Contamination in Bone Substitute Materials: A Systematic Review

Abstract

Objective: Bone augmentation has become a significant practice in various areas of bone regeneration dentistry. This systematic review analyzes the research focused on evaluating bone substitute materials for the presence of contaminants. Methods: In June 2024, an extensive electronic search was conducted using renowned databases such as PubMed, Web of Science, and Scopus. Specific keywords employed in the search included ((bone AND (substitute) AND (remnants OR (purity)) OR ((graft AND tooth) AND (remnants OR purity)) OR ((graft AND dentin) AND (remnants OR purity)). The search adhered to the PRISMA protocol and the PICO framework. The review concentrated on the origin of bone substitute materials, the processing methods used for these materials, techniques for assessing purity, and types of contamination identified. A total of 594 articles were identified of which 22 met the criteria and were incorporated into the review. Results: Investigations into allogeneic and xenogeneic bone substitute materials have revealed that, despite manufacturers’ assurances of purity, some materials still contain contaminants. Sample analyses demonstrated the presence of donor cellular remains, cellular debris, intertrabecular fat, connective tissue, and collagen. Similarly, synthetically produced bone substitute materials (alloplastic materials) contained various impurities, such as polyvinyl alcohol (PVA), CaO phases, calcium-deficient HAp phases, oily substances containing carbon and silicone, cellulose derivatives, alpha-tricalcium phosphate (α-TCP), and heavy metals. Conclusions: Bone-derived and bone-like graft materials can contain various organic and inorganic impurities.

1. Introduction

Bone tissue is subject to a continuous process of remodeling. The activity of cells such as osteocytes, osteoblasts, and osteoclasts, in addition to the existence of the bone-signaling pathway, bone matrix, and access to nutrients from the blood, provide the potential for the rebuilding and repair of damaged or defective bone tissue [1]. Nevertheless, natural regeneration processes are frequently insufficient in cases of significant bone deficit. In such cases, bone grafting is one of the most frequently employed surgical techniques for augmenting bone tissue [2]. Bone substitute materials are pivotal in clinical applications for bone tissue regeneration and repair. These materials must closely replicate the properties of natural bone, including chemical composition, phase composition, and microstructure [2]. Their clinical relevance spans a variety of orthopedic and dental procedures, such as bone grafting, fracture repair, and the reconstruction of bone defects resulting from trauma, tumors, or congenital conditions [2].

The purity of bone substitute materials is crucial for their clinical efficacy and safety. Impurities in these materials can significantly compromise biocompatibility, potentially leading to adverse reactions like inflammation, infection, or material rejection by the body [3]. Contaminants can also impair the material’s bioactivity, undermining its ability to support new bone formation through osteoconduction or osteoinduction. For example, residual cellular debris, foreign particles, or unintended phases might trigger immune responses, disrupting the healing process and increasing the risk of implant failure. Additionally, impurities can adversely affect the mechanical properties of bone substitutes. Unwanted phases or structural irregularities may weaken the material, reducing its durability and making it susceptible to degradation under physiological stress [2,3,4]. This is especially critical for applications requiring mechanical support, where the material’s strength and longevity are essential for the success of the implant. In clinical settings, the use of high-purity bone substitute materials is essential for ensuring effective structural and functional integration with host bone while minimizing the risk of complications. Achieving the desired biomechanical properties often necessitates precise control over the material’s chemical composition, phase purity, and microstructure. Consequently, the strategic use of combinations of structurally distinct compounds is common, allowing for the optimization of material properties to meet specific clinical requirements while maintaining high standards of purity [3].

The source from which the grafting material originates can be used to distinguish between several grafting materials. (see Figure 1) Autografts (autogenous materials) are derived from the same individual and are transferred from the donor area to the recipient area. Allografts (allogeneic materials) are obtained from another representative of the same species following the appropriate preparation through freezing, freeze-drying, autoclaving, irradiation, or chemical preservation to ensure the absence of an immunological response. Xenogeneic materials (xenografts) are derived from other species [4]. Bone graft materials can be characterized by determining their osteoinductivity, osteoconductivity, and osteogenicity. It can be observed that only autogenous materials possess all three characteristics and are currently the gold standard in surgical procedures. Allogenic materials are the second-best option, offering various forms and large quantities [1]. Xenografts rich in natural hydroksyapatite [5] are also frequently utilized as materials due to their favorable osteoconductivity. However, they are not osteogenic or osteoinductive to the same extent as the aforementioned materials.

Despite the implementation of aseptic techniques during the harvesting and transplantation of bone grafts, the possibility of contamination remains. It is possible for microbiotas to form on the material during the interval between harvesting and transplantation [6]. Furthermore, environmental exposure of the donors to osteotoxic substances, such as lead, plays a significant role in graft purity. While the data in question are not routinely available, it is nevertheless the case that exemplary levels of accumulated lead in animal bone tissue have been demonstrated to impair the healing of fractures and bone metabolism [7]. Nevertheless, the incorporation of selected ions like strontium, iron, magnesium, zinc, or lanthanide substituted or co-substituted in hydroxyapatite has the potential to enhance the properties of the bone graft such as better biocompatibility, improved angiogenesis capability, or enhanced osteoconductivity [8,9,10,11].

It is of the utmost importance that grafting materials undergo appropriate processing, as this can significantly reduce the risk of unwanted contamination. Firstly, a comprehensive donor screening process is conducted, which includes an examination of the donor’s medical and social histories, as well as microbial and viral testing. Moreover, aseptic recovery, appropriate processing techniques, and a range of disinfection methods, including antibiotics, detergents, mechanical processes, chemical solutions, and terminal sterilization, are employed [12]. The demineralization of the tissue sample increases the fusion potential of the graft. A decellularization process was employed to minimize the risk of immunological response and graft rejection. However, in the case of bone grafts, the retention of some living cellular elements may be beneficial. This can be achieved through many techniques, including refrigeration in media, freeze-drying, cryopreservation, freezing, and media storage at room temperature [12].

The objective of this study was to conduct a comprehensive review and synthesis of existing data in the scientific literature regarding contamination associated with the preparation and processing of bone graft materials. A systematic review of this topic is necessary as no studies addressing a similar question were identified in the databases searched. The authors, therefore, deemed it essential to address this gap systematically.

