanent teeth. According to Arvidson et al. (2011) [175], it is assumed that the perivascular region in the dental pulp is the niche for pulp-derived stem cells (PDSCs) of mesenchymal origin. In in vitro studies, PDSC are able to regenerate, and they have multilineage potential and plasticity [191] (including chondrocytes and osteoblasts).

Stem cell therapy demonstrates extraordinary value for many severe injuries and diseases. It includes key elements like extracellular matrix scaffolds and stem cells [192]. Furthermore, clinical trials about jaw bone regeneration applied in dental areas have demonstrated positive results [185].

Fortunately, different pre-osteogenic cells types can be used in the practice of bone regeneration. This type of cell is further differentiated into osteogenic cell lineages.

Nowadays, tissue engineering is a process focused on harvesting of multipotent stem cells from an autologous source and their successive in vitro culture. Then, the amount of such cells is increased within the injured tissue [193]. A major disadvantage of the stem cell transplantation is the need for large amounts of cells and accessibility.

There are different kinds of stem cells present in the human body; nevertheless, currently, there are only two main types of them that are used in clinical practice, namely mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs).

#### *6.1. Mesenchymal Stem Cells Use*

MSCs are multipotent stromal cells that can differentiate into a variety of cells [194], including chondrocytes and osteoblasts. Even though bone marrow was the original source of MSCs, there are alternatives that have been drawn from the other adult's tissues [195,196]. MSCs are nonhematopoietic, which results in their lack of contribution to the formation of blood cells like that of hematopoietic stem cells [197].

MSCs have been isolated from nearly every tissue possible, including brain, spleen, liver, kidney, lung, synovial membrane, or muscles [198–201]. They can be relatively easily expanded and differentiated into multiple tissue lineages, which makes them crucial in present and future tissue-engineering [202]. Another key feature of MSCs is their rapid expansion in vitro without loss of their characteristics. Bruder et al. [203] and Cancedda et al. [204] proved that bone marrow stem cells (BMSCs) are even capable of retaining their undifferentiated phenotype for 38 doublings, which eventually results in billion-fold expansion. MSCs transplanted systemically are able to migrate to specific site of injury in animals, which proves their migratory capacity.

They can be identified by the expression of several molecules, including CD105 (SH2) and CD73 (SH3/4). Additionally, they are negative for hematopoietic markers CD34, CD45, and CD14 [205].

Ashton et al. [206] have carried out an interesting experiment. They cultured freshly isolated rabbit marrow cells both in vitro and in diffusion chambers in vivo. The differentiation of osteogenic tissues in the diffusion chambers had to be divided into two categories: the first was the formation of bone in a fibrous layer surrounding cartilage, and the second was an intramembranous bone, formed directly within fibrous tissue that was not associated with cartilage. The authors suggested the presence of osteogenic precursors that had the potential to control the differentiation process via either of two major paths of skeletal development in embryo.

In the past, the MSC differentiation process in vitro involved incubating a confluent monolayer of MSCs together with β-glycerophosphate, ascorbic acid, and dexamethasone for 2–3 weeks [207]. The problem with the use of such factors in order to influence the cells was that it implausibly reflected physiological signals that MSCs received during osteogenesis in vivo. On the other hand, currently, the role of BMPs was investigated, which resulted in the promotion of bone growth in both humans and animals models [208].

#### *6.2. Hematopoietic Stem Cells Use (BMMSCs)*

Bone marrow-derived mesenchymal stem cells (BMMSCs) remain the most widely used osteogenic cells in bone tissue engineering research. They are present in adult bone marrow. They can be successfully used as an alternative for bone grafting, because of their immense replicative and differentiation capacity to form numerous connective tissue cells. BMMSCs can be isolated from the iliac crest [196]. Additionally, they can be obtained from orofacial bones, such as mandible and maxilla bone marrow suctioned during dental treatments (dental implantation), orthodontic osteotomy, cyst extirpation, or third molar extraction [209]. It has to be emphasised that, in both human and animal studies, the bone grafted from the craniofacial area was characterised with greater results and higher bone volume than when it was extracted from rib or iliac crest [210,211].

Mashimo et al. [212] positively evaluated alveolar ridge regeneration in the extraction sockets of mice. Stem cells were implanted immediately into the injured area in femur and tibia. Histological analysis proved that, after 3 and 6 weeks, the experimental group contained a greater quantity of bone marrow than the control group. BMMSCs have a number of advantages strictly connected to bone regeneration, including dynamic proliferation, relatively easy isolation, and expansion in vitro, as well as a lack of ethical controversy related to their medical use [213,214].

#### **7. Conclusions**

This review mainly focused on summing up the utility of nanomaterials and stem cells in maxillofacial bone tissue regeneration. Many recent scientific works in the fields of tissue engineering were investigated to demonstrate that both nanomaterials and stem cells can be successfully used as materials for guiding cell differentiation, proliferation, and organisation. It is important to underline that all the previously mentioned studies indicate the fact that nanomaterials alone may not be a complete answer to generate successful bone regenerative scaffolds. One of the primary challenges is to use innovative processing technologies in combination with nanomaterials. The construction of an ideal biomimetic nanocomposite would also require incorporation of a hierarchical design. Eventually, it could be possible to obtain an optimal scaffold in combination with several materials and techniques.

Recent advances in the fields of nanotechnology and tissue engineering have established great promise for finding treatments in bone defects and have led to considerable progress in designing and fabricating bone graft substitutes. For instance, impressive progress in the synthesis and functionalisation of graphene materials has opened up new possibilities for exploring their applications in tissue engineering. The primary function of an optimal biomaterial scaffold is to support the area undergoing reconstruction, providing adequate initial mechanical strength. It should also trigger a new bone formation, and later on gradually degrade, without causing an inflammatory response. The selection of the most appropriate scaffolding material is crucial in a tissue-engineered construct.

Scientific works focusing on stem cells have confirmed that mesenchymal stem cells derived from bone marrow are multipotential. They are attractive candidates for cell-based therapy, owing to self-renewal and immunosuppressive properties. Depending on culture conditions, they may differentiate into a plethora of cell types, including osteoblasts and chondrocytes. Thus, MSC comprise a readily available and abundant source of cells for tissue engineering applications. The lack of immunogenicity of MSC has opened up the potential of using those cells in tissue repair. The idea of such strategies is to take advantage of the body's natural ability to repair injured bony tissue with new bone cells, and to remodel the newly formed tissue. Regardless of cell source, live cell-based implants appear to be superior to cell-free alternatives for bone tissue regeneration. Further research should be focused on developing techniques that combine both nanomaterials and stem cells therapies in order to allow even better clinical outcomes in the future.

**Author Contributions:** Conceptualization: M.D. and R.J.W.; Methodology: W.Z., M.D., and R.J.W.; Software: W.Z., M.D., and R.J.W.; Validation: M.D. and R.J.W.; Formal analysis: W.Z., M.D., and R.J.W.; Investigation: W.Z., M.D., Z.R., M.S., and R.J.W.; Data curation: W.Z., M.D., Z.R., M.S., and R.J.W.; Writing—original draft preparation: W.Z., M.D., and. R.J.W.; Writing—review and editing: W.Z., M.D., and R.J.W.; Supervision: M.D. and R.J.W.; Project administration: R.J.W.; Funding acquisition: R.J.W. All authors read and approved the final manuscript.

**Funding:** This research was funded by National Science Centre (NCN) grant number UMO-2015/19/B/ST5/01330 entitled '*Preparation and characterisation of biocomposites based on nanoapatites for theranostics*' and grant number UMO-2019/33/B/ST5/02247 entitled '*Preparation and modulation of spectroscopic properties of YXZO4, where X and Z-P*5+*, V*5+*, As*5+*, doped with "s*2*-like" ions and co-doped with rare earth ions*'.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Application of Selected Nanomaterials and Ozone in Modern Clinical Dentistry**

**Adam Lubojanski 1, Maciej Dobrzynski 2, Nicole Nowak 3, Justyna Rewak-Soroczynska 3, Klaudia Sztyler 4, Wojciech Zakrzewski 1, Wojciech Dobrzynski 5, Maria Szymonowicz 1, Zbigniew Rybak 1, Katarzyna Wiglusz <sup>6</sup> and Rafal J. Wiglusz 3,\***


**Abstract:** This review is an attempt to summarize current research on ozone, titanium dioxide (TiO2), silver (Ag), copper oxide CuO and platinum (Pt) nanoparticles (NPs). These agents can be used in various fields of dentistry such as conservative dentistry, endodontic, prosthetic or dental surgery. Nanotechnology and ozone can facilitate the dentist's work by providing antimicrobial properties to dental materials or ensuring a decontaminated work area. However, the high potential of these agents for use in medicine should be confirmed in further research due to possible side effects, especially in long duration of observation so that the best way to apply them can be obtained.

**Keywords:** dental nanomaterials; dental implants; endodontics; prosthetic; ozone; antimicrobial activity; microorganisms

#### **1. Introduction**

Ozone is a gas composed of three oxygen atoms: the molecular weight is 47.98, it is an unstable substance, which quickly releases single oxygen atoms, and the half-life is 40 min at 20 ◦C and nearly 140 min at 0 ◦C. Ozone is colorless and has a characteristic smell. It occurs in nature as the ozone layer which protects organisms from ultraviolet rays. Ozone is heavier than air and it moves down where gas combines with pollutants, which is known as the self-cleansing phenomenon [1–3]. It is an extremely strong oxidizer, 1.5 times stronger than chloride when comparing their anti-microbial potential [4]. A gas mixture of 0.5 to 5% of ozone and 95 to 99.5% of oxygen is used in medicine. For almost a century, singlet (nascent) oxygen is known for its ability to inactivate bacteria, fungi, and viruses. Progress in medicine opens new applications for ozone therapy [5,6].

Silver particles have been demonstrated to be effective components in adhesives, implants, or prosthetic materials. They have an electron configuration of [Kr]4d105s1, and it is now possible to produce silver nanoparticles with controlled morphology and size, as well as specific target functions and homogeneity [7,8]. Additionally, they can be successfully used in orthodontics [9] as multifunctional building blocks for dental

**Citation:** Lubojanski, A.; Dobrzynski, M.; Nowak, N.; Rewak-Soroczynska, J.; Sztyler, K.; Zakrzewski, W.; Dobrzynski, W.; Szymonowicz, M.; Rybak, Z.; Wiglusz, K.; et al. Application of Selected Nanomaterials and Ozone in Modern Clinical Dentistry. *Nanomaterials* **2021**, *11*, 259. https:// doi.org/10.3390/nano11020259

Received: 21 December 2020 Accepted: 11 January 2021 Published: 20 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

materials [10], components for tissue conditioner, as well as act synergistically with several types of antibiotics [11,12]. Antifungal effects of silver particles against *Candida albicans* can be especially observed when the material is added to silicone-based liners or resins [13]. With regard to antibacterial activity, particles of silver influence the permeability of bacterial membrane, leading to its disruption. The material is responsible for the stimulation of oxidative stress resulting in thedestruction of cellular structures, such as DNA [14], lipids or proteins, eventually causing the destruction of the entire bacterial cell. Abroad range of aspects of the synthesis, as well as application and toxicology of silver particles, have been covered in recent reviews [15,16].The time needed to fully release Ag<sup>+</sup> from a particle depends on dissolution processes which depend on pH, the concentration of the dissolved O2, surface coating or ionic strength of the medium [17].

Copper oxide (CuO) plays a significant role as a bactericidal and antifungal agent [18]. It is the simplest element of Cu compounds, that reveal a range of potential physical properties such as high-temperature superconductivity, spin dynamics or electron correlation effects [19], and is cheaper than silver oxide. It has high surface areas with uncommon crystalline structures [20,21] and additionally can improve fluid viscosity and enhance thermal conductivity. These characteristics make CuO a potentially useful energy-saving material. Its nanoparticles are successfullyused as additives in lubricants, metallic coatings or polymers [22]. The extremely high surface areas and atypical morphologies make CuO nanoparticles enhance the shear bond strength of adhesives, additionally influencing their antimicrobial characteristics, which allow them to dose-dependently inhibit, for example, *Escherichia coli* bacilli, but not *Salmonella* Typhimurium [23]. *Streptococcus mutans* cocci, on the other hand, are affected by CuO in a similar way as they are affected by particles of silver [24].According to other studies, CuO can decrease biofilm formation from 70% to up to 80% [25].

Titanium dioxide (TiO2) is a photocatalyst and is widely used as a self-disinfecting and self-cleaning material for surface coating in a variety of applications [26].Currently, titanium and its alloys are broadly used in dental implantology due to their excellent biocompatibility and good mechanical characteristics. Thanks to its properties, including nontoxicity and super-hydrophobicity, it has been applied in removing bacteria and harmful organic materials from both air and water, acting as a sterilizing agent in places such as medical centers [27]. However, TiO2 can be activated only in the low-ultraviolet (LUV)range (<400 nm). Apart from its disinfecting characteristics, recent research has proven that TiO2 allows localized drug delivery with the use of nanotubes [28].

Metal nanoparticles can be obtained by converting metals into fine particles with a diameter smaller than 100 nm. Platinum nanoparticles (PtNPs) have been reported in several studies [29,30] to have antibacterial and anti-inflammatory effects, and because PtNPs were demonstrated to be a potent antioxidant in vitro, their addition to resinbased materials may also improve their biocompatibility [31]. Additionally, platinum particles mixed with a 4-methacryloyloxyethyl trimellitic anhydride (4-META)/methyl methacrylate (MMA) adhesive increase the dentin bond strength twice when compared to the regular material [32]. Contact between Pt particles and bacteria has reportedly led to the decomposition of the latter [33]. Yang et al. [34] confirmed that platinum particles are unlikely to cause allergy, have a potential for clinical application and do not cause genotoxic potential.This review is focused on using nanotechnology and ozone in modern dentistry. Moreover, the antibacterial properties of the agents can allow for more effective methods of treatment with fewer complications.

#### **2. Antimicrobial Properties**

#### *2.1. Gaseous Ozone, Ozonated Water and Ozonated Oil*

In stomatology, three different forms of ozone are used: gaseous ozone, ozonated water and ozonated oil (sunflower oil, olive oil, groundnut oil) [35].

