**2. Bio-inspired synthesis processes: hybrid biomimetic scaffolds through biomineralization**

In the last decade *bio-nanocomposites* emerged as a new class of materials including a natural polymer (biopolymer) in combination with an inorganic phase, rather than using synthetic polymers. Indeed the need of biomimetic materials and the limitations of the current fabrica‐ tion methods are increasingly stimulating material scientists to explore this new class of compounds, thus benefitting of the presence of a polymer matrix that can be subjected to physiological, cell-mediated resorption in vivo, rather than to processes of chemical dissolu‐ tion. In fact, the chemical leaching of polymeric scaffolds is one of the possible cause of failure in vivo, as the dissolution process can be too fast with respect to the new bone formation process, and also, the degradation products of many polymers can result in harmful effects, jeopardizing the regenerative cascade so that fibrous scars may form, rather than healthy, organized bone tissue. The inspiration for the design and development of bio-nanocomposites takes place from living organisms that are able to produce natural nanocomposites showing an amazing hierarchical arrangement of their organic and inorganic components from the nano to the macro scale (**Figure 1**).

**Figure 1.** Natural bio-nanocomposites.

**Keywords:** bone regeneration, bioinspired materials, biomineralization, biomorphic

Materials science today is experiencing a paradigmatic change in the development of new smart devices for biomedical applications. Particularly, the regeneration of hard tissues (i.e. bone, cartilage, tooth) is one of the most demanding issues in medicine and requires smart devices showing high mimicry of the host tissues and ability to instruct and drive progen‐ itor cells to activate the regenerative cascade. Therefore, among the various approaches pursued so far for the synthesis of bone biomaterials, wide consensus is now consolidated around the concept of "*biomimetics*". Such a definition indicates the ability of a synthetic material to closely reproduce the chemical composition, physical properties, and architec‐ ture of native tissues, with the purpose to create 3-D environments able to deliver signals stimulating cell chemotaxis and specific differentiation of autologous stem cells [1]. In this way, the main concept is that bone regeneration can be greatly aided by the fact that, by implantation of a biomimetic scaffold, the patient body acts as a natural bioreactor guid‐ ing proper tissue regeneration without the need of complicated tissue engineering proce‐ dures or of the use of biological factors, thus improving the safety of clinical approaches. In this respect the chapter highlights some emerging concepts related to the development of bio-inspired materials addressed to hard tissue regeneration. In particular, the focus is on assembling/mineralization techniques that reproduce the cascade of phenomena acting in the formation of hybrid nanocomposites such as bone and shells, that can generate hybrid fibrous structures with excellent regenerative ability. This process, pinning on the exchange of information stored in the structure of natural polymers, is characterized by great versatility that enable the synthesis of smart multifunctional scaffolds for regeneration of tissue com‐

On the other side, the chapter is focused on the emerging concept of biomorphic transforma‐ tions by which natural structures with hierarchic architecture are converted into apatitic biomaterials with unpreceded bioactivity and structure, by multi-step chemical processes. In fact, as the process bases on heterogeneous reactions at the interface between a solid template and a gaseous phase, the obtained scaffolds result well consolidated without the need of sintering treatments and exhibit enhanced mechanical properties, due to the hierarchical architecture, thus being very promising for regeneration of load-bearing bones such as those of the limbs. Finally, the chapter highlights the recent development of an iron-substituted hydroxyapatite (HA) nanophase that, thanks to its excellent biocompatibility and intrinsic magnetic properties, demonstrated ability to be activated by remote magnetic signalling, thus representing a new switching tool for the development of a multifunctional platform gener‐ ating smart bio-devices for various applications in regenerative medicine and theranostics. This new material, overcoming the limitations of toxic iron oxide nanoparticles currently used

transformation, magnetic activation

130 Advanced Techniques in Bone Regeneration

plexes such as joints and periodontium.