2. Materials and Methods

2.1. Focused Question

This systematic review followed the PICO framework [13] as follows:

In patients needing bone substitute materials (Population), does the preparation and processing of these materials (Intervention) affect the contamination levels of alloplastic materials (Outcome) compared to bone-derived materials (Comparison)?

2.2. Protocol

The exact description of article selection for this systematic review was structured by the PRISMA protocol [14]. (Figure 2) The systematic review was registered on the Open Science Framework under the following link: https://osf.io/rz2hs (accessed on 3 August 2024).

2.3. Eligibility Criteria

The reviewers agreed to include only the articles which met the following criteria listed below [13,15,16,17,18,19,20]:

• Studies that focused on bone substitute materials processing;
• Examination of purity of bone substitute materials;
• In vitro studies;
• Full-text articles;
• Studies in English.

The exclusion criteria the reviewers agreed upon were as follows [13,15,16,17,18,19,20]:

• Studies which did not concentrate on bone substitute material preparation;
• Studies examining the properties of bone substitution materials;
• Studies which did not examine the purity of processed materials;
• Non-English studies;
• Systematic review articles;
• Reviews;
• Meta-analysis.

No restrictions were imposed regarding the year of publication.

2.4. Information Sources, Search Strategy, and Study Selection

In June 2024, an extensive electronic search was conducted using renowned databases such as PubMed, Web of Science, and Scopus. Specific keywords employed in the search included ((bone AND (substitute) AND (remnants OR (purity)) OR ((graft AND tooth) AND (remnants OR purity)) OR ((graft AND dentin) AND (remnants OR purity)). The search was conducted in accordance with the PRISMA protocol and the PICO framework. The review focused on the provenance of bone substitute materials, the processing techniques employed for these materials, methods for assessing purity, and the types of contamination identified. In the Scopus database, the results were refined to titles, abstracts, and keywords, while in PubMed, they were narrowed down to titles and abstracts. In WoS, the results were refined only to abstracts. The search parameters were constrained to studies meeting the eligibility criteria. Only articles with full-text versions were included in the analysis. A total of 594 articles were identified of which 22 met the criteria and were incorporated into the review.

2.5. Data Collection and Data Items

Seven reviewers (N.S., T.H., J.K., P.P., Ł.K., J.K., and S.K.) carefully selected the articles that met the inclusion criteria. The extracted data were then introduced into a standardized Excel file.

2.6. Assessing Risk of Bias in Individual Studies

In the preliminary phase of selecting studies, the authors independently examined the titles and abstracts of each study to reduce the possibility of reviewer bias. They evaluated the level of consensus among reviewers using Cohen’s κ test [21] (Watson, P.F). The authors resolved any disagreements about whether to include or exclude a study through discussions.

2.7. Quality Assessment

Two independent evaluators (J.M. and P.J.P.) assessed the procedural quality of each study included in the article. The criteria used to evaluate study design, implementation, and analysis included the physicochemical and/or microscopic characterization of evaluated alloplastic material, the quantitative assessment of contamination in alloplastic material, the presence of control group, a detailed description of material preparation/decontamination, a sample size calculation, and a biological assessment. Studies were scored on a scale of 0 to 6 points, where a higher score indicated better study quality. The risk of bias was categorized as follows: 0–1 points denoted a high risk, 2–4 points denoted a moderate risk, and 5–6 points indicated a low risk. Any discrepancies in scoring were resolved through discussion until a consensus was reached [13,15,16,17,18,19,20].

3. Results

3.1. Study Selection

A search of the electronic databases PubMed, Scopus, and WoS yielded 594 records. Of these, 161 were duplicates and were thus removed. The remaining 433 articles were subjected to abstract screening, which resulted in the exclusion of 549 articles that did not meet the inclusion criteria. A thorough analysis of the 45 full texts yielded a further 23 exclusions. The final number of articles included in this review was 22.

3.2. General Characteristics of the Included Studies

The studies included in the systematic review present research focused on evaluating bone substitute materials for the presence of contaminants. The general characteristics of the studies is presented in

Table 1. General characteristics of the selected studies.