There are numerous research studies describing the antibacterial activity of ozone against oral pathogens. One of such studies evaluated the antibacterial effect of ozone

against the pathogenic *Enterococcus faecalis* culture on a model of human teeth prepared using a specific protocol [36]. Samples were divided into four groups and for each of them, a different treatment was applied (ozone, photo-activated disinfection, saline as a positive control and NaOCl as a negative control). After 7 days of incubation, CFU (colony-forming unit) values were calculated. Both ozonation and photo-activated disinfection were revealed to be effective because the numbers of grown colonies were reduced [36]. Similar research was performed by Camacho-Alonso et al., who compared the activity of ozone with that of NaOCl solution, chlorhexidine, tri-antibiotic mixture, propolis and photodynamic therapy against *E. faecalis*. All the applied procedures were effective [37]. A comparison of a greater group of antibacterial procedures was conducted by Sancakli et al., who analyzed not only ozonation but also chlorhexidine and laser therapy Er:YAG (Erbiumdoped Yttrium Aluminium Garnet laser), KTP crystal (PotassiumTitanylPhosphate crystal (KTiOPO4)), alone or in combination, against *Streptococcus mutans*. The obtained results revealed that the most efficient procedures were chlorhexidine, the combination of Er:YAG laser with ozone, Er:YAG with ozone and chlorhexidine (but its effect was weaker than for chlorhexidine alone) and the combination of KTP laser with ozone and ozone and chlorhexidine. Generally, ozonation and laser therapy applied alone were insufficient and required combining with other factors [38]. Besides gaseous ozone, also the antibacterial activity of ozonated olive oil was investigated using the direct contact agar diffusion test (the measurement of inhibition zones) on *Aggregatibacter actinomycetemcomitans*, *S. mutans* and *Prevotella intermedia* isolates. Ozonated olive oil inhibited the growth of all the tested strains but antibacterial effect was weaker than for chlorhexidine, which was used as a reference sample. The authors also evaluated minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) using the standard broth dilution method. Unfortunately, only a 10% solution of ozonated olive was able to inhibit the growth of *A. actinomycetencomitans,* and for *S. mutans* and *P. intermedia*, no MIC values could be determined using 0.01–10% solutions of the oil. Consequently, MBC values were also not determined. This result is not satisfactory, especially when compared with the formulation of chlorhexidine, where a 0.01% solution was already bactericidal [35]. There are also numerous research studies describing clinical trials with ozone. In the work of Kruni´c et al., ozone treatment was applied to patients with deep carious lesions. Chlorhexidine was applied in other patients as a reference. The experiments were performed on isolated *Lactobacillus* spp. as well as on the total population of bacteria. The data demonstrate that both applied treatmentprocedures caused a reduction in the number of bacteria, however, chlorhexidine was more effective [39]. In vivo experiments were also performed by Ajeti et al. The antibacterial efficacy of ozone combined with 0.9% NaCl, 2% chlorhexidine or 2.5% NaOCl was compared. The most effective of all of the tested combinations was the combination of ozone with NaOCl [40]. Apart from gaseous ozone and ozonated olive oil, ozonated water is also applied to reduce the risk of a microbial infection. In a clinical study by Anumula et al., patients were divided into two groups. One group received chlorhexidine, and the other ozonated water as an oral rinse. The saliva samples after 0, 7 and 14 days were diluted and cultivated to calculate CFU/mL values. The obtained results were very promising because the colony number of *S. mutans* decreased, and for ozonated water, the effect was even more visible than for chlorhexidine [41]. The findings of the research studies listed above confirm the antibacterial potential of different forms of ozone but, if possible, it should be combined with other techniques to obtain better results.

#### *2.2. Titanium Dioxide Nanoparticles*

Titanium oxide can reduce bacterial growth by damaging bacterial cell membranes, which leads to enhanced permeability and, as a consequence ofthe loss of vital cellular components, to death [42]. Antibacterial and antibiofilm effect of pure TiO2 coated on the surface of metal washers was investigated using the common strains inhabiting human oral cavity: *Streptococcus sanguinis*, *S. mutans* and *L. acidophilus*. In dentistry, biofilm contributes to formation of manyoral diseases (e.g. caries, periodontis, periimplantitis) therefore it is needed

to ensure that necessary prevention and rapid reaction capabilities are in place to deal with any such problems. The simplified process of biofilm formation is presented in Figure 1.

**Figure 1.** A scheme showing the stages of biofilm formation.

The smallest reduction in biofilm formation was observed for *L. acidophilus,* but for *S. sanguinis* and *S. mutans,* the antibiofilm effect was more significant and dose-dependent—it increased with dopant enhancement [42]. A combination of TiO2 with metals is frequently described. In a work by Chen et al., the antibacterial activity against *S. mutans* was also investigated. TiO2 as well as Ag/TiO2 were mixed with polymethyl methacrylate (PMMA), silanized aluminum borate whiskers (ABWs) and nano-ZrO2 to obtain a composite. An additive of pure titanium oxide and its silver-modified derivative to the resin decreased bacterial growth without increasing the cytotoxicity of the material. Nevertheless, it should be noted that the sample with incorporated silver was much more effective [43]. Titanium dioxide, alone and combined with silver, was also combined with polyacrylate resin polymer to obtain nanohybrid coatings on titanium discs. These materials have been tested against *Streptococcus salivarius* and the obtained results are promising [44]. The antibacterial

effect of TiO2 combined with silver was also investigated on *S. sanguinis* along with evaluating bacterial adhesion to the material in the form of titanium discs coated with TiO2 or Ag (alone or combined with hydroxyapatite). The results indicate that the discs coated with silver combined with hydroxyapatite were the most effective of all the tested samples [45]. In the work of Lavaee et al., *S. mutans* and *S. sanguinis* were isolated from saliva samples of patients and antimicrobial activity of TiO2, alone and combined with nano-Ag or nano-Fe3O4, was tested. The combination of these three nanoparticles (nano-TiO2 + nano-Ag + nano-Fe3O4) turned out to be the most effective of all the tested materials and this effect was observed for both tested strains. Moreover, the antibiofilm activity of this mixture was also observed [46]. Apart from silver, copper is another material that can be combined with titanium oxide to enhance its antimicrobial effect [47]. Titanium dioxide is also a promising material due to its photocatalytic properties: after UV exposure, it produces ROS, which enhances its antibacterial properties [48].

#### *2.3. Silver Nanoparticles and Ions*

Silver, especially in its ionic form, has the highest antimicrobial activity among metals. Its mode of action is complex and involves protein impairment by forming Ag-S bonds, which leads to respiratory chain dysfunction and damage to membrane pumps. Ag+ionsare also genotoxic—they interact with DNA which causes its condensation and inhibition of the replication process. Silver also stimulates ROS production, which is characteristic of metals [49]. Besides releasing ions, silver nanoparticles can also affect bacterial cells by themselves. One mode of action is based on attaching to the surface of a cell and leading to the denaturation of cell membrane as a result ofthe accumulation of particles. Due to their nano-size, they can also penetrate through the cell wall, and disturb metabolic processes and bacterial signal transduction [50]. There is an extensive body of research describing antibacterial activity of silver-based nanomaterials against oral pathogens. In the work of Liu et al., polyetheretherketone (PEEK) was coated with 3–12 nm of nano-silver coatings. The antimicrobial potential of the obtained materials was evaluated usingan *S. mutans* strain. The obtained results indicate that efficacy increases with the thickness of the silver layer, however even a 3 nm-thick sample effectively prevented bacterial growth. The number of bacterial colonies attached to the surface of silver-coated materials was also reduced [51]. *S. mutans* was also used in the research of Kim et al., who evaluated the antimicrobial potential of feldspathic porcelain combined with nano-sized silver at concentrations of 0%, 5%, 10%, 20% and 30%. All the tested samples, even the non-doped one, inhibited bacterial growth, but the most significant reduction was observed for the highest concentration of silver (30%) [52]. In order to evaluate antibacterial activity of silver nanoparticles, a model of human teeth (after extraction) was prepared and, after cleansing, contaminated with *E. faecalis*. After 21 days of incubation, the samples were divided into three groups treated with different intracanal dressings: Ca(OH)2 paste, Ca(OH)2 paste mixed with chlorhexidine and Ca(OH)2 paste supplemented with a suspension of silver nanoparticles. Samples prepared in such a way were incubated for 1 week and 1 month and then the colonies were counted. Moreover, SEM (Scannicelectron Microscopy) images were measured. The obtained data revealed that all of the applied dressings effectively reduced bacterial growth and adhesion, and the silver additive improved this effect [53]. Interesting results were also obtained by Yin et al., who prepared sodium fluoride solution mixed with different concentrations of polyethylene glycol-coated silver nanoparticles (PEG-AgNPs). Antibacterial activity (half maximal inhibitory concentration, IC50) of these materials was examined using an *S. mutans* strain. The obtained data indicate that PEG-AgNPs effectively kill bacteria at a concentration of 21.16 ppm ofsilver and this concentration was non-cytotoxic for a human gingival fibroblast cell line (HGF-1) [54]. In the work by Bacali et al., antibacterial activity of polymethyl methacrylate (PMMA) denture resin combined with graphene and Ag nanoparticles was tested against, among others, *S. mutans*. Interestingly, pure PMMA also inhibited *S. mutans* growth but the addition of graphene and silver nanoparticles

intensified this effect [55]. Three oral microbes (*E. faecalis*, *S. mutans* and *Streptococcus oralis*) were used to evaluate the antimicrobial activity of bioactive glass combined with silver nanoparticles and tetracycline. The prepared materials effectively released the loaded drug, which led to the inhibition of bacterial growth. However, materials without the addition of tetracycline were not so effective. Silver-combined bio-glass slightly inhibited the growth of *E. faecalis* but no such effect was observed for *S. mutans* [56]. Chitosan was also used as a carrier of drug (gentamicin) and silver nanoparticles. Pure chitosan slightly reduced bacterial growth (*S. mutans*) but adding gentamicin or the combination of Ag/chitosan or Ag/chitosan/gentamicin enhanced the antibacterial effect. Surprisingly, no differences between the action of these three materials were detected [57]. Epigallocatechin gallate was another material used as a carrier for nanoparticles. Antimicrobial efficacy was evaluated for silver nanoparticles and compared with ionic silver in the form of AgNO3. The results indicate that the effectiveness of silver nanoparticles against *S. mutans* is higher than that of the AgNO3solution. The red-to-green ratio calculated from confocal laser microscopy (dead/live cells proportion) was also higher for AgNPs. Moreover, in microscopic images, it can be seen that bacteria did not form a biofilm structure in the AgNPs sample, whilst for AgNO3 and control (water), a fully grown biofilm can be noted. Additionally, AgNPs significantly reduced the production of lactic acid and polysaccharides by *S. mutans* biofilms [58].

#### *2.4. Platinum Nanoparticles*

The antibacterial effect of platinum is based on the ability to decompose bacterial cell structure. The exact mechanism is uncertain and, as for metals in general, probably connected with the increase of reactive oxygen species (ROS) production, which results in cellular damage via oxidative stress [33]. Platinum nanoparticles (PtNPs) have an antimicrobial effect against oral pathogens. In the work of Itohiya et al., PtNPs were obtained via direct irradiation of platinum with an infrared pulsed laser and tested on common dental bacteria: *S.mutans*, *E. faecalis* and *Porphyromonasgingivalis*. The range of applied concentrations of PtNPs was 1–20 ppm and the concentrations above 5 ppm completely inhibited the growth of all the tested strains [33]. Platinum nanoparticles were also combined with polymeric PMMA and the antibacterial activity of such a combination was evaluated using *S. mutans* and *Streptococcus sobrinus* reference strains. The results indicate that platinum nanoparticles exhibit antimicrobial activity against the planktonic form of both tested strains (growth reduction). Moreover, platinum-combined polymer turned out to be less prone to bacterial adhesion than pure PMMA [59].

#### *2.5. Copper Oxide*

Copper oxide nanoparticles (CuONPs) can be obtainedwith the use of various methods of synthesis. By using different preparation methods, particles with different sizes can be produced; for example, through colloidal-thermal synthesis, precipitation synthesis, microwave irradiation or sol-gel techniques, very small particles can be obtained which are estimated from 3 to 10 nm. Larger particles from 10 to even 30 nm can be received via sonochemical synthesis, spinning disk reactor, solid-state reaction, microemulsion system or thermal decomposition [60].

CuONPs, due to their small size, are well known for their optical and magnetic properties and electrical conductivity. However, copper oxide nanoparticles are also known for their biological properties, in particular for their antimicrobial activity; nevertheless, cytotoxic properties were well estimated in both in vitro and in vivo models [60].

Skin and the respiratory system are parts of the human body which are the most exposed to CuOPNs. The cytotoxicity of copper oxide nanoparticles against normal cell lines such as human keratinocytes (HaCaT)and mouse embryonic fibroblasts (MEF) was estimated by Luo et al. Copper oxide nanoparticles, in a dose-dependent manner (from a concentration of 20 μg/mL), distinctly induced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk); moreover, level of p53 was significantly decreased

in HaCaT and MEF cell lines, and a reduction of the amounts of viable cells was also observed in a concentration-dependent way, which clearly indicates the toxic effect on the above-mentioned cell types [61]. Another study confirmed the cytotoxic effect of CuONPs on human lung epithelial cells (A549). Concentration from 10mg/mL to 100μg/mL drastically decreased cell viability. CuONPs particles were compared with CuO bulk particles and it was clearly demonstrated that CuO bulk particles exhibitedaweaker toxic effect (concentration 58 mg/mL) than CuO nanoparticles (concentration of 15 mg/mL). TEM images (Transmission Electron Microscopy) show the entry of CuOPNs into the intracellular environment, but also into the nucleus, mitochondria and lysosomes. Mitochondrial influx of CuO nanoparticles may lead to inducing its depolarization and probably caused the generation of reactive oxygen species (ROS) as well, which elevated oxidative stress [62]. The cytotoxic effect of CuONPson the A549 cell line was also confirmed by an independent research of Akhtar et al. [63]. The toxic effect of CuO nanoparticles against human blood lymphocytes was also evaluated by estimating cell viability, ROS generation, peroxidation of lipids, but also by estimating lysosomal and mitochondrial disruption. The data clearly indicated the disintegration of mitochondrial membrane and an elevated generation of reactive oxygen species (ROS), and lysosomal damage was also visible. The viability of human blood lymphocytes drastically dropped and was estimated at IC50 after the treatment with CuO nanoparticles at a concentration of 385 μM [64]. Sun et al. compared the cytotoxic properties of several different metal nanoparticles, such as Fe3O4, Fe2O3, SiO2, TiO2 and CuO, and exposed to them a human type II alveolar epithelial cell line (A549), human non-small cell lung cancer (H1650) cells and human nasopharyngeal carcinoma (CNE-2Z). Copper nanoparticles were determined to be the most toxic among all the tested nanoparticles and on all the tested cell lines, and the toxic effect was estimated at a concentration of 30 μg/mL [65]. Additionally, Beltrán-Partida et al. estimated the potential cytotoxic effect of CuNPs, CuCO3 and the antifungal drug triclosan on primary human gingival fibroblasts (HGF) isolated from a clinically healthy young (15 years old) male patient as a suitable model for this type of test. Among all the tested compounds, CuCO3 was the most toxic on HGF when compared to CuNPs which exhibited IC50 at a concentration of 137.4 μg/mL [66].

The antimicrobial potential of copper has been known for ages but, as a result ofthe development of nanotechnology, nano-sized copper oxide also became an issue of interest. The antibacterial effect of CuO was described by Ramazanzadeh et al., who prepared brackets coated with CuO, ZnO or a 1:1 combination of the materials which was tested on *S. mutans* after 0, 2, 4, 6 and 24 h. No effect was observed for ZnO but CuO completely inhibited bacterial growth after 4 h of incubation. The ZnO/CuO mixture was even more effective because the bacterial growth was affected after 2 h of treatment [67]. The susceptibility of *S. mutans* was also tested by Toodehzaeim et al., who prepared nanocomposites doped with 0.01%, 0.5% and 1% of CuO and the inhibition zones were measured around discs placed on the surface of agar plates with bacteria [68]. MIC50 values were determined for CuO by Amiri et al. on the *S. mutans*, *Lactobacillus acidophilus* and *Lactobacillus casei* strains. The obtained values are as follows: <1 μg/mL for *L. acidophilus*, 1–10 μg/mL for *S. mutans* and 10 μg/mL for *L. casei*. At the concentration of 1000 μg/mL, a complete reduction of growth was observed for all the tested bacteria [21].

#### **3. The Use of Ozone and Nanoparticles in Dentistry**

#### *3.1. Restorative Dentistry*

Tooth decay is a very common disease: The Global Burden of Disease Study 2017 reported that there are 2.4 billion people suffering from this illness, withcaries of primary teeth as a problem concerning 530 million children [69]. Ozonation is an efficient treatment that can help to reduce the amounts of microorganisms in oral cavity preventing to dental caries—the most common disease worldwide. In Figure 2 has been shown the number of oral diseases worldwide indicateing a serious problem for many people.

**Figure 2.** A graph showing the number of patients affected by oral diseases. Teeth caries is one of the most common illnesses in the world.