**1. Introduction**

These outstanding architectures are the key of the insuperable performance of natural structures, particularly by a mechanic perspective: nacre, shells, bones, ligaments, tooth enamel, and dentine are just some examples of hierarchical, hybrid bio-nanocomposites found in nature. The mechanism at the basis of this outstanding structural arrangement is the establishment of hybrid building blocks formed upon heterogeneous nucleation of inorganic nanophases (such as carbonates and apatites) onto natural polymers, driven by several control mechanisms acting at the molecular scale [2]. In particular, during new bone formation, type I collagen, extruded by fibroblast cells, acts as a template for the nucleation of the mineral phase through a hierarchical assembly of collagen molecules into fibrils and ever thicker fibres, whereas HA nano-nuclei nucleate onto specific positively charged sites located in the collagen molecules. This process is governed by several control mechanisms inherent in the molecular structure of collagen that guide the formation of new bone at all scale sizes: a) the chemical interaction of HA with collagen prevents the crystallization and growth of the mineral phase, which results in a nearly amorphous material characterized by an apatite-like lattice; b) the growth of mineral nuclei is controlled by the organic matrix, so that the size of the nuclei are constrained up to few nanometers; c) the topotactic interaction induces specific crystal orientation of the mineral phase growing on the collagen fibers and evolving into lamellae; d) finally, lamellae are organized through different hierarchical levels to form the structure of the macroscopic bone [3–8]. The use of scaffolds able to guide cells to the re-growth of new bone tissue is an approach now considered as necessary for bone regeneration. Native extra-cellular matrix contains multiple signals whose presentation follows precise spatial and temporal patterns. In designing scaffolds for hard tissue regeneration, such signals must be reproduced so as to give chemical, physical, structural, and morphological information to cells and compel them to express specific phenotypes. Besides, ideal scaffolds guiding tissue regeneration should also have adequate properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, and mechanical performance. In particular, the essential characteristics of regenerative bone scaffolds are: surface activity enabling the establishment of a tight interface between the scaffold and the new tissue; osteoconductivity i.e. the ability to function as a template for 3D cell colonization; appropriate degradation profile without host tissue responses such as inflammation or fibrous encapsulation of the implant [9].

The reproduction of the bone biomineralization process in laboratory enabled the synthesis of hybrid HA/collagen composites reproducing most of the relevant features of newly formed bone and osteochondral tissues [10, 11]. Type I collagen extracted by equine tendon and dispersed into acetic acid in the form of nanofibrils can be subjected to controlled assembly in aqueous environment by pH variation, simultaneously to the mineralization with apatite nanophases where the content of foreign ions can be tailored to reach bio-competent compo‐ sitions. In fact, the maintenance of a disordered crystal structure allows the entrapment of ions naturally present in the physiological environment (i.e. Mg2+, CO3 2−, Sr2+, Na+ , K+ , SiO4 4−) into the structure of the mineral phase. The molecular habitus of type I collagen acts as a 3D substrate for heterogeneous nucleation of the mineral phase but also as a constraint for the growth and long-range ordering of the mineral crystals (**Figure 2**).

Nature-Inspired Nanotechnology and Smart Magnetic Activation: Two Groundbreaking Approaches Toward a New Generation of Biomaterials for Hard Tissue Regeneration http://dx.doi.org/10.5772/63229 133

**Figure 2.** Scheme of collagen assembling and mineralization.

These outstanding architectures are the key of the insuperable performance of natural structures, particularly by a mechanic perspective: nacre, shells, bones, ligaments, tooth enamel, and dentine are just some examples of hierarchical, hybrid bio-nanocomposites found in nature. The mechanism at the basis of this outstanding structural arrangement is the establishment of hybrid building blocks formed upon heterogeneous nucleation of inorganic nanophases (such as carbonates and apatites) onto natural polymers, driven by several control mechanisms acting at the molecular scale [2]. In particular, during new bone formation, type I collagen, extruded by fibroblast cells, acts as a template for the nucleation of the mineral phase through a hierarchical assembly of collagen molecules into fibrils and ever thicker fibres, whereas HA nano-nuclei nucleate onto specific positively charged sites located in the collagen molecules. This process is governed by several control mechanisms inherent in the molecular structure of collagen that guide the formation of new bone at all scale sizes: a) the chemical interaction of HA with collagen prevents the crystallization and growth of the mineral phase, which results in a nearly amorphous material characterized by an apatite-like lattice; b) the growth of mineral nuclei is controlled by the organic matrix, so that the size of the nuclei are constrained up to few nanometers; c) the topotactic interaction induces specific crystal orientation of the mineral phase growing on the collagen fibers and evolving into lamellae; d) finally, lamellae are organized through different hierarchical levels to form the structure of the macroscopic bone [3–8]. The use of scaffolds able to guide cells to the re-growth of new bone tissue is an approach now considered as necessary for bone regeneration. Native extra-cellular matrix contains multiple signals whose presentation follows precise spatial and temporal patterns. In designing scaffolds for hard tissue regeneration, such signals must be reproduced so as to give chemical, physical, structural, and morphological information to cells and compel them to express specific phenotypes. Besides, ideal scaffolds guiding tissue regeneration should also have adequate properties with respect to degradation, cell binding, cellular uptake, non-immunogenicity, and mechanical performance. In particular, the essential characteristics of regenerative bone scaffolds are: surface activity enabling the establishment of a tight interface between the scaffold and the new tissue; osteoconductivity i.e. the ability to function as a template for 3D cell colonization; appropriate degradation profile without host tissue

132 Advanced Techniques in Bone Regeneration

responses such as inflammation or fibrous encapsulation of the implant [9].

naturally present in the physiological environment (i.e. Mg2+, CO3

growth and long-range ordering of the mineral crystals (**Figure 2**).