Study	Aim of the Study	Material and Methods	Results	Conclusions
Hamidi et al. [22]	Synthesis of hydroxyapatite from eggshells, which can be used as a bone substitute.	Preparation of the hen eggshells: - Cleaned; - Crushed; - Ground; - Heated. CaCO ↓ CaO + organic substances - Activated mechanochemically in a quartz jar (obtaining HAp).	- Samples contained a CO32− substitutional group; - Heat-treated EHA samples had a chemically purer HAp phase. Heating to 1100 °C effectively produced highly crystalline HAp powder with β-TCP as a secondary phase.	- HAp derived from eggshells can be used for bone replacement; - Higher eggshell milling speeds improved HAp’s stability, purity, and crystallinity but caused its agglomeration; - Heat treatment at higher temperatures led to smoother particles and their spherical shape. 800 °C—Irregular, clustered particles. 1100 °C—Smoother and spherical.
Barbeck et al. [30]	Analysis of the ultrastructure of two available on the market bone blocks: allogeneic Maxgraft and xenogenic SMATRBONE.	Materials submitted to the following: - SEM; - Histological analyses. Cytocompatibility analyses with the use of fibroblast cultures.	Xenogeneic bone block—minor remnants of collagenous structures. Allogeneic bone matrix—high amounts of collagen. Both bone blocks free of other cell remnants.	- No cells or cellular remnants found in neither of the materials. Microscopic analyses revealed that the purification of both bone blocks differed depending on their collagen contents.
Ullah et al. [9]	Synthesis of strontium(II)/iron(III) (Sr2+/Fe3+) co-doped hydroxyapatite (HAp) bioceramics.	Bioceramics preparation: Sonication-assisted aqueous chemical precipitation method; X-ray diffractometer (XRD) analysis; Further measurements and assays.	Material partially pure. XRD analysis confirmed single-phase purity, indicating the stability of HAp when sintered at 1100 °C. The presence of peaks corresponding to β-TCP in the Sr2+/Fe3+ co-doped HAp bioceramics suggests that these samples were not entirely pure HAp, as they contained a secondary β-TCP phase.	Sr2+/Fe3+: HAp bioceramics promote bone formation functions in the following ways: - The inhibition of osteoclast activity (decreased bone resorption); Stimulating activity of preosteoblastic cell division and osteoblast differentiation.
Le et al. [24]	Synthesis of alpha-calcium sulphate hemihydrate (α-CSH) from calcium sulphate dihydrate.	- Dehydration and hydration of calcium sulphate; Dihydrate powder to obtain the (α-CSH); - Assessment in simulated body fluid, - assessment under SEM. Cytotoxicity analyses.	XRD analysis confirmed the formation of calcium sulphate hemihydrate (CSH) with traces of dihydrate (CSD) remnants. The synthesized hemihydrate was identified as α-CSH (purity of 98.62%).	The presented method was successful in synthesizing high-purity α-CSH (surgical-grade quality), although not without some disadvantages.
Ismail et al. [25]	Synthesis and obtaining a pure hydroxyapatite from green mussel shells.	Processing the green mussel shells; Obtaining precipitated calcium carbonate and the final hydroxyapatite product. The powders at each stage of production were subjected to SEM-EDX and XRD analyses.	- HAp production at each stage—powders contaminated with different CaCO3 phases or Ca(OH)2. HAp obtained after 18 h of synthesis in the autoclave tube with a hydrothermal reactor—pure.	PCC method resulted mainly in the crystallization of various CaCO3 phases, and 18 h of hydrothermal treatment allowed the obtaining of high-purity hydroxyapatite, free from other calcium phosphate compounds and amorphous phases.
Vojevodova et al. [26]	Synthesis of hydroxyapatite/polyvinyl alcohol (n-HAp/PVA) composite material and the inorganic phase purity after applying different synthesis parameters.	- Synthesis of n-HAp/PVA composite material; - XRD analysis. Transform infrared spectroscopy.	- Unreacted reactants and side products (β-TCP and CaO) found in n-HAp/PVA composite materials. ↓ CaO content higher than desired, depending on synthesis parameters. - Higher Mg content in the CaO precursor. ↓ Decreased β-TCP formation	- n-HAp/PVA composite materials have some purity issues (the presence of β-TCP, free CaO, and calcium-deficient HAp phases). The presence of PVA has the largest impact on inorganic phase purity.
Ghanaati et al. [31]	Histological analysis of two allogeneic and three xenogeneic bone blocks used in dental surgery. Assessment of the presence of components recommended by the manufacturer, focusing on organic and inorganic components within the bone blocks.	Preparation of bone graft materials (DIZG Human-Spongiosa, Tutobone, Puros Allograft, OsteoBiol Sp, Bio-Oss Spongiosa). Division into two parts; Decalcification; Dehydration; Embedding in paraffin; Preparation of five 4 um thick sections from every block; Staining of the selected sections (H+E); One section used to identify osteoclasts by histochemical staining for tartrate-resistant acid phosphatase. Examination under a microscope.	- DIZG Human-Spongiosa: no connective tissue remnants; - Tutobone: organic/cell remnants in the trabeculae; - Puros Allograft Spongiosa: organic/cell remnants in and on the trabeculae; - OsteoBiol Sp: organic/cell remnants in and on the trabeculae; - Bio-Oss: no organic cellular or extracellular components like connective tissue remnants.	- Three of the five bone blocks contained organic/cellular remnants; - In all five bone replacement materials, the manufacturers ensured that their blocks were free from such residues.
Piccinini et al. [32]	Comparison of the purity, chemical composition, and physical properties of OsproLife and Cerasorb.	Beta-tricalcium phosphate (β-TCP) granules (OsproLife) and Cerasorb were subjected to the following: - XRD; - Spectrometry; - Microscopy; - Cytotoxicity analysis; - In vivo bone performance in a rabbit model. Histological analysis and micro-computed tomography analysis were performed after implantation at 6, 12, and 52 weeks.	The XRD analysis showed the following: OsproLife (β-TCP) contains minor impurities within acceptable limits (hydroxyapatite (HAp), tetracalcium phosphate (TTCP), and alpha-tricalcium phosphate (α-TCP).	β-TCP OsproLife - Promising material for bone substitute use as it shows all desired chemical and physical properties; - Positive in vivo response—no local or systemic reaction was described.
Lee et al. [37]	Investigation of the microstructure, physical characteristics, and bone regeneration effect of β-TCP nanogranules using structural and physical properties and histological analysis.	Preparation of β-TCP powders by liquid–solid mixture precipitation method; Assessment of the morphology, pore structure, and phase composition of the β-TCP granules using the following: - FT-IR; - X-ray diffraction; - SEM; - Particle size analyzer; - Micro-CT. In vivo histological evaluation on Covance Beagles.	XRD and FT-IR tests showed the following: 99% purity of the material (no remnants of calcium pyrophosphate). In vivo study—no signs of inflammation, and the newly formed bone at the defect site had a larger volume compared to the control group.	Synthesized nanometric β-TCP: - Higher purity (99%); - Higher compressive strength (N2.22 MPa); - Higher porosity (N75%); - Specific surface area (N2.50 m2/g).
Lorenz et al. [38]	Histological analysis of allogeneic Maxgraft bone blocks available on the market to investigate the presence of other organic components.	Preparation of Maxgraft bone blocks: - Decalcified; - Dehydrated; - Embedded in paraffin; - Histological and histochemical staining. Microscope examination assessing the structure of the bone matrix and other components (collagen and cells/cellular debris)	- Cell remnants found within the osteocyte lacunae; - Mono- and multinucleated cells present. Intertrabecular fat, connective tissue, and collagen detected within the intratrabecular interspaces.	Some certified purification techniques do not allow the production of allogeneic material without organic cells and tissue components.
Hsu et al. [40]	Testing of α-calcium sulphate hemihydrate bioceramic as a bone substitute material.	α-calcium sulphate hemihydrate was synthesized and analyzed with the following: - SEM; - XRD; - Calorimeter; - GBX DGD-DI contact angle goniometer. In vitro cytotoxicity tests and the test on the CAM model were performed.	- Purer α-calcium sulphate hemihydrate when synthesized using a higher temperature with only single CSH (hexagonal) diffraction peaks present (no other precipitates or impurity compounds found). In vivo study: angiogenesis and osteogenesis.	Microwave-synthesized a-CSH is characterized by the following: - Good biocompatibility; - Angiogenesis; - Osteoconduction properties. Potential bone graft substitute for clinical applications.
Kubosch et al. [33]	Comparison of the clinical efficacy of allogeneic cancellous bone grafts (ACB) and synthetic or highly processed xenogeneic bone substitutes (SBS) in the treatment of bone defects.	- A total of 232 patients treated for bone lesions were divided into two groups—one treated with ACB (DIZG,) and the other treated with SBS (mainly BioOss) over a 10-year period. Both materials were seeded with human osteoblasts and later subjected to SEM analysis.	Histologically, - a similar bone structure was demonstrated in both allogeneic and xenogeneic materials. Cellular remnants (adipocytes and fibrocytes) were present only in allograft blocks.	- Both materials were similarly suitable for the treatment of bone defects. The treatment outcome was influenced by the site of implantation and epidemiological parameters. - No statistically significant differences were observed between ACB and synthetic or highly processed xenogeneic bone substitutes (SBS) in terms of complications or consolidation failure rates.