The consequence of primary tooth caries is a high risk of infection in permanent teeth; for this reason, it is important to prevent early childhood caries (ECC). Ozone therapy seems to be an appreciable method to supervise carious lesions. Ximenes, Cardoso, and Astorga et al. examined the potential of gaseous and aqueous ozone in the reduction of *S. mutans*, *L. acidophilus* and *E. Faecalis*: scientists took note of the last-mentioned bacteria, which is not a typical pathogen for ECC, but it is the species with the highest resistance to antimicrobial factors. Researchers point out good effectiveness of both methods in the evaluation of ECC [70]. Other studies show theefficacy of gaseous ozone in the reduction of *Lactobacillus* sp. in deep carious lesions to be similar to the efficacy of chlorhexidine [39]. On the other hand, Hauser-Gerspach et al. point out in their research that there is no significant difference in the reduction of viable bacteria in cavitated carious lesions in children in vivo, while aqueous ozone seems to be more effective in some cases [71]. Duangthip et al. also indicate a lack of efficacy of ozone in controlling dentin caries [72]. Another study points to ozone therapy as a good alternative for patients experiencing anxiety [6]. Kalnina [73] found no significant difference in the efficacy of ozone and fluoride varnish and sealants in reducing the probability of caries in permanent tooth. Similar study results were observed in the remineralization of initial caries in enamel. Due to these properties, ozone can be a powerful alternative for the prevention and treatment of caries. The author drew attention to the high cost of ozone generator compared to fluoride varnish and sealants [73]. Microleakage is a common problem in dentistry, withsecondary caries as its frequently observed effect. According to the study, ozone has no negative influence on pit and fissure sealants which protect the surface in more than 85% of caries in children. Ozone can be used to disinfect a previously prepared surface without affecting the adhesion of pit and fissure sealants [74]. A similar observation on the subject of initial micro-tensile bond strength of a self-etch adhesive to dentin was provided by Dalkilic et al., whoindicated a safe use of ozone as a disinfectant [75]. Another advantage of ozone is the absence of pain during the therapy, which makes it easier to work with anxious patients [76]. Almaz et al. noted the wide range of applications of ozone and effective properties in caries treatment, prevention, and remineralization. However, some researchers indicate inefficacy of ozone [77].

Mineral Trioxide Aggregate (MTA) is often used in the case of operator errors, such as root perforation or exposure of the pulp. This material can be modified with TiO2 and other nanoparticles to improve the properties. TiO2 provides an antimicrobial effect, self-cleaning and photo-elastic properties [78]. Another material commonly used in dentistry is glass ionomer (GIO), whose main components are calcium or strontium alumino-fluoro-silicate glass powder, which reacts with water-soluble acidic polymer. The resultant material is valued for the ability to release fluoride ions to the surface which stop the cariogenic process. Addition of TiO2 nanoparticles increases the durability of GIO (especially to compressive strength), which is a significant disadvantage of the material. Garcia-Contreras et al. [79] mentioned a better antibacterial effect of GIO modified with TiO2 nanoparticles. However, the sensitivity of GIO to water has increased, which caused the loss of material. Ferrando-Magraner et al. concluded in their scientific research that the most beneficial application of titania nanoparticles is for dental bonding material [80].

Cavities filled with dental material after inaccurate preparation can cause secondary caries. Theaddition of nanoparticles to materials can prevent the need of replacement. Composite resin modified with Ag doped with ZnO nanoparticles demonstrates antibacterial properties. Studies indicate that compressive strength does not change significantly in comparison to unenriched composite resin. Another property of nanoparticles is the reduction of bacterial biofilm, which is more resistant than planktonic bacterial cells [81], [82]. Koohpeima et al. used Ag nanoparticles as a coating before the application of etch-and-rinse and self-etch adhesive systems. The strength of bond did not change negatively, Ag NPs did not affect the color, which could have been the case under the influence of metal NPs. Together with Ag NPs, it increases the chances of proper treatment [83]. Vazquez-Garcia et al. indicates that calcium silicate cements, white MTA and Portland cement with 30% of ZrO2, after manipulation with AgNPs solution, have good effectiveness against *E. faecalis* in both forms of biofilm and planktonic cells. Both materials have similar application, and addition of NPs does not negatively affect their properties [84]. Elgamily et al., in studies concerning a cavity disinfectant containing AgNPs and AuNPs in comparison to chlorhexidine (CHX), examined their impact on *S. mutans*. Both agents reduced the amounts of bacteria, but CHX was more effective [85]. Silver diamine fluoride (SDF) is an effective anticariotic agent which currently includes AgNPsas a result ofthe development of nanotechnology. Scientists recommend it, especially for children, as the agent has good antimicrobial properties [86]. On the other hand, Fakhruddin et al. [87] point out that even though in vitro results are positive, there is no evidence of good effectiveness in in vivo studies.

Amiri et al. examined the influence of CuO Np on oral bacteria and *Candida* species. According to research, CuO NPs have good antibacterial properties but they have a weak impact on *Candida* species. Due to antibiotic resistance of bacteria, this nanoparticle may be a good control measure for preventing caries [21].

#### *3.2. Endodontics*

Endodontic treatment consists of proper canal preparation followed by hermetic filling. It can be achieved with the use of chemo-mechanical procedures which include abundant irrigation and effective canal shaping. Root canal instruments widen the main root canal space and remove most of the canal content, at the same time, mechanical interference produces smear layer which forecloses proper disinfection and prevents tight sealing of the root canal system. Chemical cleansing removes the smear layer and plays a major role in the overall success of endodontic treatment. The main reason why chemical interference and disinfection protocols are so crucial is the complexity of the root canal system and rich network of channels which are unreachable to instruments. The most important characteristics of irrigators are their tissue-dissolving ability and low toxicity, combined with antimicrobial effect [88]. The goal of modern endodontic therapy and modern endodontic materials is to achieve maximal disinfection of the root canal system, smallest possible area of preparation which will provide long-term success and no reinfections.Ozone is considered as a beneficial choice of canal antiseptic. Its main advantages are high antimicrobial activity, low toxicity, and the fact that they do not generate drug resistance [89]. In root canal treatment, ozone is useful to eliminate *Enteroccocus fecalis*, *Peptostreptococcus micros*,

*Pseudomonas aeruginosa* and *Candida albicans*. Ozone is used in many forms such as gas, water, and oil. The water and gas forms can be used in the rinsing protocol. The oil form can be used as a medical insert in cases of dental pulp necrosis. The gas form provides high penetrability to lateral channels and root deltas which increases the chance of maximal disinfection. The concept of the device is simple, ozone is generated by the machine, then it is channeled through the handpiece to the affected root canals [90]. In Figure 3 has been shown an application of ozone therapy unit. Ozone is generated in the unit and then delivered to the root canal with an endodontics cannula. Silicone cap prevents ozone leakage. It has been shown deep penetration of ozone providing maximum disinfection rate.

**Figure 3.** Aschemeof application of ozone treatment.

After treatment, the gas is converted back into oxygen by the ozone neutralizer. According to the studies, the suggested method of ozone treatment in the root canal system is at the end of chemo-mechanical preparation. To improve its effect, the amount of organic debris left inside the root canal should be reduced to a minimum [91]. Many studies were conducted to verify assumptions connected with disinfection strength of ozone. Some of them noted that ozone water efficiency was similar tothat of 5.25% NaOCl [91–96], some of them obtained the same results with ozone gas, and in their case, the efficiency was similar to that of 3% NaOCl (Sodium hypochlorite) [97]. Results to the contrary were reported in some studies, in which after using ozone water irrigation, the canal content contained bacteria [98,99]. Ozone was investigated in many disinfection protocol combinations and according to a study of Hubbezoglu et al., the best action was accomplished when ozone was used at a concentration of 16 ppm with ultrasonic agitation [94]. Noites et al. achieved the best bacteria reduction effect with 2% chlorhexidine followed by 24 s of ozone [100]. This combination can be considered in the treatment of a resorbed apex, wide open foramen. Nevertheless, these indications should be estimated with caution [101]. Recaizan et al. compared the reduction of bacteria with the use of ozone to their reduction using other antiseptics. Bacteria reduction using ozone was 90.4% and it turned out to be more efficient than using Er.YAG and KTP lasers [99]. Scanning electron microscope examination was used in order tocomparedifferent treatment protocol results including a control group, NaOCl + EDTA (Ethylenediaminetetraacetic acid), NaOCl + citric acid, and NaOCl + ozonated water groups. Indentation distance increase turned out to be comparable in every protocol. Tubule densities, tubule diameters, total surface free energy and the Lewis

base polar force were higher using NaOCl + EDTA treatment compared to the control group, NaOCl + citric acid and NaOCl + ozonated water treatments. The comparison of contact angle and wettability of three sealer materials shows that the use of NaOCl + ozonated water groups significantly increases the wettability of Acroseal on the root canal dentine and decreases contact angle compared to the control group. The results for Apexite and Endomethasone material yield distinctive results, in which the usage of NaOCl + ozonated water increases the contact angle compared to the control group [102]. The Study of Bojar et al. represented results in which AH- 26 and EX fill root canal sealers shows an improved shear bond after root canal ozone treatment [103]. The conclusion is that ozone has a certain antibacterial effect, its major advantage is non-toxicity and penetrability, however ozone cannot be used alone as acanal antiseptic, although studies show that it might improve root canal therapy and should be included in the treatment protocol.

Nanotechnology can also be applied in endodontic treatment. It is considered to be a stimulating and up-to-date topic that needs to be elaborated. Nanoparticles are minute solid particles with a diameter of 1–100 nm [104]. Their advantages are ultra-small sizes, high surface area to mass ratio and high chemical reactivity. Their antimicrobial effect is the result of large surface area and high charge density. AgNPs is one of the nanomaterials that can be considered in endodontic treatment. It has antibacterial effect on both Gram-positive and Gram-negative bacteria. It also shows antiviral and antifungal action. The mechanism responsible for the antimicrobial effect is still not fully understood. The most plausible mechanism is the interruption of ATP particle and preventing DNA replication by free silver ions, production of reactive oxygen or direct damage of cell membrane by Ag+. It is certain that free silver ions play a crucial role in antibacterial action. Scientific evidence shows that release of Ag+ from smaller particles (<10 nm) is higher than from largerones. One of the disadvantages of the AgNPs is possible toxicity. AgNPs above 80 μg/mL could be considered cytotoxic [24].Preclinical studies on rats show effectssuch as accumulation in organs, discoloration, cytotoxicity or even sperm cell production disturbance [105,106].

AgNPs can be used for coating gutta-percha. Shantiaee et al. compared nanosilvercoated gutta-percha to an uncoated one and it resulted in slightly less leakage [107]. Lotfi et al. compared AgNPs solution to NaOCl. The size of silver particle used in this examination was 35 nm. Their results report a similar antibacterial effect of AgNPs and 5.25% NaOCl [108]. González-Luna et al. obtained the same results but with comparison to 2.25% NaOCl [109]. A study of Abbaszadegan et al. shows satisfactory annihilation of *E. faecalis* [110]. Nevertheless, some studies show that AgNPs were less effective against *E. faecalis* than NaOCl, therefore, at the moment, AgNPs can not be considered as a substitute for NaOCl [104]. In Figure 4 has been shown, in simplification, the use of Ag+ ions in irrigation solution for endodontic application. Their advantages are ultra-small size, high chemical reactivity, and antimicrobial properties. The solution enables good disinfectant penetration and good antibacterial effect.

Another interesting application of AgNPs is adding them to the mineral trioxide aggregate (MTA). The idea was to improve activity against strict anaerobes. The outcome showed efficiency against different bacteria and *C. albicans* higher than in the case of unmodified MTA [111]. Another tested combination was Ca(OH)2 and AgNPs. The result was that Ca(OH)2 showed better antibacterial effect in comparison to AgNPs alone or AgNPs + Ca(OH)2 [109]. The addition of 0.15% AgNPs and 2.5% dimethylaminohexadecyl methacrylate (DMAHDM) to the AH Plus paste significantly increased antibacterial activity against *E. Faecalis* [112]. Chlorhexidine (CHX)-AgNPs containing lyotropic liquid crystals showed excellent sterilization and inhibitory effect on *E. faecalis* lasting for more than one month with a bacterial annihilation rate of 98.5%.

**Figure 4.** A scheme of irrigation with AgNPs solution. AgNPsreleaseAg+ions.

Nano-MgO can also be included in endodontic treatment. Its advantages are antimicrobial activity and non-toxicity. In one of the studies, nano-MgO was compared to different disinfectants such as CHX and NaOCl. There was no substantial difference between nano-MgO solutions, 5.25% NaOCl and 2% CHX gluconate regarding the time required to inhibit *E. faecalis* and *S. aureus* growth. On the other hand, 5 mg/L nano-MgO presented better overall antibacterial efficacy in the elimination of *E. faecalis* than 5.25% NaOCl [113]. Copper and copper oxide nanoparticles present both antibacterial and antifungal effect. They produce superoxide ions, reactive oxygen and cause leakage of intracellular components that causes cell death. Antibacterial effect may be provided by hydroxyl radicals, damage of DNA and important proteins [114]. A crucial stage to obtain desirable antibacterial effect is the synthesis of NPs followed by stability in the medium, which is usually a polymeric matrix. A study by Hajipour et al. shows that the antibacterial strength of copper nanoparticles depends on both nanoparticle and bacterial concentration, temperature, pH and aeration [115]. Except for *S. aureus*, CuO NPs show higher strength compared to other nanoparticles such as Sb2O3, ZnO or NiO. The role of CuO nanoparticles is to create an enriched surface of the inorganic antibacterial agents. Copper NPs show bactericidal effect similar to triclosan and decrease the level of many bacteria populations, such as *S. aureus*, *E. coli* and *B. subtilis*. Cu NPs show potential as a component in endodontic materials, however further studies are needed [116]. A layer of nanometric TiO2 can be used as a coating on super-elastic rotary NiTi instruments. The idea is to improve mechanical and chemical properties without losing cutting effectiveness. The study showed that TiO2 coating improved cutting capacity, corrosion behavior and fatigue resistance [117].

#### *3.3. Dental Surgery*

Ozone has a wide range of applications in treatments such as implant placement, hemisections, extractions, bone regeneration and tissue healing. Often, it depends on a patient's condition, in these therapies' antibiotics are used prophylactically. It isavery important factor in dentistry to ensure that the presence of microbes in the field of work is reduced to a minimum. Researchshow significantly faster regeneration, reduction of complications and pain. Even in difficult cases of patients with diabetes and microangiopathy, ozone therapy

after topical application improves treatment. Animal research show better osseointegration in cases of reduced immunity [118]. Peri-implantitis is a frequently occurring inflammatory process caused by biofilm, it leads to bone loss and other complications [119]. In some research [120], scientists reported positive influence on the clinical attachment loss (CAL) and defect bone fill, which points to ozone decontamination as an effective method in bone healing in reconstructive surgery. Third molar surgery can cause pain, swelling and trismus.Kazancioglu et al. examined the influence of ozone. Due to its antimicrobial and analgetic properties, reduction of pain was observed when the gas was used one week before and one week after the extraction. However, ozone had no impact on trismus and swelling [121]. Other studies compared ozone and laser therapies: ozone was effective in pain treatment, as opposed to low-level laser therapies (LLLT), and it was also effective in reducing swelling and trismus. These methods can be alternative to corticosteroids and nonsteroidal anti-inflammatory drugs, which can cause side effects [122].

Titanium is commonly used in implantology owing to its adequate biocompatibility, however daily hygiene activities cause harm to the surface of implants. TiN coating enhances hardness, hydrothermal and ozone treatment boosts osteoconductivity and provides decontamination. This activity increases the chance of successful treatment outcome [123]. Efficacy of ozone treatment at implant surfaces also depends on material roughness, since higher roughness increases bacterial adhesion.