The reproduction of the bone biomineralization process in laboratory enabled the synthesis of hybrid HA/collagen composites reproducing most of the relevant features of newly formed bone and osteochondral tissues [10, 11]. Type I collagen extracted by equine tendon and dispersed into acetic acid in the form of nanofibrils can be subjected to controlled assembly in aqueous environment by pH variation, simultaneously to the mineralization with apatite nanophases where the content of foreign ions can be tailored to reach bio-competent compo‐ sitions. In fact, the maintenance of a disordered crystal structure allows the entrapment of ions

the structure of the mineral phase. The molecular habitus of type I collagen acts as a 3D substrate for heterogeneous nucleation of the mineral phase but also as a constraint for the

2−, Sr2+, Na+

, K+ , SiO4

4−) into

By this process CO3 2− ions can be introduced to preferably occupy the phosphate site of the HA lattice (B type position) [4], thus providing the mineral phase with enhanced activity for cell adhesion and resorbability. Carbonate ions are abundant in young and newly formed bone tissue, and decrease in mature bone, thus evidencing their role in bone development. Among the foreign ions present in biologic apatite, Mg2+ have the marked property of increasing the nucleation kinetics of HA on collagen fibres but, in the meantime, hampering crystal growth, thus generating nano-size HA nuclei, strongly enhancing the bioavailability of the mineral phase. In fact, magnesium is found in much higher concentrations in young and newly formed mineralized tissues and is considered today as a fundamental element governing the first stages of bone formation [12]. Silicon is a minor element, essential for healthy skeletal devel‐ opment in higher biological organisms [6, 13], in particular for its role in the formation of crosslinks between collagen and proteoglycans [14], that provide stabilization of the new bone matrix and prevent enzymatic degradation.

The bone-like features of HA/Collagen hybrid composites reflect in bio-resorbability at physiological pH and high surface activity, particularly referred to the crystal size (i.e. ranging from 30–50 nm long, 15–30 nm wide, and 2–10 nm thick) [7, 8, 15–17] and to the specific orientation of the apatite nuclei, in respect to the long axis of collagen. The preferential growth of apatite nuclei along the *c* axis, as induced by the presence of particular functional chemical groups on the surface of the organic template, affects the surface polarity of the final hybrid composites, and consequently protein adhesion and cell attachment. The hierarchical assembly of these nano-size building blocks into macroscopic objects occurs upon supramolecular arrangement of collagen fibrils into thicker fibres, thus resulting into a final hybrid composite where, on a macroscopic scale, the mineral phase assumes a complex and hierarchical architecture, strictly dependent on the combination of the various above-described phenom‐ ena, which hierarchically occur on different dimensional scales in correspondence with the sites of heterogeneous nucleation.

The HA/Col composites assume a fibrous structure as well as high and interconnected porosity, the amount and morphology of which can be tailored by customized freeze drying processes (**Figure 3**).

**Figure 3.** Bio-hybrid HA/Collagen composites.

The final dried scaffolds exhibit high activity towards cells; therefore they can be easily resorbed in vivo whereas new tissue forms. However, to limit the enzymatic degradation possibly preventing successful cell colonization and integration, cross-linking methods can be applied by using physical or chemical approaches addressing specific links among functional groups of collagen, thus enabling fibre bridging and tailored stability against resorption.

The in-lab reproduction of the phenomena occurring in biological processes can be considered as a conceptually new approach for nanotechnology and may pave the way to the development of new devices with outstanding properties. On the basis of the recognition of the different requirements to regenerate cartilaginous and bony part, such processes can be directed to graded scaffolds reproducing different histological areas in the osteochondral tissue by simply varying the degree of mineralization and the alignment of collagen fibres [11]. Therefore, hydrogels with designed features can be engineered into three-layered devices reproducing the sub-chondral bone (mineralization = 60-70 wt%), mineralized cartilage (mineralization = 30-40 wt%), and the hyaline cartilage (mineralization = 0 wt%) (**Figure 4**).