Chandran et al. [11]	To explore the impact of lanthanum (La3+) and praseodymium (Pr3+) ion substitutions in hydroxyapatite (HAP) on its phase purity, morphology, antibacterial efficiency, and biocompatibility.	Synthesis of hydroxyapatite (HAp) with co-doping of La3+ and Pr3+ ions exploring its physicochemical and biological properties.	The newly synthesized hydroxyapatite powders HLC (lanthanum co-substituted), HPC (praseodymium co-substituted), and HPLC lanthanum and praseodymium) were subjected to the following: - Cytotoxicity analysis; - XRD - Spectrometer analysis. The structure was analyzed using SEM.	- All synthesized samples exhibited phase purity; - XRD patterns confirmed the crystallization of the HAp structure without the presence of secondary phases. Substitutions of La3+ and Pr3+ ions into the HAp lattice did not introduce additional phases or impurities that would alter the primary HAP crystal structure.
Fathi et al. [27]	To produce a hydroxyapatite nanosized powder with increased bio-resorption properties and to evaluate its in vitro behavior.	The hydroxyapatite nanosized powder was prepared via sol-gel method and characterized for the following: - Phase purity; - Chemical homogeneity; - Bioactivity. This was done using the following: - FTIR; - Spectroscopy; - XRD. The in vitro test was performed in a simulated body fluid (SBF) medium.	The XRD results indicated the following: - Obtained hydroxyapatite was highly pure, and no extraneous phases were found; - CaO phase was found in the case of the sintering process at 700 °C.	Hydroxyapatite nanosized powders—pure materials that exhibit a higher dissolution rate than conventional hydroxyapatite powders. Nanosized powders possess superior bio-resorption capabilities and maintain a chemical and crystal structure similar to natural bone apatite.
Amouriq et al. [36]	To assess the skin sensitization potential of pharmaceutical-grade hydroxypropyl methylcellulose (HPMC) types: Benecel and E4M, in guinea pigs.	Preparation and purification of the materials; Analysis of physicochemical properties carried out using the following: - Gas–liquid chromatography; - FTIR analysis. SEM analysis was conducted. The sensitivity test was performed on Dunkin Hartley guinea pigs by intradermal sensitization.	The FTIR analysis showed the following: - Impurities were largely cellulose derivatives; - Silicon and carbon were found in Bencel materials, which turned to cause extreme sensibility reactions.	Benecel and E4M showed different grades of purity. E4M is better choice as an injectable bone substitute material as it does not cause extreme sensibility reactions as Bencel material.
Kanchana et al. [41]	To investigate the influence of sodium fluoride on the synthesis of hydroxyapatite using the gel method, focusing on how fluoride doping affects the crystallization, structure, and properties of hydroxyapatite crystals.	Hydroxyapatite growth carried out at room temperature under the physiological pH; NaF was added to half of the samples; Samples of pure and fluoride-doped HAp were sintered at 600, 900, and 1200 °C in the ambient atmosphere. All samples were subjected to SEM, XRD, TG, and FTIR.	Pure hydroxyapatite—no impurities (only samples sintered at 1200 °C with secondary products such as β-TCP or CaO). Fluoride-doped HA sintered in higher temperatures did not show secondary products besides pure HAp.	Hydroxyapatite synthesized via the gel method showed altered morphology: - Fibrous; - Granular (when fluoride present it hinders its formation and increases crystallinity). Fluoride-doped HAp maintained purity after high-temperature sintering, unlike pure HAp, which decomposed into β-TCP and CaO.
Murugan et al. [28]	To prepare a heat-deproteinated xenogeneic material from the slaughterhouse waste.	The adult tibias were collected and heat-deproteinated in appropriate conditions. Then, the material was subjected to the following: - Thermogravimetric analysis; - FTIR; - XRD.	- No secondary phase transformations observed in heat-deproteinated bone—the presence of pure hydroxyapatite (HAp) even at elevated temperatures confirmed. FTIR spectra—organic macromolecules in raw bone and bone heated to 300 °C, which disappeared in samples heated to 500 °C, 700 °C, and 900 °C—removal of antigenic organic substances around 500 °C.	The study shows that the heat-deproteinated bone can be used as a bone substitute material. However, it has to be treated with a temperature minimum 500 °C to eliminate all the organic macromolecules.
Fretwurst et al. [34]	Comparing different allogeneic bone substitute materials for alveolar ridge reconstruction.	Four different allograft specimen blocks from different suppliers were cut into two halves and subjected to histological and biochemical analysis.	- Organic tissue remnants (adipocytes, osteocytes) found in all samples; - Chondroblasts and fibroblasts found in Puros samples. Biochemical analysis allowed for successful DNA isolation and purification.	Some allografts may contain impurities such as cells of different types that may carry antigens (when detected by the recipient organism, they can lead to bone reconstruction failure).
Sandeep et al. [35]	The aim of the study was to assess the biocompatibility of in-house synthesized hydroxyapatite (HA) and novel bioactive glass-coated hydroxyapatite (BGHA) through in vitro experiments, and to compare their effectiveness in promoting osseous regeneration in vivo using a lapine femoral model with critical size defects.	The BGHA and HAp were grown in adequate conditions and then subjected to a series of analyses: - SEM; - TEM; - XRD; - FTIR; - EDAX; - Cytotoxicity test. In vivo experiment: 20 New Zealand rabbits—defects in the right femur were implanted with BGHA, and the left femur was implanted with HAp.	The XRD analysis showed the following: BGHA - Some impurities can be found (calcium silicate and tricalcium phosphate). Earlier trabeculae formation and faster cellular infiltration compared to HAp.	Bioactive glass-coated hydroxyapatite granules offer superior potential as a non-loading bone substitute for replacing missing bone.
Shepherd et al. [29]	The aim of this study was to explore how human osteoclasts develop and function on dense discs made of hydroxyapatite (HAp) and zinc-doped hydroxyapatite (Zn2+:HAp), aiming to discern the impact of zinc substitution.	The powders were treated as follows: Synthesized following the procedure; Subjected to XRD analysis to evaluate their purity. Pressed into discs and subjected to a cell culture experiment.	In both cases of HAp and Zn2+:Hap, the material purity was confirmed (a single phase was detected).The in vitro cell study showed the following: - Zinc substitution caused a decrease in osteoblasts cells after 21 days. Higher levels of zinc in HAp decreased the resorption level.	Zn2+:HAp could be a beneficial alternative for implant coatings, potentially reducing bone loss around implants and lowering the risk of implant loosening.
Lorenz et al. [39]	Histological evaluation of allogeneic cancellous bone block for horizontal and vertical alveolar ridge augmentation in humans.	- A total of 14 patients received augmentation with an allogeneic cancellous bone block; - After 6 months of healing, 28 implants were placed, and a bone biopsy was simultaneously taken for histological and histomorphometric analysis; - The formation of new bone, connective tissue, and remaining bone replacement material, as well as vascularization and the formation of multinucleated giant cells (MNCG) within the augmentation bed were analyzed. A blank bone block was also subjected to histological analysis.	- New bone formation visible near the bone block; - The histomorphometric analysis showed the following: o 18.65 ± 12.20% of newly formed bone; o 25.93 ± 12.36% of allogeneic cancellous bone block; o 53.45 ± 10.34% of connective tissue. - MNCG was observed on the surface of the biomaterial. In empty bone blocks, cell remains associated with the donor were found in the osteocyte lacunae.	The bone block could serve as a scaffold for new bone formation despite the presence of organic residues from the donor because they had no effect on bone formation or the recipient in the long term.
Ullah et al. [23]	Doping with Sr2+ and Fe3+ ions into HAp nanoparticles for potential biomedical applications.	HAp co-doped with Sr2+ and Fe3+ ions nanosized particles were systematically synthesized by a sonication-assisted aqueous precipitation method. Nanoparticles were evaluated for various physicochemical and biological properties.	X-ray diffraction showed the following: - Phase purity confirmed; - Hexagonal structure. SEM showed agglomerated, rod-like morphology of HAp nanoparticles that contained pores composed of small spheroids. Nanoparticles showed a dependence of magnetization on the loading level in mol% and showed tunable porosity and microhardness after heat treatment and hemolysis below 5%.	The multifunctional properties of the synthesized nanoparticles make them candidates for various biomedical applications such as the following: - Bone grafts; - Guided bone regeneration; - Targeted drug delivery; - Magnetic resonance imaging. Hyperthermia-based cancer treatment.