In the case of titanium and zirconia, both of which were polished, and zirconia was additionally acid etched, research showed high efficiency of gaseous ozone treatment. The study demonstrated reduction of *P. gingivalis* by more than 99.94% and *S. sanguinis* bymore than 90%, ozone was applied for 24 s at 140 ppm [124]. Isler et al. mention periimplantitis as a serious problem in dentistry, research demonstrates ozone therapy as an effective adjunction to surgical regenerative treatment. Better results of PI, GI, PD and the amount of bacteria in individual quadrants were observed by scientists. Additionally, no infraction of surfaces, in this case made of titanium and zirconia, was observed [125].

Antimicrobial properties of metal nanoparticles such as titanium dioxide, silver or copper dioxide make it possible to use them as implant modification. Amount of plaque biofilm is an important factor of implantation failure [104]. Titanium alloy is commonly used in dentistry; however, it does not show antimicrobial properties by itself [126].These properties can be improved by applying a surface coating, e.g., visible light active TiO2. This material can also be doped with metals and non-metal particles which improve its properties. However, further research is needed to design toxic-free nanoparticles [127]. The TiO2 nanoparticle has very beneficial antimicrobial properties which can prevent dental plaque formation [128]. Wettability of surface is important for correct osseointegration but, on the other hand, there are studies that point out better antimicrobial properties at the hydrophobic surface which characterizes TiO2 nanotubes. They have good biocompatibility which makes them suitable material for dental implants, they are also suitable for drugdelivery. These properties increase chances of successful treatment [129]. Nano-titania coating also has a positive effect on the peri-implant tissue whose osseointegration process is accelerated [130]. There are many microbes in the oral cavity which is why antibiotics are not effective enough for every pathogen; in addition, bacterial resistance reduces the efficacy of chemotherapy. In TiO2 nanotubes can be suspended in various substances, which further enhance its well known microbicidal properties. For better biocompability of implant nanohydroxyapatite, a coating can be added which does not affect the desired properties of titania nanotubes coated with Ag nanoparticles [126,131]. *S. aureus*, *S. epidermidis*, *E. coli* and *P. aeruginosa* are common causes of dental implant complications, such as peri-implantitis. Bacterial infection takes place even 30 min after dental treatment and nanoparticles can help in ensuring correct treatment [132]. Some research demonstrate that nanoporous titania has better antimicrobial properties than nanotube titania material, also the former ensures the release of the loaded drug for 7 days [133]. In Figure 5 has been shown the advantages of using nanoporous TiO2 loaded with LL37 which gives positive effect on osseointegration.

**Figure 5.** Ascheme showing difference in osseointegration between implant surface with and without coating.

Silver NP has a high antimicrobial and osseointegration value which makes this material good for implant coating. It reduces bacterial adhesion up to 50%. The previously mentioned titanium implant has good biocompatibility, which with the addition of silver NP coating, providesan outstanding antimicrobial effect. Nanoporous or nanotube titania coating in combination with silver NP ensure a wide range of antimicrobial properties [133,134]. In addition to the antimicrobial effect which depends on the size of nanoparticle (the smaller it is, the stronger its effect), silver NP coating applied to a titanium implant increases the density of bone [50]. Platinum nanoparticles have a wide range of applications in medicine, such as cancer treatment, bone allograft, bone loss and others. Due to its properties (antimicrobial, anti-inflammatory), PtNP may be suitable for the dental surgeries of guided bone regeneration and guided tissue regeneration [134].

#### *3.4. Prosthetic*

For removable partial dentures affect, i.e., periodontal tissues, maintaining their hygiene is a very important aspect that affects the long-term use of the prosthetic in the oral cavity by the patient. Therefore, new sterilization methods are needed that help maintain the hygiene of the prosthesis, and thus eliminate the formation of plaque (control its formation). Recently, the use of ozone, which has antibacterial properties, seems to be very promising. The plaque on the prosthesis that causes periodontitis consists mainly of *Lactobacillus*, *Propionibacterium* and *Arcalinia* bacteria. The *Propionibacterium* species were the dominant part of the flora. An equally important factor influencing inflammation of the oral mucosa from the prosthesis are yeasts of the *Candida albicans* genus. *C. albicans* produces large amounts of acetic and pyruvic acid, which causes inflammation. The yeast adheres well to the denture base materials in vitro, although the adhesion depends on the strains and environmental conditions. Denture-induced stomatitis is routinely encountered in clinical practice as a symptom of the plaque build-up on the dental surface (dentures), therefore effective plaque control should be initiated to prevent these consequences. An effective method is the use of ozone as a denture cleaning agent. The advantages of ozone in the water phase are its potency, ease of application, lack of mutagenicity, quick bactericidal action and suitability for use as a solution for soaking

medical and dental instruments [134,135]. The use of ozone as a denture cleaner is effective against the methicillin-resistant *S. aureus* and viruses [136].Ozone can be applied (used) to clean the surface of alloys in removable partial dentures with little effect on alloy quality in terms of reflectance, surface roughness and weight. Direct exposure to ozone gas was a more effective germicide compared to ozonated water. Therefore, ozone gas can be clinically useful for the disinfection of removable dentures [137]. In dentistry, ulcers can occur as a result of using dentures. They are usually located in the vestibular furrows of the maxilla and mandible, causing pain. The treatment of ulcers requires the discontinuation of the use of the prosthesis, its correction, laser therapy and oral and prosthesis hygiene. Treatment also includes the use of topical medications such as chlorhexidine mouthwashes, topical hydrogels, hydrogel dressings, and sometimes topical cortisone. The use of ozone in the treatment of ulcers also seems to be an interesting method due to its properties. Research by Bader et al. on a group of women and men wearing prostheses showed that ozone reduces the size and pain of traumatic ulcer, and accelerates the regeneration of diseased tissue, and thus shortens the treatment [138].

The field of prosthetics is another branch of dentistry in which nanoparticles can be implemented. Their main role is to provide better antimicrobial properties of materials used for making removable dentures. Without proper hygiene, these restorative alternatives can lead to denture stomatitis and cause both reversible and irreversible consequences. One of the studiescompared antifungal activity between a standard denture base (polymethylmethacrylate—PMMA) and one modified with AgNPs. The conclusion was that the addition of AgNPs to the denture base provides 105 less *C. albicans* adhesion than the control group after 24 and 48 h incubation [139]. Another study examined incorporation of silver-sulfadiazine-loaded MSNs into PMMA at up to 5%. Researchers examined mechanical properties and microbial effect against *Candida albicans* and *Streptococcus oralis* on removable and provisional dental restoratives. Results showed that the addition of Ag-MSNs decreased the adhesion of microbes and at the same time improvedmechanical properties of PMMA. The inorganic part of NPs provided better mechanical properties, at the same time, silver ions provided a microbial anti-adhesive effect. Li et al. also examined the addition of silver NPs and their effect on *Candida albicans* adhesion and biofilm formation. Their results demonstrated that the minimal concentration of silver NPs which provides the anti-adhesion effect on *C. albicans* is 5%. Lower concentrations do not ensure this effect [140]. Another study examined the addition of AgNPs to irreversible hydrocolloid impression materials such as Zelgan or Tropicalgin. Results showed better antimicrobial activity compared to standard materials. Adding silver NPs can also provide an increase of gel strength, permanent deformation or the flow of the material, although many of mechanical properties show different variations depending on the wt% of AgNPs [141].AgNPs can also be added to alginate impression powder to increase antimicrobial activity and reduce the risk of cross-contamination by bacteria, viruses or fungi [142]. Köro ˘glu et al. investigated the effect of adding solution of AgNPs to acryl liquid used for mixing with the powder part of acrylic material. In their study, 0.3, 0.8 and 1.6 wt% of AgNPs were used. Results showed that the addition of 0.8 and 1.6 wt% AgNPs decreased the flexural strength and elastic modulus of microwave-polymerized acrylic resin [143]. In the study of Oei et al., silver ions improved both antimicrobial and mechanical properties of PMMA and the antibacterial activity increased to 28. The study of Matsuura et al. showed that AgNPs implemented in tissue conditioners support antifungal activity against *C. albicans* and antibacterial activity against *S. aureus*, *Pseudomonas aeruginosa* for 4 weeks [144]. Many studies indicate that the addition of AgNPs increases antimicrobial properties of the material and at the same time, doesnotaffect its mechanical properties. It can be considered as a promising component of denture base, dental impression material or tissue conditioner, although further studies are needed. AgNPs can also be considered as a component of permanent prosthetic restoration materials such as porcelain. The idea is to improve fracture toughness and prevention of crack propagation. One of the studies revealed that AgNPs increase the fatigue parameter, shorten the time

required for slow crack growth and reduce crack growth rate [145]. Fujieda et al. proved that fracture toughness and Young's modulus increased with addition of AgNPs and Pt-NPs [146]. Another study showed that the addition of silver ions improved mechanical properties of Computer Aided Design/Computer Aided Manufacturing (CAD/CAM) blocks and at the same time, decreased crack length [147]. Mohsen et al. examined the effect of AgNPs on the color of ceramics. Their results showed that the addition of AgNPs affected the color quality of dental ceramic. According to numerous studies, AgNPs should be considered as an addition to porcelain, due to improvement of mechanical properties. Another nanoparticle tested in the field of prosthetics is Pt nanoparticles. One of the studies examined modified PMMA denture acrylic containing platinum nanoparticles. The size of the processed Pt NPs was less than 5 nm. Results showed an anti-adherent effect provided by reducing the area available for bacterial adhesion, at the same time, no Pt ion leaching was observed, orit was observed at extremely small amounts [59]. The addition of TiO2 nanoparticles in prosthetic treatment has been investigated in many studies. Researchers examined the physical, mechanical and biological behavior of a PMMA/TiO2. In one of studies, PMMA/TiO2 nanocomposite specimen containing 3 wt. % TiO2 NPs was examined. The results revealed that TiO2 NPs improved stiffness and mechanical behavior of the PMMA matrix. They also provided both antibacterial and anti-adhesive effects [148]. Some studies suggest that TiO2 antimicrobial effect is based on the photocatalytic effect, that is attributable to deactivation of cellular enzymes that lead to higher permeability and to cell death [149]. The study by Tsuji et al. showed that an increased amount of TiO2 can weaken the material and cause internal decomposition, therefore adequate amounts of NPs needs to be used [150].

#### **4. Cytotoxicity**

#### *4.1. Ozone*

As it was mentioned before, there are three different therapeutic ways in which ozone canbe used in dentistry and other fields of medicine. In dentistry, ozone can be applied in the form of ozonated oil (sunflower oil etc.), ozonated water or simple gaseous ozone.

An in vitro study provided by Borges et al. showed the safety of ozone treatment towards a human skin keratinocyte cell line (HaCaT) and a murine fibroblast cell line (L929). Data demonstrated increased relative cell number of murine fibroblasts and human keratinocytes after treatment with ozone at a concentration of 8 μg/mL.With the use ofa wound healing assay and a scratch assay, it can be clearlyseen that the rate of the wound healing process is the same as in the control group for both cell lines [151]. Another study conducted by Huth et al. evaluated the cytotoxicity of gaseous and aqueous ozone (in PBS) and other antimicrobial agents such as sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), hydrogen peroxide 3% (H2O2) for 1min exposure (a clinically relevant time period for ozonated water and gaseous ozone application) and metronidazole for 24 h exposure to gingival fibroblasts (HGF-1) and human oral epithelial (BHY) cells. Results reveal that less toxic features were observed in HGF-1 and BHY cells treated with ozonated water (1.25 μg/mL), which showed the most biocompatible properties, when compared to gaseous ozone, NaOCl, CHX or H2O2, which occurred to be the most toxic. An apoptotic assay, used to measure caspase-3 and -7 activity, revealed no alteration after treatment with ozonated water (ozonated PBS) when compared to control in both cell lines. Additionally, metronidazole demonstrated no significant cytotoxic effect towards gingival fibroblasts nor human oral epithelial cells [152]. Similar data was provided later by Colombo et al., who used ozonated olive oil and the CHX agents Corsodyl Dental Gel® and Plak Gel®. They evaluated their biocompatibility on immortalized human gingival fibroblasts (HGFs). Each agent was diluted 1:10 four times in Dulbecco's Modified Eagle's Medium (DMEM), which resulted in receiving different concentrations of the tested compounds (1:10, 1:102, 1:103, 1:104). After 2 and 24 h of treatment with the aforementioned agents, cells showed 100% viability when treated with ozonated olive oil in all tested concentrations when compared to the control. CHX compounds showed severe cytotoxicity in the highest

concentration (1:10) for Corsodyl Dental Gel® and Plak Gel® and moderate cytotoxicity in lower concentrations (1:102, 1:103, 1:104) [153]. Kashiwazaki et al. tested ozonated water and hand disinfectants which contained 83% ethanol, 1% chlorhexidine, 1% chlorhexidine ethanol, 0.2% benzalkonium chloride and 0.5% povidone-iodine on a human keratinocyte cell line in a three-dimensional cultured human epidermis model. Cells were divided into two groups, the first group, 1-week cultured, which developed an immature stratum corneum (SC), and the second group,2-week cultured, which developed a mature SC. Histological changes, cell viability and release of interleukin 1α were evaluated after treatment with ozonated water and hand disinfectant. There was no histological alteration after treatment in 1-week cultured cells and 2-week cultured cells, and also, no vacuolar cell formation was observed. A viability assay showed no cytotoxic effect of ozonated water and interleukin 8 activation was not observed, which indicatesthe high biocompatibility and immuno-compatibility features of the tested ozonated water [154]. Another study evaluated non-toxic and anti-tumor properties of ozonated water in anin vivo mouse model with the useof tumor-bearing mice and the control group. No injurious effect on normal tissue, such as spleen, small intestine, liver, kidney and muscle, was observed after 24 h direct application of ozonated water, even at a relatively high concentration (208 mM). Nonetheless, tumor tissue exhibited a decreased proliferation rate, inhibited growth and showed features of necrosis, which indicatesan increase of ROS levels [155].

#### *4.2. Titanium Dioxide*

Titanium dioxide nanoparticles (TiO2 NPs) can be synthesized via numerous methods such as the sol-gel method, the hydrothermal method, the co-precipitation method, sluggish precipitation, hydrolysis, and simple precipitation [156], but also with the use of the solvothermal or direct oxidation method [157]. With the use of different synthesis methods, we can obtain various sizes of nanoparticles which oscillate from ≤10 to ≥100 nm and even from 200 to 300 nm, and different polymorphic phases such as rutile, brookite or anatase [156,158–160].

TiO2 NPs are widely used in physics and chemistry due to their photochemical activity and physicochemical stability [158]. Titanium dioxide nanoparticles find their application in gas sensors, solar energy converters, pigments, ceramic supports, but can also be used to purify wastewater [159,161]. Moreover, TiO2 NPs are extensively used in UV filters in sunscreens and other everyday cosmeticssuch as lip balm, creams, foundations and also toothpaste [162]. In the food industry, TiO2NPs are used as a white pigment in food or food supplements [160]. In dentistry, titanium dioxide nanoparticles can be found in toothbleaching gels or dental composites used in orthodontics, in dental acryl resins [163,164].

Due to the many applications of TiO2 NPs, it is crucial to evaluate their potential cytotoxic effect on cells and tissues. Skin, gastrointestinal tract and respiratory system are the most exposed to TiO2 NPs, especially when applied in cosmetics such as sunscreens, food, or when used in the field of dentistry.

