**2. Features of Biomimetic and Hybrid Biomaterials**

From its first understanding, calcium-based biomineralization process uniqueness has attracted high attention due to its applicability in many different fields, especially for bone tissue engineering and regenerative medicine as well as the mild conditions, i.e., physiological temperature, pressure, and pH, in which this process occurs. Thus, the first step has been trying to translate the natural process into an in-lab process, mimicking the formation of the natural hierarchically structured organic–inorganic composites. Natural mineralization is commonly divided into two different groups recognized as "biologically induced" and "biologically controlled".

As reported by Weiner and Dove [35], the precipitation of minerals that occurs as result of interactions between biological components and the environment is termed "biologically induced" mineralization. In this situation, the chemical conditions of the environment, such as pH and CO2, indirectly favor the formation of a specific mineral type. The mineral composition varies consistently with the variation of the environments in which they form, resulting in different morphologies, water content, element composition, structure, and particle size.

In "biologically controlled" mineralization, the organism uses cells that actively take part in the nucleation, growth, morphology, and final location of the mineral that is deposited. While the degree of control varies across species, almost all controlled mineralization processes occur in an isolated environment. The result can be remarkably sophisticated, species-specific products that give the organism specialized biological functions. Calcium-based biomineralization can be described with subsequent stages, the first of which consist of inorganic molecules pre-assembling into ordered structures. Then, the molecular recognition among the organic and inorganic interfaces controls crystal nucleation and growth, allowing the formation of subunits. Finally, cells will take part in the process, forming biominerals with multilevel structure by assembling subunit minerals. All these steps are controlled by synergistic action of various environmental factors such as pH, temperature, and organic matrix chemistry [2]. Regarding the matrix, the interaction between inorganic and organic matrices leads the entire process, resulting in widely different results depending on the stereo-chemical and physical interaction occurring among the two components. This process is affected by different parameters; nevertheless, taking into account the influencing factors, the process can be transferred into an "in-lab" process and can even be controlled and directed towards the development of novel materials endowed with intriguing features. Speaking of organic matrices, it has been proven that the structural organization complexity highly affects the final biomineralization product, which might result in a three-dimensional scaffold in the case of highly organized matrices, or well as flakes or powders in the case of poorly organized matrices [36]. A clear example of this is given by the mineralization of collagen compared to gelatin. Collagen, with its multi-level organization, can undergo the biomineralization process without losing its structure, resulting in a three-dimensional scaffold with different properties depending on the biomineralization grade [10]. Gelatin, on the other hand, being a lower assembled polymer, can be exploited to form low structured biomineralized materials, composed of hybrid micro-flakes made of clusters of nano-particles [37].

In addition to collagen and gelatin, it is possible to find examples in the literature of mineralization with different polymer matrices as cellulose, chitosan, alginate, and silk (Table 1). Ahmed Salama has shown, for instance, how cellulose and its derivatives are promising candidates for developing and constructing smart organic/inorganic hybrid biomaterials through a calcium-based biomineralization process [38]. Cellulose/calcium phosphate hybrid materials, for example, combine the properties of both components, the functionality and flexibility of the cellulose with the heat resistance and stability of the inorganic material, to generate compounds that are being exploited for different applications such as bone regeneration, drug delivery vehicles, dental repair, and adsorption. Chitosan can be efficiently exploited to produce hybrid composites based on hydroxyapatite [39,40]. These hybrid composites are mainly devised for bone regeneration. Indeed, chitosan shares some peculiar features with collagen (e.g., a comparable role in the exo- vs. endo-skeleton and flexibility) [41]. Regarding alginate, a recent work [42]. reported the effect of alginate and its well-defined oligomers with defined structure on brushite nucleation and growth for the synthesis of hybrid materials, useful in bioactive agent delivery, wound healing, and tissue engineering. Growth experiments showed that molecular weight and additives of alginate affect the crystal growth rates and the growth mechanisms. Finally, Yang and collaborators [22] have described as silk fibroin as able to facilitate nucleation of the hydroxyapatite crystals through its molecular self-assembly, and to create efficient biomaterial with mechanical and functional properties for biomedical applications.