. A total of 10 studies aimed to synthesize new materials [9,11,22,23,24,25,26,27,28,29]. Seven of the studies compared materials [30,31,32,33,34,35,36]. Five studies analyzed materials [37,38,39,40,41]. The authors of the studies analyzed materials of various origins. Thirteen of the studies involved synthetic materials [9,11,23,24,26,27,29,32,35,36,37,40,41]. Four studies dealt with animal materials [22,25,28,38], and two with human materials [34,39]. Also, two studies analyzed both human and animal materials [30,31]. In contrast, one study compared human, animal, and synthetic materials [33].

3.3. Main Study Outcomes

The detailed characterization of selected studies is presented in

Table 2. Detailed characteristics of the included studies.

Study	Origin of Material	Type of Material	Preparation Method	Type of Remnants
Hamidi et al. [22]	Animal origin.	Eggshells.	The eggshells were treated as follows: - Rinsed with water; - Dried at 60 °C for 3 h; - Crushed into powder; - Sieved with 325 mesh. Uniform powder was then calcined at 900 °C for 3 h at a heating rate of 5 c/min.	Substitutional group of CO32−.
Barbeck et al. [30]	Human and animal origin.	Allogenic bone: Maxgraft.Xenogenic bone: SMATRBONE.	As described by the manufacturer.	Cellular and collagen-like structure remnants.
Ullah et al. [9]	Synthetic origin.	Pure HAp and Sr2+/Fe3+ co-doped HApbioceramics.	Sr2+/Fe3+ co-doped hydroxyapatite (HAp) nanoparticles were synthesized using a sonication-assisted chemical aqueous precipitation method: - Adjusting the pH to ≥9.5; - Adding diammonium hydrogen phosphate dropwise; - Stirring; - Sonication; - Settling for 24 h; - Washing; - Drying. Synthesized nanoparticles and pristine HAp were then heat-treated at 1100 °C for 2 h.	Contamination with other hydroxyapatite phases.
Le et al. [24]	Synthetic origin.	Alpha-calcium sulphate hemihydrate.	Calcium sulphate dihydrate powder was treated as follows: - Stirred in 200 mL of distilled water at 600 rpm for 15 min; - Subjected to 140 °C and 2.7 MPa for 4 h in an autoclave. The obtained suspension was filtered and rinsed with boiling distilled water, washed with acetone, and dried at 55 °C for 16 h.	Other hemihydrate or dihydrate phases.
Ismail et al. [25]	Animal origin.	Hydroxyapatite synthesized from green mussel shells.	The shells were treated as follows: - Cleaned and sun-dried; - milled and filtered with a mesh—100; - Calcined at 900 °C for 5 h; - Powder was mixed with 300 mL of 2 M HNO3 at 60 °C for 30 min; - The mixture was neutralized and filtered - CO2 gas was slowly introduced to precipitate the filtrate; - Washed with distilled water; - Dried at 110 °C for 2 h to obtain a pure precipitated calcium carbonate (PCC); - PCC was mixed with (NH4)2HPO4; - Hydrothermally treated at 160 °C for 14–18 h; - Filtrated and dried at 110 °C for 2 h to obtain a hydroxyapatite.	CaCO3 (aragonite, vaterite, and calcite phases), Ca(OH)2.
Vojevodova et al. [26]	Synthetic origin.	Nanosized hydroxyapatite/polyvinyl alcohol composite material.	- Polyvinyl alcohol (PVA) was dissolved in deionized water at 60 °C to make a 5% solution; - Suspension was prepared by adding 0.45 M Ca(OH)2 to the PVA solution; - A total of 2 M H3PO4 was slowly added into this suspension while stirring at 45 °C; - After 24 h, the suspension was dried at 50 °C for about 72 h to produce the as-synthesized composite obtained material was subsequently sintered in air at 1100 °C for 1 h.	Pure hydroxyapatite (HAp) phase and polyvinyl alcohol (PVA) phase, β-TCP, CaO.
Ghanaati et al. [31]	Human and animal origin.	Allogenic bone: DIZG Human-Spongiosa, Puros Allograft Xenogenic bone: Tutobone, OsteoBiol Sp, Bio-Oss Spongiosa	As described by the manufacturer	Matrix components remnants, cellular remnants, fibrous remnants, connective tissue remnants
Piccinini et al. [32]	Synthetic origin.	OsproLife- Beta-tricalcium phosphate (bTCP) granules.	As described by the manufacturer.	Hydroxyapatite (HAp), tetracalcium phosphate (TTCP), and alpha-tricalcium phosphate (α-TCP), heavy metals.
Lee et al. [37]	Synthetic origin.	Beta-tricalcium phosphate (β-TCP).	- A total of 0.5 M (NH4)2HPO4 solution was mixed with Ca(NO3)2⋅4H2O powder; - Stirred for 5 min (calcium-deficient apatite precipitates were formed); - Mixture was aged for 2 h; - Filtered; - Dried at 80 °C for 20 h; - Calcined at 800 °C for 12 h; - Formed β-TCP powders were sieved; - Mixed with 55.% polyethylene glycol (PEG) particulates; - Pressed into disks; - Heat-treated at 600 °C to remove PEG; - Sintered at 850 °C, and cooled. The resulting porous β-TCP scaffold was then fractured into granules of 500–1000 μm.	Calcium pyrophosphate remnants.
Lorenz et al. [38]	Animal origin.	Allogenic bone: Maxgraft.	As described by the manufacturer.	Cells, cell remnants, connective tissue remnants, fatty tissue-like structures.
Hsu et al. [40]	Synthetic origin.	alfa-calcium sulphate hemihydrate bioceramic.	- Calcium sulphate dihydrate CSD was mixed with deionized water; - Stirred; - The mixture was sealed and microwaved at different temperatures (100 °C, 130 °C, and 160 °C) for 10 min under 800 W power while stirring; - Samples were filtered; - Washed five times with high-purity ethanol; - Dried at 60 °C for 8 h samples were ground into powder.	CSD (monoclinic) and CSH (hexagonal).
Kubosch et al. [33]	Human, animal, and synthetic origin.	Allograft bone:DIZG Human Bone Blocks. Xenograft bone: BioOss, Endobone, Pyrost, Tutobone. Synthetic: Norian SRS, Chronos, Atlantik, Alaska, Nanostim, Actifuse, PerOssal.	As described by the manufacturer.	Cellular remnants (adipocytes and fibroblasts).