The cytotoxic and genotoxic effect of titanium dioxide nanoparticles was evaluated by Meena et al. The results obtained by them pointed out thedose-dependent (50, 100 and 200 μg/mL) and time-dependent (24, 48 and 72 h) toxic effect of TiO2 NPs on human embryonic kidney cells (HEK-293). TiO2 nanoparticles induced elevated lactate dehydrogenase releasedfrom the cell, and damage of cell membrane which led to cell death. Moreover, cells treated with 100 and 200 μg/mL of TiO2 nanoparticles exhibited an increased level of reactive oxygen species and elevated level of proapoptotic proteins such as Bax, caspase-3 and upregulation of the p53 protein in response to DNA breakage [165]. Another study conducted by Shukla et al. confirmed the cytotoxic and genotoxic effect of nano-TiO2 on a human liver cell line (HepG2). Relatively low concentrations of nanoparticles (20, 40, 80 μg/mL) increased the level of ROS, which led to DNA breakage via the oxidative stress-dependent pathway. Additionally, upregulation of p53, Bax, caspase-9 and caspase-3 was observed, however Bcl-2 expression was reduced, which points to apoptosis via the mitochondrial, and thus by the caspase-dependent pathway [166]. This indicates that

titanium dioxide nanoparticles should be used with caution and in very low concentrations. Due to the fact that the respiratory system is also exposed to the inhalation of TiO2 NPs, it may lead to an increased risk for lung health. TiO2 NPs were tested towardsa human lung cancer cell line (A549) to evaluate its potential genotoxic and cytotoxic characteristics. A relatively low concentration of the tested titanium dioxide NPs (10 and 50 μg/mL) after 6to 24 h of incubation led to an increase in the level of ROS, p53 and p21. Similar to the above-mentioned research, Bcl-2 level was downregulated at the mRNA and protein level; moreover, the cleavage of caspase-3 was observed. This data suggests that TiO2 NPs cause alteration in gene expression which leads to apoptotic changes and, as a consequence, to human lung cancer cell death [167]. Another research compared five different TiO2 NPs: 9 nm rutile (R9), 5 nm rutile (R5) and 14 nm anatase (A14), the commercially available 60 nm anatase (A60) and P25, which contained 80% anatase and 20% rutile, and their potential cytotoxic effect on a human bronchial epithelial cell line (BEAS-2B), a human type II alveolar epithelial cell line (A549) and human bronchial epithelial cells (NHBE). It occurred that the level of ROS was increased especially after 24 h of incubation in all five different TiO2 NPs; however, the strongest effect was observed in the P25 sample in NHBE and BEAS-2B cells after 2 and 24 h. For A549 cells, smaller particles (5 and 9 nm) of TiO2induced elevated release of intracellular ROS. Cell viability did not drastically alter allofthe tested cell lines, however, after incubation with a relatively high concentration (400 μg/mL), the viability of NHBE and A549 cells slightly dropped. Interleukin 8 (IL-8) plays a key role as a chemoattractant for neutrophils and other granulocytes and stimulates their migration to the infection site. After treatment with five different types of TiO2 nanoparticles, the level of the pro-inflammatory mediator IL-8 was substantially elevatedin all the cell lines when compared to the control group. The strongest effect was observed for anatase (A60), however A14 seems not to induce IL-8 upregulation [168]. Titanium dioxide nanoparticles (TiO2 NPs) were also tested against a human epidermal cell line (A431) to establish their potential risk to human skin cells. At low concentrations of 8 and 80 μg/mL and short time of incubation (6 h), a decreased level of glutathione and strongly increased level of reactive oxygen species was observed, which led to apoptosis. Genotoxic characteristics, evaluated with the use of a commitment assay, showed damage of DNA at the concentrations of 8 and 80 μg/mL after 6 h of treatment. These data clearly indicate that TiO2 nanoparticles should be used at very low concentrations and with high caution [169].

Independent studies conducted by Xue et al., Gao et al. and Wright et al. [170–172] have proven the cytotoxic effect of titanium dioxide nanoparticles on the human skin keratinocyte cell line. Xue tested different sizes (4, 10, 21, 25 and 60 nm) and different forms (anatase/rutile, rutile and anatase) under UVA radiation. Results clearly pointed to the toxic effect on HaCaT cells and an increased amount of apoptotic cells, which was induced in a dose-dependent manner, inwhen treated with 10 and 25 nm particles at a concentration of 200 μg/mL. However, UVA radiation alone did not affect cell viability. Elevated levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were also observed and, at the same time, there was a decreased amount of superoxide dismutase, which indicates cell damage [170]. TiO2 nanoparticles (25 nm) when compared to nanosized bismuth oxybromide (BiOBr) demonstrated a stronger toxic effect on ahuman keratinocyte cell line. Enhanced ROS was also confirmed at a concentration of 25 μg/mL of TiO2NPs. Titanium dioxide nanoparticles also induced early and late apoptosis of HaCaT cells and the cell cycle was found to be arrested after treatment with TiO2NPs [171]. Wright analyzed three different sizes of particles (a 1 mm particle composed of 100% rutile, a 21 nm particle composed 80% of anatase and 20% of rutile and a 12 nm particle which contained only anatase). Data demonstrated dose-dependent elevated caspase-8 and caspase-9 activity and increased apoptosis of the tested cells. It is worth noting that cells did not exhibit malignant transformation when treated with TiO2 NPs and exposed to UVC radiation [172]. In vivo studies conducted on mice and rat models also confirmed toxic characteristics and increased inflammatory response, which indicates oxidative tissue damage in rats [173]. Mouse model showed in vivo toxicity and DNA disruption of liver tissue and a decrease of glutathione peroxidase, which clearly indicate liver tissue damage [174].

#### *4.3. Silver*

Many methods are known for the synthesis of silver nanoparticles (AgNPs). With the use of physical, chemical and biological methods, various AgNPs with desirable morphology can be obtained in order to achieve the best properties [175]. Therefore, silver nanoparticles can be used in different fields of science; for example, for single molecule detection [176] or by enhancing electrical conductivity, AgNPs can be intended for high-frequency electronic applications [177]. However, in the biological approach, silver nanoparticles have been known mostly for their antibacterial properties. Due to these characteristics, they can be used as an efficient disinfectant to sterilize surfaces or medical equipment, and devices in the industry during the food packaging process or in environmental usage as air and water disinfectant [178]. AgNPs can also be used to impregnate and functionalize textiles [179], but they can also be used as coating for wood flooring [180].

Despite their various properties, silver nanoparticles exhibit a toxic effect in vitro and in vivo. Many studies revealed that silver nanoparticles increase the level of ROS in cells and therefore increase oxidative stress and cause defects in DNA structure [181,182]. AshaRani et al. [183] evaluated the cytotoxicity effect on human cell lines using human glioblastoma cells (U251) and normal human lung fibroblast cells (IMR-90). The size of the tested Ag nanoparticles oscillated from 6 to 20 nm, however cell viability and ATP level drastically dropped in a time- and concentration-dependent manner, especially after 48 and 72 h incubation. Further investigation revealed mitochondrial damage and increased level of ROS, as well as arrest of the cell cycle in the G2/M phase and disruption of DNA structure [183]. Another study confirmed a dose-dependent and time-dependent toxic effect of silver nanoparticles and silver ions on human osteoblasts (OB) and primary human mesenchymal stem cells (MSC). A severe decrease of cell viability as well as increased oxidative stress was observed already in 10 μg/g of the tested AgNPs after 21 days of incubation with the tested compounds. The human osteoblast (OB) cell line proved to be more sensitive to exposition to silver nanoparticles [184]. Another research confirmed toxic properties of both silver ions and silver nanoparticles towards the human lymphoblastoid TK6 cell line. It occurred that both forms of AgNPs induced cytotoxicity and genotoxicity at comparable concentrations, which oscillated from 1.00 to 1.75 μg/mL of the tested compounds. Additionally, genotoxicity was confirmed with the use of the micronucleus assay and the results clearly showed an increased level of reactive oxygen species (ROS) at relatively low concentrations of both forms of silver after 24 h treatment. The expression of genes involved in oxidative stress, such as glutathione peroxidase 7, thyroid peroxidase and heme oxygenase, and expression of genes in response to cell cycle arrest caused by DNA damage, such as cyclin-dependent kinase inhibitor 1A, were substantially elevated. This study clearly indicates that both silver ions and silver nanoparticles are toxic by leading to an increase in cellular stress and DNA structure damage [185].

The main problem is that in vitro toxicity of silver nanoparticles and silver ions occurs at relatively low concentrations and, on the other hand, antimicrobial properties demand substantially elevated concentrations of AgNPs. Therefore, it is of immense importance to estimate the most favorable conditions, ensuring both antibacterial properties and safe use with regard to cell lines and, even further, for in vivo applications. Hence, Albers et al. [184] estimated in vitro cytotoxicity of silver ions and nanoparticles in osteoclast (OCs) and osteoblast (OBs) cells at antibacterial concentrations against *S. epidermidis*. The study showed a size-dependent and concentration-dependent toxic effect of AgNPs. The most cytotoxic effect was observed after treating OBs and OCs cells with silver nanoparticles sized 50 nm, and the larger the particles were, the weaker cytotoxic effect was observed. This suggests that the smaller the size of the nanoparticle surface is, the more silver is released to the environment. Moreover, OBs cells were more sensitive to the used nanoparticles. As compared with antibacterial activity, which was obtained at a concentration of 8 mg/mL for

AgNPs towards *S. epidermidis*, 50% of cell viability was maintained at a concentration of 0.048 mM for osteoclast cells, and for the same concentration, the viability of osteoblast cells was highly below 50%. Thus, at the present moment, there are no possibilities to combine antimicrobial and cyto-safety properties [184]. Pérez-Díaz et al. showed similar data but compared the viability of dermal human fibroblasts at an antimicrobial concentration of sliver nanoparticles against *Streptococcus mutans*. The data clearly indicated that AgNPs concentrations higher than 10 ppm represented acytotoxic level, while reduction of biofilm growth was observed at the concentration of 100 ppm [186].

On the other hand, combined materials, such as Ag NP-coated titanium dental implants with hydroxyapatite (HA) applied to the surface, seem to be promising solutions, because after 7 days, primary human osteoblasts showed biocompatibility with the tested material. Osteoblasts also demonstrated adhesive properties toward the tested material and there were no alterations in cell morphology, and elevated levels of alkaline phosphatase and lactate dehydrogenase were not observed either [187]. This suggests that complex materials such as coating titanium implants with nanoparticles may be the best solution to obtain cyto-safety properties in vivo and in vitro.

#### *4.4. Platinum*

Platinum, due to its high biocompatibility, its corrosion resistance and the possibility to visualize it under X-rays, is widely used in the field of biomedical sciencesinsurgical instruments, implantable electronic devices and implantssuch as cardioverter-defibrillators, stents, knee or hip implants as well as dental implants [188].

However, platinum nanoparticles (PtNPs) demonstrate different features when compared to platinum solid implants. PtNPs can be obtained via a variety of methods, usually through the reduction of Pt ions in the liquid phase with a stabilizing or capping agent to produce nanoparticles in the form of colloids, eventually via the microemulsion method or via reduction and impregnation of Pt ions into micro-porous base [189]. Due to their method of synthesis, different shapes can be obtained, such as isolated nanoparticles, dendrites or crystalline nanowires with various sizes, which can be used in the industrial application, in optical fields, as a catalyst in fuel cells and biosensors [190–193].

Some researchers pointed out a size-dependent toxic effect on macrophage cell Raw 264.7 viability. An increased cytotoxic effect was observed when cells were treated with platinum nanoparticles sized 5 nm as compared to PtNPs sized 30 nm, asa smaller size of nanoparticles can be easily taken upby cells; nevertheless, both samples of nanoparticles exhibit a toxic effect. A dose-dependent toxic effect was also observed, cell viability decreased drastically at a concentration of 5 ppm of the tested nanoparticles. Moreover, data indicated that density and cell morphology were altered after treatment with PtNPs. Additionally, platinum nanoparticles induced the activation of caspase-3 and caspase-7, which led to nucleus fragmentation and apoptosis, which also indicates that an elevated level of caspases arrested the DNA repair process [194]. Another study conducted by Konieczny et al. [195] confirmed the cytotoxic effect of platinum nanoparticles on human skin cells. The authors have used PtNPs with an estimated size of around 5.8 and 57 nm, and both were protected with polyvinylpyrrolidone. Nanoparticles were used with three different concentrations of 6.25, 12.5 and 25 μg/mL and, after 24 and 48 h treatment, the viability of normal human epidermal keratinocytes (NheKs) slightly decreased, especially when treated with smaller particles. Nonetheless, cell viability seems to be unaffected, and genotoxicity and DNA damage via activation of caspase-3 and caspase-7 were observed, primarily caused by smaller PtNPs. This study indicated dose-dependent and concentration-dependent toxic properties of platinum nanoparticles [195]. In Labrador-Rached et al.'s research, it was demonstrated that 70 nm PtNPs in a concentration of 100 μg/mL exhibit a relatively high cytotoxic effect, however a toxic effect observed at lower concentrations of 5 and 25 μg/mL was indiscernible. They also obtained elevated ROS levels in response to an upregulated release of pro-inflammatory factors such as IL-1β, IL-8 and TNF-α in a HepG2 liver model [196].

An in vivo model provided by Lin et al. showed that small platinum nanoparticles of 5 nm and larger, up to 70 nm, by acting on ion channels by the extracellular site of ventricular cardiomyocytes of neonatal mice, disrupted cardiac electrophysiology, which can lead to the threat of cardiac conduction block. However, a relatively high dose of 5 and 70 nm PtNPs did not meaningfully elevate ROS generation and lactate dehydrogenase level [197].

To compare toxic nanoparticles, green synthesis of platinum nanoparticles could be a good solution to reduce their toxic properties [198]. Some studies still demonstrate toxicity and genotoxic effect on various cell lines, such as human embryonic kidney (HEK293) cells [199] or human breast cancer (MCF-7) cells [200]. However, novel studies reveal a selective cytotoxic effect on cancer cells (MCF-7) when compared to normal human embryonic kidney cells (HEK293) [201], which could be a good direction for the usage of this particles.

#### **5. Discussion**

Ozone has a wide range of applications. In dentistry, its properties are not only antimicrobial, but immuno-stimulating, analgesic and anti-inflammatory. The use of ozone in dental surgery seems promising, especially because of its wound-healing properties. There are many caries prevention agents on the market: ozone seems to be effective against microbes after cavity preparation, but the opinions of scientists are divided [71,72]. The problem in the case of ozone is its efficacy against biofilm. Ozone therapy can help maximize disinfection effect, at the same time providing low toxicity and low possibility of drug resistance. Although ozone effectiveness shows a wide range in many studies, it can be considered as an additional disinfection protocol step. New technologies in dentistry are most often associated with a high cost of their introduction to the dental office. What also poses a problem in the case of ozone generators are substitutes that do not require investment and are at the same time effective, examples are fluoride varnish and sealants for caries prevention or NaOCl used in endodontics [73]. Apart from the discussed ozone applications in dentistry, there are many other fields in medicine where ozone can have a significant impact. Acceleration of wound-healing of oral mucosa is an example of positive properties, which offers a wide range of application possibilities [202].

Ag NPs are the most frequently discussed NPsin the dentistry literature. Due to their properties, they are widely used in medicine, for example in the form of coating in dental implants or as a modifier of dental materials. Also, many studies suggest that AgNPs can be considered in endodontic protocols as an additional irrigation or amplifier of the antimicrobial effect of other agents, such as MTA and CHX. However, the problem is to find the most favorable ratio between cytotoxicity and antimicrobial properties, which depends on the size of NPs. TiO2 are used especially as coatings of implants or rotary NiTi instruments. They can be combined with other NPs such as Ag or ZrO2NPs to obtain better properties of materials. Nanoporous and nanotube titania coating may contain various substances, such as antibiotics, silver and zirconia, which improve their properties [126]. TiO2 NPs appear to have a promising reduction effect on biofilm which is a serious problem in dentistry. However, the problem of cytotoxicity arises again. Silver and titania NPs appear more often in literature and their antimicrobial properties seem to be well proven in contrast to CuO and Pt NPs, which do notappear frequently in articles. Pt NPs have promising properties, especially in guided bone and tissue regeneration. Also, in the field of prosthetics, theyare used in modified PMMA denture acrylic. However, again the problem is their cytotoxicity and the possibility of using other NPs [203]. CuO nanoparticles are used in conservative dentistry as a caries preventing agent and they seem to have a potential for application in endodontic materials. Unfortunately, cytotoxicity is a significant problem.