The ideal scaffold useful to obtain a robust in vivo affinity must be designed with the final purpose to resemble the 3D architecture, nanostructure, chemical composition and mechanical properties of the extracellular matrix (ECM) of the native tissue [21]. The scaffold must be composed of biocompatible materials able to guarantee cells viability, support of cellular functions and induction of molecular and mechanical signals without eliciting adverse effects on cells and, consequently, local or systemic undesired responses in the host [43,44]. Biomineralized substrates influence cells biochemistry by the exchange in proteins and ions that, subsequently, conditions the 3D microenvironment. This implies that proteins and ions composition (e.g., calcium and phosphate ratio, collagen, proteoglycans, etc.) of native tissue is a crucial parameter to be consider for scaffold design and synthesis [45]. If materials quickly degrade in vivo, the scaffold fails in its mechanical support role; conversely, an inflammatory response could be provoked by the foreign material if the scaffold has an excessively long biodegradation time. Therefore, a controlled biodegradability is crucial in scaffolds fabrication [46]. Overall, the microarchitecture of the support should be highly porous and interconnected to provide inwards diffusion of oxygen and nutrients and elimination of waste products in order to meet cellular requirements for adhesion, growth, differentiation and migration [47].

Porosity generally supports cell migration into the scaffold by promoting interaction between cells and the available surface area of the scaffold. Pores density and size influence cellular behavior in a inversely proportional way [48]; as pores size decreases, pores density increases as well as the surface area available on the scaffold for cells interactions. However, if pores are too small, cells are not able to penetrate and migrate inside the structure. Indeed, the pores dimension is able to affect vascularization of scaffolds. A different vascularization can consequently tune cells differentiation [49–51]. Consequently, scaffolds composition, but also scaffold porosity, need to be optimized on dependence of the tissue type to interact with; as example, for bone tissue engineering scaffolds need to contain a mixture of macroand micro-pores that allow cells to grow in vivo, facilitating cell-material interaction and complete scaffold colonization, respectively [52].

Additionally, in vivo mechanical signals (Young's modulus, compressive strength and fatigue strength mechanical forces) for cells must be replicated in the scaffold to induce the correct cellular differentiation pathway. Mechanical stiffness and porosity are often conflicting physical properties, as the first is inversely related to the other. Consequently, finding a good compromise between the scaffold properties for to promote the correct cellular activity and mechanical integrity is often a hard challenge [53]. The scaffold surface

can be bio-decorated with specific proteins/biomolecules or biomotives to improve its outcomes on cell adhesion, proliferation, and differentiation. Tissue formation and ECM deposition are regulated and performed by a wide variety of biomolecules. For this reason, scaffolds can be enriched by growth factors during their production, assuming a significant role in tissue engineering applications [54–56], such as improving tissue formation or the reward of specific biomolecules in pathological conditions [21].

The set of all above-mentioned properties makes the scaffold bioactive, osteoconductive and osteoinductive in tissue engineering, regeneration and modelling of mineralized tissue (bones, tooth, tendons, and cartilage). Osteoconductive scaffold can support the ingrowth of cells into pores, channel or pipes, while osteoinduction refers to the activity of a contact or soluble material to induce the differentiation of pluripotent stem cells towards specific cells lineage. Achieving these demands, calcium-based biomineralization process represents an excellent biomimetic manufacturing strategy to obtain hierarchically designed scaffolds with appropriate mechanical stability, flexibility and a highly porous and interconnected structure [12,15,57].

Biomineralized nanomaterials represent also a promising tool for different clinical applications. Nanoparticles as drug carriers for drug delivery involves the linking of drug molecules to the nanomaterial to guarantee a controlled distribution and release rate of the drug [58,59]. In this case a good biological activity and biodegradability together with good stability under close physiological conditions are required. Most of the components of biomineralized nanomaterials (e.g., calcium phosphate, carbonate and iron oxide) are naturally present in the body and easily and normally metabolized and absorbed by it; (3) good biological activity. Biomineralized nanomaterials facilitate loading of active molecules into the mineral phase by the interaction between biominerals and biomolecules, making possible to use them for drugs and molecules loading to be used inseveral biomedical practices [1].