Chandran et al. [11]	Synthetic origin.	Apatite matrix substituted with lanthanum and praseodymium.	- Nitrate solutions containing Ca2+, Pr3+, and La3+ ions were prepared; - (NH4)2HPO4 solution was added under constant stirring at 90 °C; - A total of 2% CTAB (cetyltrimethylammonium bromide) was added relative to the cationic mixture concentration to enhance dispersion; - Mixture was stirred for 2 h; - Dried at 120 °C; - Sintered at 900 °C. Throughout the synthesis, three different compositions were obtained: - HLC (La3+ substituted); - HPC (Pr3+ ion’s doping). HPLC (co-doped with La3+ and Pr3+ ions).	Ionic remnants, pure hydroxyapatite remnants.
Fathi et al. [27]	Synthetic origin.	Hydroxyapatite nanosized powder.	- Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and a phosphoric pentoxide (P2O5) were dissolved in absolute ethanol; - Solution was gradually transformed into a gel during 24 h of continuous stirring at room temperature; - Gel was aged for 24 h and then dried at 80 °C for 24 h; - Dried gel underwent sintering in a muffle furnace at a ramp rate of 5 °C/min up to different temperatures: 600 °C, 700 °C, 800 °C, and 900 °C. Samples were cooled to ambient temperature.	Impurity phases, CaO.
Amouriq et al. [36]	Synthetic origin.	Hydroxypropyl methylcellulose polymer vector with biphasic calcium phosphate (BCP-Bencel and E4M).	- Bencel and E4M were prepared as a 3% aqueous solution; - BCP was subjected to a purification process including the following: o Centrifugation at 4000 rpm for 1 h; o Ultracentrifugation at 20,000 rpm for 1 h to separate insoluble components. - Purified HPMC was filtered through a 0.2-mm-pore acetate cellulose filter dried at 50 °C for 15 h.	An oily substance containing carbon and silicone, cellulose derivative.
Kanchana et al. [41]	Synthetic origin.	Hydroxyapatite synthesized in the presence of sodium fluoride.	- The solution of sodium metasilicate gel (SMS) was adjusted to pH 7.4 using 10% glacial acetic acid and a 0.6 M Na2HPO4 solution; - Solution was poured into test tubes and allowed to gel; - Calcium chloride was added; HAp samples doped with sodium fluoride; - Supernatant was prepared by mixing 1 M calcium chloride with 10% sodium fluoride; - Growth of both pure HAp and doped samples was monitored through transparent gel media; - After three weeks, the grown HAp samples were harvested; - HAp samples, with and without fluoride, were heated at temperatures of 600, 900, and 1200 °C, with a heating rate of 5 °C/min in an air atmosphere. After holding for 5 h at the target temperature, the furnace was cooled to room temperature.	Granular structure, β-TCP, and CaO phases.
Murugan et al. [28]	Animal origin.	Xenograft bone (bovine bone).	- Samples rinsed with water and then boiled in distilled water for 12 h - immersed in 2% NaCl for 12 subjected to thermal processes at 300, 500, 700, and 1200 °C.	Organic remnants.
Fretwurst et al. [34]	Human origin.	Allogenic bone: Puros, Maxgraft, Osteograft, Allograft CTBA.	As described by the manufacturer.	Organic material consisting of cells, cell debris, and fibrous tissue between the bone trabeculae.
Sandeep et al. [35]	Synthetic origin.	Hydroxyapatite (HAp) and bioactive glass-coated hydroxyapatite granules (BGHAp).	- HAp powder was synthesized via a precipitation reaction between aqueous calcium and phosphate solutions in stoichiometric proportions under basic conditions; - The resulting precipitate was aged, washed, centrifuged, calcined, and ball-milled to obtain a fine powder; - Powder was then mixed with a pore former and compacted into cylinders, which were fired and subsequently crushed into granules. To produce BGHAp, the following procedure was utilized: - Granules were coated with a thin silica sol, dried, and sintered at 1200 °C. Sintered granules were cleaned in acetone and deionized water using an ultrasonicator, followed by sterilization in an autoclave.	Calcium silicate and tricalcium phosphate.
Shepherd et al. [29]	Synthetic origin.	Hydroxyapatite (HAp) and zinc-doped hydroxyapatite (Zn2+:HAp).	- HAp and Zn2+: HAp powders were produced through a precipitation route using calcium hydroxide (Ca(OH)2) and orthophosphoric acid (H3PO4) For Zn2+:HAp; - Zinc nitrate hexahydrate was added to the Ca(OH)2 to achieve zinc substitution; - Precipitation involved slowly adding the acid solution to the calcium solution in deionized water; - Washing; - Filtering; - Drying the precipitate at 60 °C overnight; - Small samples of each powder underwent heat treatment at 1100 °C in an air atmosphere for 1 h, with the temperature ramping up at 10 °C/min. Powders were ground and sieved to less than 75 μm.	Zinc remnants.
Lorenz et al. [39]	Human origin.	Allogenic bone: Tutobone.	As described by the manufacturer.	Organic/extracellular remnants.
Ullah et al. [23]	Synthetic origin.	Sr2+/Fe3+ co-doped hydroxyapatite.	- Sr2+/Fe3+ co-doped hydroxyapatite (HAp) nanosized particles were synthesized via aqueous precipitation and sonication; - Calcium nitrate tetrahydrate, diammonium hydrogen phosphate, strontium nitrate, and ferric chloride hexahydrate were dissolved in deionized water; - Ammonium hydroxide (NH4OH) adjusted the pH to ≥9; - (NH4)2HPO4 was added dropwise to form a white precipitate, which was stirred at 65–70 °C for 4 h, sonicated for 20 min, and allowed to settle for 24 h; - Washing and drying at 60 °C; - Sr2+/Fe3+ co-doped HAp nanoparticles were obtained. Pure HAp nanoparticles were synthesized similarly without Sr(NO3)2 or FeCl3·6H2O.	Elements and substitutional group remnants.