Different antimicrobial agents are reported to be used as the components of orthodontic adhesives. Among them are metal nanoparticles which, regarding the growing bacterial resistance to the commonly used drugs, may be considered as an alternative treatment applied as a peri-implant infection prevention measure [68]. Generally, nanoparticles,

due to their small size as well as large surface area, possess unique features and, especially when combined with metal ions, can help prevent bacterial growth in the vicinity of the material [68]. The efficacy of antimicrobial treatment with inorganic nanoparticles is highly correlated with their physicochemical properties. In general, the smaller size of grains the material has, the more effective it is. Another important factor, apart from the size of particles, is their morphology, and it was established that needle-shaped particles are more likely to damage bacterial cells than spherical ones [204]. Metal nanoparticles may have bactericidal or bacteriostatic effects. The result of the former is bacterial death and the latter leads to the inhibition of growth or multiplication [204]. Most frequently, the antimicrobial effect of applied nanoparticles is based on the release of free metal ions, although other modes of action have also been noted, such as direct mechanical damage of bacterial cell after the internalization of the particles into the cell. Another mechanism is connected with the production of reactive oxygen species (ROS) [204]. Nanoparticles can also adsorb to the cell wall and, as a result of depolarization, increaseits permeability [205]. Moreover, metals lead to protein dysfunction and impair the enzymatic activity, which results in cellular metabolism malfunctioning [206]. Nanotechnology provides a wide range of modifications in dentistry, where new, better materials or implants are constantly being sought. The addition of nanoparticles can improve their properties such as their bactericidal, adhesion, or osseointegration properties. The discussed materials have a variety of applications in many fields of dentistry. The issue which is mainly elaborated in this reviewisantimicrobial properties. However, nanomaterials offer many more possibilities, as they have even more properties which enhance materials and tools used in medicine.

#### **6. Conclusions**

In summary, both ozone treatment and nanotechnology seem to have a prosperous future in dentistry, offering a wide range of applications. Ozone can be used in every field of dentistry due to its efficient antibacterial properties. Treatment with ozone may be more appropriate for people with visit anxiety. The role of Ag and TiO2 NPs in dentistry seems to be promising. In the case of CuO and Pt NPs, the problem may be the cytotoxicity and the presence of alternative materials, e.g., other NPs. It is important to compare the ratio of the effectiveness of the discussed agents to those commonly used. Their price may also pose a problem, especially in the case of prophylactic treatments, such asozonehygienization and povidone iodine impregnation.Both ozone and nanoparticles discussed in this review have antimicrobial properties. For this reason, they can be used as an improvement to treatment methods or materials. Another advantage is the prevention of complications such as peri-implantitis and secondary caries. Nanotechnology gives a huge field for the development of new materials and methods of treatment in modern dentistry. Further research is needed for each of the agents to rediscover or find the most advantageous method of obtaining the material or using it in dentistry.

**Author Contributions:** Conceptualization: M.D. and R.J.W.; Methodology: A.L., N.N., J.R.-S.,W.D. and R.J.W.; Software: K.S., M.D., W.D. and R.J.W.; Validation: M.D. and R.J.W.; Formal analysis: W.Z., M.D., M.S., Z.R., K.W. and R.J.W.; Investigation: W.Z., W.D., M.D., Z.R., M.S. and R.J.W.; Data curation: A.L., N.N., J.R.-S., W.D., W.Z., M.D., Z.R., M.S., K.W. and R.J.W.; Writing—original draft preparation: A.L., N.N., J.R.-S., W.Z., M.D. and. R.J.W.; Writing—review and editing: K.W., M.D. and R.J.W.; Supervision: M.D. and R.J.W.; Project administration: R.J.W.; Funding acquisition: R.J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Science Centre (NCN), grant number UMO-2015/19/B/ST5/01330, entitled 'Preparation and characterisation of biocomposites based on nanoapatites for theranostics'.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **Nanomaterials Application in Orthodontics**

**Wojciech Zakrzewski 1, Maciej Dobrzynski 2,\*, Wojciech Dobrzynski 3, Anna Zawadzka-Knefel 4, Mateusz Janecki 5, Karolina Kurek 6, Adam Lubojanski 1, Maria Szymonowicz 1, Zbigniew Rybak <sup>1</sup> and Rafal J. Wiglusz 7,8,\***


**Abstract:** Nanotechnology has gained importance in recent years due to its ability to enhance material properties, including antimicrobial characteristics. Nanotechnology is applicable in various aspects of orthodontics. This scientific work focuses on the concept of nanotechnology and its applications in the field of orthodontics, including, among others, enhancement of antimicrobial characteristics of orthodontic resins, leading to reduction of enamel demineralization or control of friction force during orthodontic movement. The latter one enables effective orthodontic treatment while using less force. Emphasis is put on antimicrobial and mechanical characteristics of nanomaterials during orthodontic treatment. The manuscript sums up the current knowledge about nanomaterials' influence on orthodontic appliances.

**Keywords:** nanomaterials; orthodontics; brackets; wires; antimicrobial effect

### **1. Introduction**

Nanomaterials are widely used in modern clinical dentistry. They improve various properties, such as antimicrobial properties, durability of materials. These particles do not exceed 100 nm, due to they obtain a better ratio between the surface and mass. The larger the surface area of the material, the greater its reactivity. It is also easier to absorb them in the body, which can also result in high cytotoxicity [1]. Nanomaterials are used in many areas of dentistry, such as conservative dentistry, endodontics, oral, and maxillofacial surgery, periodontics, orthodontics, and prosthetics [2]. Orthodontics is a branch of dentistry dealing with the improvement of occlusal conditions and facial aesthetics in both children and adults. In cooperation with other specialists (such as dental surgeons, maxillofacial surgeons, periodontists), the orthodontist is able to significantly improve the patient's quality of life [3]. Nanotechnology is used, among others, in brackets, archives, elastomeric ligatures, orthodontic adhesives. Improving the microbicidal properties, reducing friction

**Citation:** Zakrzewski, W.; Dobrzynski, M.; Dobrzynski, W.; Zawadzka-Knefel, A.; Janecki, M.; Kurek, K.; Lubojanski, A.; Szymonowicz, M.; Rybak, Z.; Wiglusz, R.J Nanomaterials Application in Orthodontics. *Nanomaterials* **2021**, *11*, 337. https://doi.org/10.3390/ nano11020337

Academic Editor: Maria Letizia Manca Received: 31 December 2020 Accepted: 24 January 2021 Published: 28 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and increasing the strength of the material are some of the advantages. However, a significant problem is the potential cytotoxicity of nanomaterials, therefore further research is needed [2].

The prolonged process of wearing orthodontic braces results in increased accumulation of dental plaque and eventually results in a greater risk of caries. Its development is generally associated with the activity of cariogenous bacteria due to prolonged dental plaque accumulation on teeth surfaces, deficiencies, avitaminosis, and diet. The demineralization process that starts the caries is called a white spot lesion (WSL), meaning, that decalcification of enamel surfaces adjacent to the orthodontic appliances is directly associated with orthodontic treatment [4]. Several studies confirm the accelerated accumulation of WLS in orthodontic treatments. Such tendency creates clinical problems leading to unacceptable esthetic alterations that, in some cases, might lead to conservative, restorative treatment. Research shows that more plaque can accumulate around composites compared to other restorative materials, which results in an increased percentage of secondary caries [5]. Moreover, resin composites do not have bacteriostatic properties.

Promising results in the prevention of pathological changes associated with orthodontic treatment are obtained through the use of nanotechnology. According to the European Commission states that: "Nanomaterial is defined as a natural, incidental, or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1–100 nm. In specific cases and where warranted by concerns for the environment, health, safety, or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1% and 50%" [6]. Implication of nanotechnology is beneficial to humans, it has been broadly used in the modern dentistry in in restorative dentistry as an additive nanoparticle with remineralizing properties in composite resins, dental adhesives, oral care products, in the control of bacterial biofilm as an antibacterial and antimineralizing additive in dental hygiene products such as toothpaste, mouth rinses, and composite resins. Nanotechnology is useful in the diagnosis of malignant and precancerous cavity diseases, periodontal diseases, and is also used in implantology—as a modification of the implant surface [7] and in the use of impression materials [8]. The development of technology gives better opportunities to both patient and orthodontist due to new physicochemical, mechanical and antibacterial properties of nanosized materials and can be used in coating orthodontic wires, elastomeric ligatures, and brackets, producing shape memory polymers and orthodontic bonding materials. Not only can we control biofilm formation, reduce bacterial activity and act anticariogenic, but also, through the desired tooth movement, shorten the treatment time.

There are many advantages in medicine of using nanotechnology; however, it creates many doubts regarding the safety for humans and the environment. Nanoparticles can easily penetrate tissues and can affect biological behaviors at different levels. It is necessary to conduct detailed research on the environmental and toxicological properties in order to assess the risk and lead a sustainable application of nanomaterials. The aim of this work was to describe and summarize the current use of nanoparticles and their antibacterial activity in orthodontics, including resin, brackets, and archwires.

#### **2. Nano-Coatings in Orthodontic Archwires**

Minimizing the frictional forces between the orthodontic wire and brackets has the potential to increase the desired tooth movement and thus shorten treatment time. In recent years, nanoparticles have been used as a component of dry lubricants. These solid-phase materials are capable of reducing the friction between two sliding surfaces without the need for a liquid medium. One of the many examples are Inorganic fullerene-like tungsten sulfide nanoparticles (IF-WS2) that are used as self-lubricating coatings for orthodontic stainless steel wires [9]. Friction tests simulating the performance of coated and uncoated wires were carried out on an Instron machine, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis of the coated wires showed a clear impregnation of IF-WS2 nanoparticles in the Ni-P matrix.

Atomic force microscopy (AFM) was used as a tool to assess the surface roughness of stainless steel (SS), beta-titanium (β-Ti), and nickel-titanium(NiTi) wires [10]. The surface roughness measurement of the AFM method confirmed the fact that the roughness of the measures on the effectiveness of sliding mechanics, the corrosion behavior, and aesthetics of orthodontic arches. The influence of decontamination and clinical exposure on the modulus of elasticity, hardness and surface roughness of SS and NiTi arches, and AFM paper coupled with a nanoindenter were assessed [11]. The results of the AFM popularity assessment that the decontamination regimen and clinical exposure had no statistically significant effect on NiTi wires, but had a statistically significant effect on SS wires. In a diagnostic study, the clinical significance of statistical studies, analysis, and testing of the arch equipment on orthodontic movement is not predicted.

#### *2.1. Nano Coatings Reducing Friction on Orthodontic Archwires*

Orthodontic arches are used to generate biomechanical forces that are transmitted through the brackets to move the teeth and correct malocclusion, spacing, or crowding. They are also used for retention purposes, i.e., to keep the teeth in their current position. Currently, orthodontic arches are made of non-precious metal alloys. The most common types of wire are SS, NiTi, and β-Ti alloy wires. In the case of sliding mechanics, friction between the wire and the lock is one of the major factors influencing tooth movement. When one moving object makes contact with another, friction occurs on the contact surface, which causes resistance to the movement of the teeth. This frictional force is proportional to the force with which the contacting surfaces are pressed against each other and is governed by the interface surface characteristics (smooth/rough, chemically reactive/passive, or lubricant modified). Minimizing the frictional forces between the orthodontic wire and brackets will accelerate the desired tooth movement and thus shorten the treatment time.

NiTi substrates can be coated with cobalt and a layer of IF-WS2 nanoparticles using the electrodeposition method. The coated substrates showed friction reduction of up to 66% when compared to the uncoated ones. The results of such studies may have potential applications in reducing friction when using NiTi orthodontic wires. On the other hand, allergic reactions in patients with nickel sensitivity may be the disadvantage of introducing nickel into this type of coating. Therefore, the effect of such NiP coatings on stainless steel and NiTi wires should be assessed for biocompatibility in animal models and further human trials.

#### *2.2. Delivering Nanoparticles from an Elastomeric Ligature*

Elastomeric ligatures can serve as a support scaffold to deliver nanoparticles that can be anti-cariogenic or anti-inflammatory. They may also carry embedded antibiotic drug molecules. The release of anti-cariogenic fluoride from elastomeric ligatures has already been described in the literature [12,13]. Research has shown that fluoride release is characterized by an initial burst of fluoride in the first few days followed by a logarithmic fall. The whole process is effective against common enamel demineralization around the orthodontic bracket during treatment [14].

#### *2.3. Shape Memory Polymers (SMP) in Orthodontics*

In the last decade, there has been a growing interest in the production of aesthetic orthodontic wires to complement brackets in the color of the teeth. Shape memory polymers (SMPs) are materials that can remember equilibrium shapes and then manipulate and fix them into a temporary or dormant shape under certain temperature and stress conditions. They can later relax to their original, stress-free state under thermal, electrical, or environmental conditions. This relaxation is related to the elastic deformation stored in the previous manipulation. Recovery of SMP into equilibrium shape can be accompanied by an appropriate and prescribed force, useful for orthodontic tooth movement, or a macroscopic change in shape that is useful in ligation mechanisms. Due to the ability of SMP to have two shapes, these devices meet requirements unattainable by modern orthodontic materials, allowing the orthodontist to insert them into the patient's mouth more easily and comfortably [15].

When placed in the oral cavity, these polymers can be activated by body temperature or light-activated photoactive nanoparticles thereby causing tooth movement. SMP orthodontic wires can provide an improvement over traditional orthodontic materials as they provide lighter, more consistent forces which, in turn, can cause less pain to patients. Also, SMP materials are transparent, stainable, and stain-resistant, providing the patient with a more aesthetic apparatus during treatment. High percent elongation of the SMP apparatus (up to about 300%) allows for the application of continuous forces over a large range of tooth movement, and thus, fewer patient visits [16,17]. Future directions of research on shape—nanocomposite polymers with memory for the production of aesthetic orthodontic wires may have interesting potential in the research of orthodontic biomaterials.

#### *2.4. Control of Oral Biofilms during Orthodontic Treatment*

Nanoparticles have a larger surface area to volume ratio (per unit mass) compared to non-nano scale particles, interacting more closely with microbial membranes and providing a much larger surface area for antimicrobial activity. In particular, metal nanoparticles with a size of 1–10 nm showed the highest biocidal effect on bacteria [18]. Silver has a long history of use in medicine as an antibacterial agent [19]. The antimicrobial properties of nanoparticles have been exploited through the mechanism of joining dental materials with nanoparticles or coating the surface with nanoparticles to prevent adhesion of microbes to reduce biofilm formation [20,21]. It was found that resin composites containing fillers implanted with silver ions that release silver ions have an antibacterial effect on oral *streptococci* [22].

Ahn et al. [16] compared an experimental composite adhesive (ECA) containing silica nanofillers and silver nanoparticles with two conventional composite adhesives and a resin-modified glass ionomer (RMGI) to investigate the surface characteristics, physical properties, and antimicrobial activity against cariogenic *streptococci*. The results suggest that the ECAs had rougher surfaces than conventional adhesives due to the addition of silver nanoparticles. Bacterial adhesion to ECA was lower than to traditional adhesives, which was not affected by saliva. Bacterial suspensions containing ECA show slower growth of bacteria than those containing conventional adhesives. There is no significant difference in the shear bond strength and fracture strength of the bond between ECA and conventional adhesives.