**Physic-Chemical Parameter Scaffold Nanosystems** Biocompatibility Absence of cytotoxicity [44]; Support and stimulation of cellular activity [43] Absence of cytotoxicity Support and stimulation of cellular activity [1] Biodegradability Controlled biodegradability [46] High bioabsorption and biodegradability Absence of bioaccumulation of ions [1] Architecture Stability under physiological condition Highly porous and interconnected [47] Hierarchical design structure [9,57] Stability under physiological conditions [1] Porosity and pore size Mixture of macro- and micro-porosity [48–52] / Mechanical properties Mechanical integrity [53] Stability under physiological conditions [1] Surface properties Support and stimulation of cellular activity Tissue-specific functionalization [54–56] Tissue-specific functionalization Target-specific functionalization [1] Bioactivity Osteoinductive Osteoconductive [6,9,57] Controlled drug release and distribution [1,58,59]

**Table 1.** Influence of physical chemical properties of hybrid materials on in vitro/in vivo behavior.

#### **3. Applications in Biomedical Field: Tissue Regeneration and Many More**

Bone is the second most transplanted tissue with four million operations per year, using different bone alternatives worldwide [21,60]. Calcium-based biomineralization process is responsible for the formation of natural hard tissues such as bone and teeth [2,9,36,61] that are composed of extracellular matrix (ECM), several cells types, and water, which through a fascinating process are able to create a highly organized and hierarchically structured nanocomposite [13,36,62]. In detail, biomineralized ECM is mainly composed of plate-like nanocrystalline hydroxyapatite (the inorganic phase) and several organic components among which collagen is prevailing [63–65].

ECM highly influences adhesion, proliferation and differentiation of several cells types such as osteoblasts, bone lining cells, osteocytes as well as osteoclasts [65–67]. Several factors determine the fascinating and unique characteristic of bone, starting from composition arriving to structure and porosity, features essential for bone remodeling process. More precisely, bone tissue is characterized by a dynamic nature which allows to preserve its healthy condition, thanks to bone remodeling which consist in a dynamic and fine equilibrium between bone resorption by osteoclasts and bone formation by osteoblasts in response to biomechanical stimuli [68,69].

However, although this process preserve bone, its self-healing capacity is enough only to repair small bone damages. Subsequently, in case of extensive bone damage, bone substitutes are often required. For several decades, the gold standards were autografts or allografts [36,44]. In the last decade, instead, tissue engineering and regenerative medicine attention shifted towards development of biological replacements able to mimic and regenerate the tissues itself. In this perspective, scaffolds are becoming a fundamental tool for tissue engineering and regeneration, they indeed act as substrates able to structurally guide cells and provide anchorage sites, making possible to develop engineered structure made of a combination of materials and living cells. More precisely, the ideal bone scaffold is a transient implant that must create an adequate environment for cells to stay viable, attach, proliferate, differentiate and deposit ECM to replace the damaged or impaired one [70–72]. To do what describe above, scaffolds should resemble the natural bone tissue under different points of view, such as composition, structure, mechanical requirements and biological features. As consequence, to mimic the chemistry and structure of natural bone, biomineralization process is absolutely the most suitable in-lab process to produce biomimetic biomaterials, in which low-crystalline hydroxyapatite is mineralized on collagen fibrils, and highly porous scaffolds suitable to promote bone tissue regeneration [13,16,73].

Although this process has touched several fields of our life, biomedical engineering has surely received more specific attention, ending to be the most investigated. Nonetheless, recently, the attention has moved towards new advanced applications of mineralized materials. These applications include: (i) development of 3D predictive models for cancer study or drug testing, (ii) study of physical filters against solar radiation, (iii) creation of nano and micro controlled drug delivery systems. Additionally, the introduction of new fabrication technologies, e.g., 3D (bio) printing, are further extending the applications in other fields. These features will be discussed in the next sections.