. Studies selected in this systematic review varied in their comparison research focused on evaluating bone substitute materials for the presence of contaminants.

Six groups of researchers [9,22,25,27,28,40] confirmed that bone substitute materials synthesized using a higher temperature show greater purity. Ismail R. et al. [25] found that pure hydroxyapatite was produced after synthesizing for 18 hours in an autoclave tube using a hydrothermal reactor. Seven studies [28,30,31,33,34,36,38] identified the presence of organic/cellular remnants. High levels of collagen were discovered to be connected to the allogeneic bone matrix [30]. Additionally, six studies [24,25,26,27,29,35] identified the presence of nonorganic remnants. Le et al. [24] confirmed the formation of calcium sulphate hemihydrate (CSH) with traces of calcium sulphate dihydrate (CSD) remnants. Furthermore, Ismail R. et al. [25] verified that the hydroxyapatite powders were contaminated with various CaCO₃ phases or Ca(OH)₂ at each stage of production.

3.4. Quality Assessment

Among 22 articles included in the review, no entries were assigned with a high risk of bias, 16 were assigned a moderate risk of bias [9,22,23,25,26,27,28,30,31,33,34,36,38,39,40,41], and the remaining 6 with a low risk of bias [11,24,29,32,35,37] (see

Table 3. Quality assessment.

Study	Physicochemical and/or Microscopic Characterization of Evaluated Alloplastic Material	Quantitative Assessment of Contamination in Alloplastic Material	Presence of a Control Group	Detailed Description of Material Preparation/Decontamination	Sample Size Calculation	Biological Assessment	Total pts.	Risk of Bias
Hamidi et al. [22]	1	0	1	1	0	0	3	Moderate
Barbeck et al. [30]	1	0	1	1	0	1	4	Moderate
Ullah et al. [9]	1	0	1	1	0	1	4	Moderate
Le et al. [24]	1	1	1	1	0	1	5	Low
Ismail et al. [25]	1	1	0	1	0	0	3	Moderate
Vojevodova et al. [26]	1	1	0	1	0	0	3	Moderate
Ghanaati et al. [31]	0	0	1	1	0	0	2	Moderate
Piccinini et al. [32]	1	1	1	1	0	1	5	Low
Lee et al. [37]	1	1	1	1	0	1	5	Low
Lorenz et al. [38]	1	0	0	1	0	0	2	Moderate
Hsu et al. [40]	1	0	1	1	0	1	4	Moderate
Kubosch et al. [33]	1	0	0	0	0	1	2	Moderate
Chandran et al. [11]	1	1	1	1	0	1	5	Low
Fathi et al. [27]	1	1	0	1	0	0	3	Moderate
Amouriq et al. [36]	1	0	1	1	0	1	4	Moderate
Kanchana et al. [41]	1	1	1	1	0	0	4	Moderate
Murugan et al. [28]	1	1	1	1	0	0	4	Moderate
Fretwurst et al. [34]	1	0	0	1	0	1	3	Moderate
Sandeep et al. [35]	1	1	1	1	0	1	5	Low
Shepherd et al. [29]	1	1	1	1	0	1	5	Low
Lorenz et al. [39]	0	0	1	1	0	1	3	Moderate
Ullah et al. [23]	1	1	1	1	0	0	4	Moderate

).

4. Discussion

During bone augmentation procedures, due to significant bone loss, the lack of contamination of the surgical field is crucial to the success of the procedure [42,43]. Aseptic and antiseptic techniques help reduce the number of contaminants transferred to the surgical field by the operator’s tools and hands. However, all these efforts to increase the success of treatment may be in vain when using bone substitute material containing contaminants. This systematic review included 22 papers that evaluated various contaminants in bone substitute materials. These contaminants can be of organic origin, chemical compounds, or crystalline phases that hinder or prevent new bone formation. The findings indicate significant gaps in research on the contamination of bone substitute materials. Among the studies included in the review, thirteen focused on synthetic materials [9,11,23,24,26,27,29,32,35,36,37,40,41], four studies dealt with animal materials [22,25,28,38], and two investigated human materials [34,39]. Additionally, two studies analyzed both human and animal materials [30,31], and one study compared human, animal, and synthetic materials [33].