#### **3. Bracket Materials**

The development of technology for the production of orthodontic materials and products provides better opportunities for patients with functional, health, and aesthetic results. It also improves the daily technical performance of the orthodontist. To perform their function properly, the brackets should have good biocompatibility, correct hardness, and strength, smooth archwire slot to reduce frictional resistance, smooth surface to reduce plaque deposition, should be precisely manufactured for each tooth, have high corrosion resistance, and ionic release [23].

Orthodontic braces are manufactured by three main methods which may be used in combination: Casting, injection molding, and milling from different types of material including metal, plastics, ceramics, and combinations.

Among the compositions of metallic, we can distinguish stainless steel, non-nickel steel, low-nickel stainless, cobalt–chromium alloys, titanium, and its alloys, gold alloys, and platinum alloys [24].

Metallic materials and their alloys are characterized by high mechanical parameters, usually better than ceramics or polymers. The surface in contact with the wire should have a relatively high modulus of elasticity to minimize the disbursement of energy

transmitted by the wire from inexpedient plastic deformation and difficult enough to minimize expenditure wear caused by the movement of the activated wire. On the other hand, the base of the bracket must be sufficiently deformable to facilitate removal during treatment completion [24].

Stainless steel is a metallic alloy commonly used in the production of orthodontics brackets, due to its low cost, higher modulus of elasticity, and good biomechanical properties [25]. It can be classified as austenitic, martensitic, ferritic, duplex (austenitic-ferritic), and precipitation-hardening. The most commonly used alloys in orthodontic brackets are 303, 304, 316, 317, 17-4 PH [26]. Conventional type 316 L austenitic stainless steel is composed of %wt: Iron: Balance, manganese: 2.0, chromium: 16–18, nickel: 10–14, molybdenum: 2–3, and traces of phosphorus, sulfur, and carbon [27]. Although this alloy works well in clinical use, signs of corrosion have been observed.

17-4 PH alloy demonstrates improvement in corrosion resistance, frictional behavior, and cytotoxicity, more than austenitic stainless steels 303, 204, and 316/316 L [26]. Nickel stabilizes the austenitic phase; the anti-corrosive properties and the ductility are improved while the addition of chromium facilitates the formation of a passive anti-corrosion coating [28].

Although allergenic, cytotoxic, and mutagenic their content in the brackets is so small that their use is safe. Since the occurrence of adverse reactions, it was considered that exposure to these elements should be kept to a minimum. This resulted in the introduction of various non-nickel or very low nickel content stainless steel which is more resistant to corrosion and does not release nickel into the oral cavity. Compared to 316 L, Alloy 2205 is harder and less corrosive [29].

Titanium was used in the construction of brackets as a material with a proven lack of allergenicity and increased corrosion resistance. The many current dental and medical applications have made titanium the obvious choice of all the available components.

Commercially pure titanium grade 4 and Ti-6Al-4V alloy are the most widely used types for manufacturing orthodontic brackets, The different methods of obtaining the brackets result in significant differences in physical, mechanical, and bulk material properties. Corrosion resistance is achieved due to the presence of a thin passive protective layer made of titanium oxide. This layer is more stable than its counterpart chrome oxide on stainless steel [30]. Gold-coated brackets were introduced as an alternative to steel and titanium brackets. They are plated with 300 micro inches of 24 karat gold, therefore, have significantly brighter appearance. Moreover, they have better mechanical properties compared with conventional brackets made of stainless steel alloys. Gold alloy brackets are introduced as highly anticorrosive and the first choice for patients allergic to nickel (Ni) [30]. Significant side effects have not been observed clinically.

Additionally, nano-sized gold particles can be used on orthodontic appliances e.g., aligners, to increase its antibacterial activity, by preventing biofilm formation as can be seen in Figure 1. Both the gingiva and teeth are covered by aligners for almost the entire day, which is a risk factor for plaque accumulation. Gold particles also show positive biocompatibility both in vitro and in vivo.

A suitable substitute for stainless steel brackets are those coated with a platinum layer. Platinum has been found as a material totally compatible in the oral environment. Its alloys are five times more resistant to abrasion than gold and compared to stainless steel, they have excellent corrosion resistance, a harder surface which reduces friction and improves the mechanics of sliding. As a combination of the platinum layer and the unique implantation process, a barrier has been created that protects against the diffusion of nickel, cobalt, and chromium.

Similar electrochemical properties, including excellent corrosion resistance, to that of platinum brackets, are demonstrated by those made of cobalt chrome steel [31]. Regarding friction resistance, cobalt–chromium brackets are comparable, but have slightly less friction than stainless steel brackets when used with stainless steel wires; however, cobalt–chromium brackets offer more friction than titanium brackets with both stainless steel and beta-titanium wires [32].

**Figure 1.** Aligners coated with modified gold nanoparticles causing enhanced antibacterial activity against *Porphyromonas gingivalis*. Due to presence of the coated aligner, the number of bacterial cells was decreased, causing increased biofilm formation prevention.

Although metal brackets exhibit excellent mechanical properties and provide many clinical advantages the issue of aesthetics remains a challenge. Elements made from ceramics and plastics have been widely used in clinical orthodontics.

The first plastic brackets appeared in the early 1970s and were made of acrylic, then polycarbonate, but unfortunately problems related to them were quickly noticed. They had a tendency to water sorption, change color upon contact with the ultraviolet light and some food or drinks [33].

There has been observed an increased adhesion of pathogens like *Streptococcus mutans* and *Candida albicans*. In order to eliminate problems and improve their properties the following solutions are possible: Reinforcement with other materials such as ceramic or fiberglass fillers and/or metal slots, chemical modification of the polymer and alternative polymers for instance urethane dimethacrylate, high-density polyethylene, and EBP [34]. Research shows that compared to stainless steel brackets, plastic brackets are only suitable for clinical use if they have a metal slot [35].

An important issue is the biocompatibility of plastic materials, especially in terms of cytotoxic effects of particle- and fiber-reinforced polycarbonate orthodontic brackets in fibroblast and breast cancer cells through the activation of mitochondrial cell death mechanisms [36].

Although polycarbonate brackets with metal reinforced slots demonstrate a significantly lesser degree of deformation, followed by pure polyurethane, pure polycarbonate, and fiberglass reinforced polycarbonate brackets torque problems still exist. Ceramic reinforced polycarbonate brackets showed the highest deformation under torque stresses [37].

Polyoxymethylene brackets were found to be harder and less rough. Unfortunately, this material is also unattractive due to the opacity and milky color. Moreover, it appears to release some formaldehyde over time.

There is still a search for an ideal polymer that would combine the optical properties of translucency and the mechanical properties of stiffness, resistance to water absorption, and degradation. The introduction of new materials should ensure this does not release toxic compounds, in particular leaching of monomer Bis-GMA (bisphenol A-glycidyl methacrylate), TEGDMA (triethylene glycol dimethacrylate) [38]. The advantage of polymer brackets, as in the case of those from stainless steel, is the ease and safety of removing them from the tooth.

Among the brackets ensuring excellent aesthetic and optimum stable properties, we also include those made of ceramics. Their advantages are high rigidity and abrasion resistance as well as biocompatibility, and they are free from discoloration. Ceramic brackets are usually composed of aluminum oxides. There are two varieties currently available polycrystalline and monocrystalline (Saphire) forms, depending on their method of production. Another category is the polycrystalline Zirconia which has been offered as an alternative to alumina ceramic [39]. Polycrystalline zirconia brackets have the greatest toughness amongst all ceramics however are very opaque and can exhibit intrinsic colors. The monocrystal alumina brackets, which are noticeably clearer and consequently more aesthetic, along with having higher strength, than the polycrystalline alumina brackets, show low fracture toughness, due to the lack of internal grain boundaries, the presence of pores, and machining damage from milling [26]. Ceramic materials have some disadvantages associated with iatrogenic enamel damage due to their hardness, bonding and debonding, Frictional resistance. Orthodontists may experience problems with bracket breakage and fracture resistance, particularly when trying the ligature or fracture from archwires forces.

#### **4. Nanomaterials in Orthodontics**

Nanomaterials versatility allows them to be used in many situations during orthodontic clinical treatment, as can be seen in Table 1.


**Table 1.** Nanomaterials application in dentistry.

Friction is one of the major factors present during retraction or alignment of teeth during orthodontic treatment. One of the methods to overcome high friction is the application of higher forces during treatment. Such action can have one significant disadvantageundesirable anchorage loss [54]. On the other hand, there are other methods of overcoming unwanted friction, including alteration of the bracket design or wire shape and size. At last, there is a possibility of nanoparticle coating addition. To benefit from the antibacterial properties of nanoparticles, there are two main strategies in orthodontics to reduce biofilm formation. One strategy focuses on coating the surface of orthodontic brackets or wires

with nanoparticles [55]. The other is about combining nanoparticles with orthodontic adhesives or acrylic materials. The advantages of nanocomposite materials include excellent optical properties, easy handling, and excellent polishability [24]. Moreover, nanofillers can reduce the surface roughness of orthodontic adhesives, which is one of the most important factors in bacterial adhesion [25], as can be seen in Figure 2.

**Figure 2.** Comparison of teeth demineralization development with and without nanoparticles' covered brackets.

#### *4.1. Silver Nanoparticles (AgNPs) Coating*

Some studies have proposed silver nanoparticles as the most effective type of metal nanoparticles for preventing the growth of *Streptococcus mutans* [56]. Recently, silver nanoparticles (AgNPs) have been shown to be materials with excellent anti-microbial properties in a wide variety of microorganisms. In the orthodontic field, studies have incorporated AgNPs (17 nm) into orthodontic elastomeric modules, orthodontic brackets, and wires, and others, against a wide variety of bacterial species concluding that these orthodontic appliances with AgNPs could potentially combat the dental biofilm decreasing the incidence of dental enamel demineralization during and after the orthodontic treatments [57,58]. AgNPs can significantly inhibit the bacterial adherence of the *S. mutans* strain on the surfaces of the orthodontic bracket and wire appliances finding that the smaller AgNP samples demonstrated statistically to have the most important *S. mutans* antiadherence activities for orthodontic brackets and wires when compared to NiTi (nickel–titanium) and SS (stainless steel wires) [59]. It is also confirmed by several studies, that coverage of AgNPs in human dentin prevents biofilm formation on the surface of the dentin, together with bacterial growth inhibition [58,60,61]. In order for AGNPs to be a stable suspension able to limit the agglomeration, they should have zeta potential values ranging between +30 and −30 mV [62,63]. Bürgers et al. [64] confirms, that smaller AgNPs have the ability to release more silver ions, which promotes their antimicrobial effect, while the histological effect of AgNPs generally focuses on inhibition of microbial metabolism, leading to impaired production of extracellular polysaccharides and specific bacterial processes leading to its general dysfunction [65]. These studies confirm, that AgNP-coated brackets can help to decrease the spot lesions appearance during orthodontic treatment, and may be even useful in compromised patients with immune deficiency, diabetes, or elevated risk of endocarditis [66]. In addition to silver, many other nanoparticles like chitosan, copper, zinc, hydroxyapatite, and silicon dioxide can be added to composites in order to reduce bacterial activity and growth.

#### *4.2. Chitosan*

Chitosan is a naturally acquired polysaccharide that is formed by the deacetylation of chitin. It is a non-toxic, biodegradable, biocompatible, and has antibacterial properties [67], on *Agregatibacter actinomycetemcomitans*, *Porphyromonas Gingivalis,* and *Streptococcus mutans* [68,69]. Chitosan additionally has inhibiting action against fungi. This material's

application as an antibacterial chemical agent in mouthwashes is limited due to its reduced solubility in water. Nonetheless, its characteristics are highly desirable in dental materials. Chitosan could be maintained inside the materials in the oral cavity due to its insolubility in water. Histologically, inhibition is caused by inactivation of the enzyme, the substitution of lipopolysaccharides, metal ions, and formation of acidic polymer like teichoic acid. Chitosan, due to its low solubility and melting temperature, can be maintained in the oral cavity for a long period of time, unlike CHX which is released and disappears in the early phase.

#### *4.3. Copper Oxide*

It was proved by Yassaei et al. [70], that no significant difference was found between silver and copper oxide (CuO) nanoparticles, but it was noted that a curing time increased with the use of copper material when compared to the silver one. The former is cheaper and additionally both physically and chemically more stable than the latter. CuO nanoparticles affect *Streptococcus mutans* bacteria in a similar way as silver particles do [56]. It was confirmed in other studies [4], that copper and copper-zinc nanoparticles had a significant inhibitory effect on the studied microbes. According to other studies, CuO is able to decrease biofilm formation from 70 up to 80% [71]. Moreover, the similar results were achieved when CuO particles were incorporated into adhesive materials [72]. Additionally, nanoparticles like CuO can act as nano-fillers and enhance the shear bond strength of adhesive.

#### *4.4. Nitrogen-Doped Titanium Dioxide (N-Doped TiO2) Brackets*

The activation of N-doped TiO2 leads to the formation of OH. Free radicals, superoxide ions (O2), hydrogen peroxide (H2O2), and peroxyl radicals (HO2). These chemicals exert antimicrobial activity, also reacting with lipids, enzymes, and proteins. According to Poosti et al. [73], TiO2 nanoparticles of size 21 ± 5 nm can be blended to light cure orthodontic composite paste in 1, 2, and 3% and all these concentrations have similar antibacterial effects. Salehi et al. [74] proved, that nitrogen-doped TiO2 brackets have shown better antimicrobial activity when compared to the uncoated stainless steel brackets. Adding TiO2 to adhesives enhances its antibacterial activity without compromising its mechanical properties [75]. Nitrogen-doped TiO2 brackets were also reported to present antibacterial activity against normal oral pathogenic bacteria [76].

#### *4.5. Zinc Oxide (ZnO)*

It has been observed, that as the concentration of ZnO increases, the antimicrobial activity also increases, followed by shear bond strength reduction. It is important to underline, that ZnO and CuO coated brackets have been observed with better antimicrobial characteristics on Streptococcus mutans than when the brackets were coated with CuO nanoparticles alone [77]. Kachoei et al. [78], Behroozian et al. [79] and Goto et al. [80] proved, that following ZnO nanoparticle coating, the frictional forces be-tween archwires and brackets significantly decreased. Because of that effect, these na-noparticles offer new opportunities in overcoming the unwanted friction forces, better anchorage control, and reduced risk of resorption.

#### **5. Relationship between the Orthodontic Arch and Bracket Materials**

We use various brackets and arches in orthodontic treatment. The most popular materials from which the locks are stainless steel, titanium, ceramics, and plastic. The materials that arches are usually made of are: Stainless steel, nickel-titanium alloy, chrome-cobalt steel, and titanium–molybdenum alloy. Between the arch and the orthodontic bracket, we can observe the phenomenon of friction, which makes it difficult to move the bracket along the arch. Friction is one of the crucial forces in orthodontics. It acts against the traction force (TF), which can be seen in Figure 3.

**Figure 3.** Different forces acting over a body under traction on top of a surface. Body to be moved, traction force (TF), friction force (FF), contact surface (CS).

The friction observed with orthodontic sliding mechanics is a clinical challenge for orthodontists—the high levels of friction can reduce the effectiveness of the mechanics, reduce the efficiency of tooth movement and further complicate anchorage control [80]. One of the main goals of orthodontic manufacturing companies is to look for new products that would generate less friction during sliding mechanics. One of them is the use of nanoparticles. There are two variables that influence the friction generated during orthodontic treatment: Mechanical and biological [81].