Tests of allogeneic and xenogeneic bone substitute materials have shown that some materials contain contaminants, although manufacturers ensure the purity of their materials. Research by S. Ghanaati et al. [31] based on the analysis of allogenic bone blocks available on the market showed that three out of five materials available on the market were contaminated. Manufacturers ensured the purity of the materials, but contaminants such as organic and cellular residues were found. Histological analysis of the Maxgraft material by J. Lorenz et al. [38] showed cellular debris, intertrabecular fat, connective tissue, and collagen. The work of T. Fretwurst et al. [34] involved the evaluation of allografts from different donors, showing that bone block cleaning techniques did not ensure perfect cleanliness of the samples. All samples were subjected to histological and biochemical analysis. In each case, contaminants and remains of organic tissues, including adipocytes and osteocytes, were found. Additionally, chondroblasts and fibroblasts were detected in Puro’s samples. Biochemical analysis allowed for successful isolation and purification of DNA. Lorenz J et al. [39] subjected histological examination to allogeneic bone blocks implanted in patients for the purpose of regenerating the alveolar process. The collected samples contained donor cellular remains. Pollution did not negatively affect the regeneration of the alveolar process. Moreover, research by Eva Johanna Kubosch et al. [33] found that only allograft blocks contained cellular remnants such as fibrocytes and adipocytes, while xenogeneic materials were free from such impurities, indicating variability in biological contamination depending on the material source. In contrast, research by Amouriq et al. [36] revealed that Benecel and E4M materials were primarily contaminated with cellulose derivatives, with additional contaminants like carbon and silicone identified in Benecel products. These findings emphasize the importance of material origin and processing methods in determining contamination levels.

Synthetically produced bone substitute materials are also not free from contaminants. Research by A. Vojevodova et al. [26], aimed at assessing the purity of the synthetically produced hydroxyapatite/polyvinyl alcohol (n-HAp/PVA) composite material, showed the presence of β-TCP, free CaO and calcium-deficient HAp phases, and PVA, which has a negative impact on osteogenesis. Some additives used during the synthesis of bone substitute materials based on hydroxyapatite significantly improve the properties of the resulting product. L. Chandran et al. [11] assessed the synthesis of hydroxyapatite (HAp) with La³⁺ and Pr³⁺ ions’ co-doping. The results indicate that the incorporation of Pr³⁺ and La³⁺ ions into hydroxyapatite (HAp) causes slight lattice distortion without secondary phase formation, with increased bioactivity, cell viability, and moderate antibacterial efficacy, suggesting promising biomedical applications. Processing the shells by grinding and thermal treatment at 1100 °C results in the formation of smooth spherical hydroxyapatite particles that can be used as a bone substitute, as proven by Hamidi AA et al. [22]. In the research of Ismat Ullah et al. [9], synthesized hydroxyapatite bioceramics were co-substituted with strontium and iron. They exhibited good properties because they inhibit the activity of osteoclasts, which reduces bone resorption and increases the activity of preosteoblastic cell division and osteoblast differentiation, thus promoting bone formation functions. However, their structure showed remnants of the secondary β-TCP phase. Nhi Thao Ngoc Le et al. [24] confirmed the formation of calcium sulphate hemihydrate (CSH) with traces of dihydrate residue (CSD). Furthermore, Ismail R. et al. [25] verified that hydroxyapatite powders were contaminated with various phases of CaCO₃ or Ca(OH)₂ at each production stage.

The results of the included studies highlighted the critical issue of contamination in bone substitute materials and its implications for phase purity and biological performance. Sandeep G. [35] identified contaminants like calcium silicate and tricalcium phosphate in BGHA materials. However, other studies, such as that by M.H. Fathi et al. [27], showed that while hydroxyapatite can be obtained with high purity, certain processes like sintering at 700 °C can introduce a CaO phase, suggesting that processing conditions are crucial in maintaining material purity. In terms of specific bone substitute materials, Marzio Piccinini et al. [32] found that OsproLife (bTCP) contains trace amounts of hydroxyapatite (HA), tetracalcium phosphate (TTCP), and alpha-tricalcium phosphate (α-TCP) within acceptable limits, indicating controlled impurity levels. Additionally, nanometric β-tricalcium phosphate synthesized by David S.H. Lee et al. [37] exhibited high purity and desirable physical properties, such as increased surface area and compressive strength. Ionic doping in hydroxyapatite, as explored by Ullah I et al. [23] and others, demonstrated that dopants like Sr²⁺, Fe³⁺, La³⁺, and Pr³⁺ can influence phase purity, though often without introducing new phases [11]. However, some doped materials, such as Sr/Fe co-doped HAp, were not entirely pure, containing secondary β-TCP phases [9].

During the research, difficulties were encountered due to the limited number of available articles containing information on the purity of bone substitute materials. There is a need for more extensive research on the presence of contaminants in materials already introduced to the market. It is necessary to establish new methods and standards for graft cleansing in order to improve the quality of bone substitute materials. In the future, searches should focus on a broader analysis of synthetic materials that can be successfully used as bone substitutes. The research methodology on bone substitute materials should be unified for further meta-analysis. The small number of studies and their significant heterogeneity limit the possibility of conducting a meta-analysis.

5. Conclusions

This systematic review focused on analyzing bone replacement materials to assess their purity and contamination levels. The results indicate that both organic and synthetic bone replacement materials contain contaminants. Despite manufacturers’ assurances of purity, contaminants such as organic and cellular residues were detected. The evaluation of allografts from different donors revealed that current bone block cleaning techniques are insufficient for achieving complete cleanliness. Future research should prioritize evaluating contamination levels in bone substitutes across various manufacturers and processing techniques. Additionally, it is essential to develop and standardize new methods and protocols for cleaning and processing bone substitute materials to ensure their purity. Furthermore, bone substitute materials come in a range of sizes and shapes, making them suitable for various applications. As the development of biodegradable materials broadens their potential uses, further studies are needed to explore these materials comprehensively.