Mechanical factors mainly include the material of the bow and bracket. The gold standard of materials for performing sliding is the combination of stainless steel brackets and arches. Based on the research by Kusy and Whitley, the friction force is influenced by the shape and size of the arc. They claim that the friction is greater in larger diameter arches [82]. Several studies show that rectangular wires cause more friction than round wires [83]. The friction also depends on the material of the arc. It has been shown that a SS wire pulled through an SS lock produces the least resistance. NiTi wires produce a little greater friction, while titanium–molybdenum (TMA) alloys the largest (Frank and Nikolai showed that NiTi wire has less friction than SS wire) [84]. Another aspect considered in terms of the friction force is the material of the bracket and the type of the bracket. Kusy et al. [85] compared the friction level of stainless steel and titanium brackets. Titanium showed a greater coefficient of friction. Based on research [86], ceramic brackets produce almost twice as much friction as SS brackets. The new, self-ligating type of brackets appears to cause less friction, but this idea still requires scientific confirmation.

It appears that the main biological factor influencing friction is the presence of saliva which, depending on the type of bracket and arch, can act as a lubricant or as a "glue". Its action will therefore increase or decrease friction. Baker investigated the effect of saliva on friction and concluded that human saliva reduces the friction force by 15–19% [87]. The correct composition and amount of saliva are therefore important in maintaining the correct treatment. Debris that can resist on the surface of orthodontic arches also appears to be a significant variable that can increase friction during orthodontic treatment. After 8 weeks of use on orthodontic arches, significant deposits of biofilm were registered. The described nanomaterials affecting the number of bacteria can reduce their number, indirectly affecting the condition of saliva and reducing the amount of plaque on orthodontic elements. Using them could prevent increased frictional forces.

According to the studies, exposure to the oral cavity for one month can cause a significant slowdown in orthodontic movement (in this case the NiTi arches were tested) due to the accumulation of biofilm [88]. Additionally, the study suggests that the acidic pH produced by the bacteria present in the plaque increases the roughness of the arc and thus

the friction between the wire and the bracket [89,90]. One of the ways to create unfavorable conditions for plaque accumulation is to try to include in orthodontic treatment the use of nanoparticles having a proven bacteriostatic effect. Properly-applied particles can also improve the mechanical factors by reducing the friction coefficient at the arc-lock interface.

#### **6. Microbial Colonization Associated with Different Kinds of FOAs.**

Fixed orthodontic appliances inhibit oral hygiene and create new retentive areas for plaque and debris as can be seen in Figure 4. It could increase the carriage of microbes and subsequent infection and it is one of the common problems that should be avoided in orthodontic treatment.

**Figure 4.** Orthodontic bracket covered by a nano-sized film.

The most common site for bacterial adhesion and biofilm formation is at the bracket adhesive-enamel junction, an area that is difficult to clean with daily brushing. The plaque that accumulates around orthodontic brackets often results in enamel decalcification, white spot formation, and dental caries adjacent to brackets. It is also difficult to remove microbial growth around orthodontic appliances. Its adherence to the fixed appliance is largely contributed by the bracket material and also the design of orthodontic brackets and ligating method [90,91]. The quantity and the quality of the plaque are influenced by many factors, including surface roughness, and surface-free energy [92]. Electrostatic attractions and van der Waal forces influence the adhesion of microorganisms to surfaces too [93]. Many types of braces are used in orthodontics. Bonded brackets have many advantages over bands such as better aesthetics, ease of placement, and removal and accessibility for oral hygiene [94].

#### **7. Introduction of Nanofillers or NP (Silver, TiO2) to Orthodontic Adhesives**

Orthodontic adhesives showed a higher capacity to retain cariogenic *streptococci* than bracket materials. Previous short-term (24-h) in vitro studies demonstrated comparable or lower and still acceptable shear strength when nano-filled adhesives were used to fix orthodontic brackets.

Compared to traditional orthodontic adhesives, the use of nanofillers reduced the surface roughness of the adhesive; however, this was not true when silver NP was added to this mixture. Nevertheless, evaluation of the long-term effect of nanofiber adhesives on preventing enamel demineralization during orthodontic treatment, particularly around brackets and under orthodontic bands, has not yet been investigated.

Silver has been found to have antimicrobial activity against gram-positive/negative bacteria, fungi, protozoa, some viruses, and strains resistant to antibiotics [95,96], as well as cariogenic *Streptococcus mutans* [97]. Resin composites containing fillers implanted with silver ions had antibacterial properties against oral *streptococci* [22]. The addition of NP

silver significantly reduces the adhesion of cariogenic *streptococci* to orthodontic adhesive compared to traditional adhesives, without compromising physical properties (shear bond strength). Adding TiO2, SiO2, or NP silver to acrylic orthodontic materials' cold-curing acrylic resins is common during the manufacture of removable orthodontic appliances such as expanders, fixers, and functional appliances which are mainly made of polymethyl methacrylate (PMMA). Compared to natural teeth, bacterial plaque adheres to acrylic resin braces with a larger surface area [98], which may lead to the development of caries-forming flora in the oral cavity. *Candida* Stomatitis is also an inflammation of the oral mucosa characterized by erythema (reddened areas), especially on the palate mucosa [99,100], which sometimes occurs under dentures (denture stomatitis) devices, or fixers.

CA is an opportunistic pathogen, and *Candida* is carried in the oral cavity in 25–75% of the studied populations [101]. A relationship has been suggested between the presence of a removable acrylic apparatus and the *Candida* carrier state, as well as low saliva pH [102]. In one study, the incidence of CA carriers before treatment with removable appliances was 39%; this number increased to 79% after 9 months and after treatment, and decreased to 14% after treatment. Similarly, orthodontic appliances placed on tooth tissues favored a greater proliferation of CA compared to dental appliances. The increase in *Candida* proliferation in people wearing removable appliances is probably due to protection against the natural and mechanical removal of saliva and the defense system [101].

Controlling CA proliferation under removable acrylic appliances can potentially prevent the development of orthodontic stomatitis. It is essential to find alternative therapies to eliminate CA that are tolerant to conventional antifungal drugs [100]. Investigation of the antimicrobial properties of NP acrylic materials and their use in mobile appliances is at an early stage and is limited to in vitro models. Sodagar et al. [54] investigated the changes in the bending strength of PMMA acrylic resin after adding TiO2 (0.5%) and SiO2 (1%) nanoparticles. The inclusion of NP in acrylic resin adversely affected the flexural strength of the final product and this effect was correlated with the concentration of NP [103]. However, a variable was observed after the addition of silver nanoparticles to the acrylic liquid of the two PMMA resins.

The mature dental plaque is composed of glucans and various microorganisms, the most common of them is *S. mutans* (the most cariogenic) and *Candida albicans*. Researchers like Shrinivaasan Nambi Rammohan, Ahn, Papaioannou, Fournier, or Brusca explored the relationship between CFUs (*S. mutans* alone, *C. albicans* alone, *S. mutans,* and *C. albicans* in combination) on surfaces of different kinds of orthodontic materials. When *S. mutans* was evaluated alone Shrinivaasan et al. [104], Papaioannou et al. [105], Fournier et al. [106], and Brusca et al. [107] found no obvious difference in the adhesion of *S. mutans* to stainless steel, plastic, and ceramic brackets. Quite different results were obtained Ahn et al. [108] There was a greater number of CFUs on stainless steel brackets than on plastic and ceramic brackets. Titanium and gold brackets showed lesser CFUs than stainless steel brackets. In the case of CA was evaluated alone, titanium brackets had the greatest of CFUs number because of the characteristics rough surface of these brackets [108] and gold brackets had the least number of CFUs because of the inert properties of gold. Plastic and ceramic brackets revealed a greater adherence than stainless steel brackets [107]. When *S*. *mutans* and *C. albicans* were evaluated in combination the clinical situation was different than an individual examination of these microorganisms and showed an antagonistic relationship at least in the initial growth but in the established plaque, they rather seem to exert a synergistic effect. For plastic and ceramic brackets, there was a greater number of CFUs and for metal brackets was the least [107].

Summarizing, titanium had some antibacterial properties but was not effective against the fungi. They grow by hyphae formation and the rough surface helped the increased levels of *C. albicans* [109]. Gold brackets revealed a decreased number of CFUs *S. mutans* and *C. albicans* and it could be inert properties of gold. Plastic and ceramic brackets showed greater levels of CFUs when *C. albicans* were studied alone and in combination with *S. mutans*. On composite yeasts exhibited numerous cell elongations which help in the adhesion mechanism and formation of pseudohyphae. Metallic brackets increase the level of bacterial adhesion compared with ceramic brackets because of the highest critical surface tension (greater surface energy). Stainless steel had an increased potential for microorganism attachment [110]. Properly, the material with high surface free energy will attract more bacteria than material with low surface free energy [110].

#### **8. Nanomaterials in Orthodontics and Their Use in the Nearest Future**

Nanoparticles are increasingly involved in dentistry [111]. They are used more often in conservative dentistry, endodontics [112,113], and prosthetics [111], where they become an integral part of treatment. They are used in irrigating solutions, filling materials and alloy in prosthetics. Their dynamic development should also include other fields of somatology, such as orthodontics. Currently we try find a ways to improve the value of mechanical orthodontic appliances. The use of nanomaterials partially solves this problem. The improvement of the biomechanical value of the orthodontic locks and arches, as well as the interference with the bacterial flora by nanomaterials seem worth developing. In the near future, adding nanoparticles to the materials of appliances will be the gold standard, improving the quality of orthodontic treatment. In addition to determining the basic components of the components of an orthodontic appliance, it will also be necessary to use appropriate proportions of nanoparticles in alloys. In the future, nanoparticles will also partly solve the problem of increased demineralization during treatment, which could reduce the number of complications. The use of the described particles also gives better control of the anchorage. Better control results in better, more predictable treatment [114], which reduces the stress of the orthodontist and increases patient satisfaction [115]. It will be possible to more accurately pursue the goals set at the beginning of treatment—during orthodontic diagnostics. A more thorough treatment will result in a better quality of life for the patient after treatment. The level of compatibility remains a challenge for nanoparticles in the future [116]. Further research is required to determine the safety of their use. Overcoming this problem makes it possible to easily increase the quality of orthodontic treatment. The use of nanoparticles will also reduce the described number of complications during orthodontic treatment, which will result in limiting the performance of additional procedures to eliminate complications. It also reduces treatment time, which reduces the cost of treatment. The shorter treatment time also allows more patients to be healed.

#### **9. Materials in Orthodontics and Their Use in the Nearest Future**

The future of nanotechnology in orthodontics has potential to develop in a number of additional applications as well including shape-memory polymers, self-healing materials, self-cleaning materials, biometric adhesives, tooth movement using orthodontic nanobots, and nano-changes on the surfaces of temporary anchorage devices (TADs) to increase their retention but still allow them to be removed when no longer needed [117,118].

Shape memory polymers, such as dual shape materials, belong to the group of "actively moving", which can change shape from one to the other. Orthodontics can use low stiffness transparent polymer arcs that can transform into arcs with a specific modulus of elasticity when exposed to a heat or light for example. With this procedure, it is possible to increase the effectiveness of treatment and aesthetics [119]. Self-healing materials that can repair themselves similar to biological systems. Hybrid materials have been developed, made of micro-ducts containing liquids or dissolved therapeutic agents. These materials can be used in the production of locks and orthodontic arches. A breach of the buckle or wire causes the nanobubble to burst and expose the monomer to the environment, thereby filling the resulting rupture gap with the described therapeutic agents [120].

Biometric adhesives—It is an enamel-friendly bonding mechanism for orthodontic appliances. The process takes place due to the formation of localized van der Waals forces [121]. This action ensures a strong bond between the materials without the use of a chemical substance. This material is often named "geckel". It acts as like a sticky note and exhibits strong, reversible adhesion in air and in water [122]. In orthodontics, such a procedure would ensure adequate bond strength without prior conditioning of the enamel. Self-cleaning materials have been developed, by using appropriate materials, increase the safety of using orthodontic appliances. The idea was taken from aircraft, where planes are covered with a titanium oxide nanocoating. A super-hybrid layer of hydrofluoric acid forms on the surface to prevent contamination. Photocatalytic activity resulting from the reaction of titanium oxide with light has attracted attention in orthodontic materials [123]. They try to find how inducing a reaction on the alloy of Ni-Ti archwires. By appropriate procedure—thickening the titanium oxide layer, electrolytic treatment and applying heat, it is possible to obtain a crystalline structure of rutile (titanium dioxide) on the surface of the materials [124].

#### **10. Conclusions**

Nowadays, nanotechnology plays an important role in the dental field since it has the potential to bring significant innovations and benefits. The recent positive results are a stimulus for future research, especially regarding orthodontics. The range of research including orthodontic bonding materials, covering of brackets and wires, as well as their antimicrobial characteristics has a huge potential. The review focused on scientific works concerning the use of nanoparticles in orthodontics that has been published in the literature over the last few years. The physicochemical properties gained by nano-sized materials have augmented the efficiency of orthodontic treatment. In this review, due to indicating the main types of literature reviews and referring to key studies showed that the physicochemical properties gained by nano-sized materials have augmented the efficiency of orthodontic treatment. Information can be implemented by scientists and doctors involved in the orthodontic therapy is included.

This review has also showed that the nanomaterials application regarding mechanical and antibacterial properties in orthodontics [55]. Nanoparticles can be successfully added to acrylic resins, cements, or orthodontic adhesives to prevent enamel demineralization during orthodontic treatment. Their versatility in clinical orthodontics can be seen in Table 1.

This review marked, that control and coordinated management of orthodontic treatment is crucial. Dental materials often present limitations during orthodontic treatment, but recently, nanotechnology and science have helped to partially solve some of the limitations. Nanomaterials can successfully reduce friction between the wire and the bracket, which may influence the orthodontic treatment. They are also useful in increasing the antimicrobial characteristics of materials used during treatment. Adding nanoparticles to the adhesives can increase their mean shear bond strength. This review provides several perspectives for the development of use nanomaterials in orthodontic.

Firstly, it is necessary to improve and search for new opportunities in overcoming the unwanted friction forces, better anchorage control, reducing the risk of resorption all should be based on evidence-based medicine and research generating stronger evidence.

Secondly, it is necessary to monitor the treatment of patients who use orthodontic nanomaterials due to the specificity of the oral cavity environment, which is dynamically changing. Biocompatibility and cytotoxicity are important considerations when using new bioactive materials. In the available literature, the knowledge about adverse effects resulting from the use of nanomaterials in orthodontics is limited. Despite the undoubted advantages of nanomaterials, knowledge about them is still incomplete and should be verified and carefully assessed, and the potential benefits should corresponded with the risk.

The application of nanomaterials in dentistry, especially in orthodontics is anticipated to grow further, and an interdisciplinary approach focusing on expertise in dentistry and nanomaterial science is required. The future in orthodontics will benefit greatly through nanotechnology.

**Author Contributions:** Conceptualization: M.D., W.Z., and R.J.W.; Methodology: W.Z., M.D., and R.J.W.; Software: W.Z., M.D., W.D. and R.J.W.; Validation: M.D. and R.J.W.; Formal analysis: W.Z.,

M.D., M.S., Z.R., and R.J.W.; Investigation: W.Z., M.D. W.D., Z.R., M.S., and R.J.W.; Data curation: W.Z., M.D., W.D., A.Z.-K., M.J., K.K., A.L., Z.R., M.S., and R.J.W.; Writing—original draft preparation: W.Z., M.D., W.D., A.Z.-K., M.J. K.K., and. R.J.W.; Writing—review and editing: W.Z., M.D., and R.J.W.; Supervision: M.D. and R.J.W.; Project administration: M.D. and R.J.W.; Funding acquisition: R.J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to acknowledge financial support from the National Science Centre (NCN) within the project "Preparation and characterization of biocomposites based on nanoapatites for theranostics" (No. UMO-2015/19/B/ST5/01330).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

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