**Figure 4.** Scheme of osteochondral scaffolds.

In particular, the collagen-like layer, based on collagen and added with hyaluronic acid to create microstructural features improving the hydrophilic behaviour of the construct, repro‐ duces some cartilaginous environmental cues such as the formation of a columnar-like structure converging towards the external surface where it forms horizontal flat ribbons, resembling the morphology of the *lamina splendens* [10, 11].

Such composites have demonstrated enhanced cell proliferation with very spread cell mor‐ phology, as well as high osteoinductivity and regenerative potential. The HA/collagen graded composites differentially support cartilage and bone tissue formation in the different histo‐ logical layers, as demonstrated by comparative *in vivo* study carried out on adult sheep, where HA/collagen graded composites have been implanted on femoral condyles [18]. In particular, histological evaluation showed the formation of new hyaline-like tissue and good integration of scaffolds with host cartilage, with a strong proteoglycan staining and columnar rearrange‐ ment of chondrocytes, and an underlying well-ordered sub-chondral trabecular bone.

In this section it has been discussed that biologic processes pin on information exchanged at the molecular scale and on environmental boundary conditions that guide the process towards the establishment of 3-D hybrid composites with defined characteristics. This implies that bioinspired syntheses are flexible processes that can be directed to fabricate specific devices *on demand*. In this respect, hybrid HA/Col composites can be developed to assume specific 3D morphologies, thus mimicking human multifunctional tissues such as periodontal regions. Indeed, human tooth is a tissue complex formed by the periodontium, in turn including alveolar bone and cementum, linked together by the periodontal ligament firmly bound to the root, and the dentin, a highly mineralized collagen matrix with tubular organization that is protected by the enamel (**Figure 5**).

**Figure 5.** Scheme of dental tissues.

**Figure 3.** Bio-hybrid HA/Collagen composites.

134 Advanced Techniques in Bone Regeneration

**Figure 4.** Scheme of osteochondral scaffolds.

The final dried scaffolds exhibit high activity towards cells; therefore they can be easily resorbed in vivo whereas new tissue forms. However, to limit the enzymatic degradation possibly preventing successful cell colonization and integration, cross-linking methods can be applied by using physical or chemical approaches addressing specific links among functional groups of collagen, thus enabling fibre bridging and tailored stability against resorption.

The in-lab reproduction of the phenomena occurring in biological processes can be considered as a conceptually new approach for nanotechnology and may pave the way to the development of new devices with outstanding properties. On the basis of the recognition of the different requirements to regenerate cartilaginous and bony part, such processes can be directed to graded scaffolds reproducing different histological areas in the osteochondral tissue by simply varying the degree of mineralization and the alignment of collagen fibres [11]. Therefore, hydrogels with designed features can be engineered into three-layered devices reproducing the sub-chondral bone (mineralization = 60-70 wt%), mineralized cartilage (mineralization =

30-40 wt%), and the hyaline cartilage (mineralization = 0 wt%) (**Figure 4**).

All the components of tooth form upon biologic phenomena close to those leading to formation of bone and cartilage [19]. The relevant differences are related to the mineralization extent of the different tissues (i.e. alveolar bone ~70 wt%, cementum ~50 wt%, dentine ~75 wt%, enamel ~98%), the degree of aggregation of collagen fibres, and the structural organization. Therefore, bio-inspired in-lab mineralization can be directed to develop new biomimetic scaffolds mimicking the different parts of the tooth by varying the concentration of calcium and phosphate ions with respect to collagen thus achieving the desired mineralization extent. Then, oriented channel-like porosity mimicking the tubular organization of dentine can be obtained by ionotropic gelation techniques applied to the as-synthesized hydrogels (**Figure 6**).

**Figure 6.** Dentin-like scaffolds.

Preliminary research shows that the application of bio-inspired synthesis techniques can enable the development of new implantable devices for the complete regeneration of dental tissues. This is a major and highly demanding clinical need and a target of high impact for materials science and medicine.

In perspective, the in-lab biomineralization process may be in principle translated to wider applications, possibly extending the range of natural polymers that can be combined to form composite matrices activating self-assembly and mineralization with specific inorganic phases. Non-mineralized constructs can be used as scaffolds for soft tissues and organs, where the biologic and mechanical performance can be tailored by combining various raw materials such as gelatin, nanocellulose, chitosan, alginate, and fibroin characterized by different hydrophilic behaviour and stiffness. On the other hand, the simultaneous mineralization of composite polymeric matrices with nano-apatites can generate scaffolds with improved mechanical performance, thus enabling wider applications in bone surgery, particularly referred to loadbearing applications where the soft nature of hybrid scaffolds does not allow to withstand strong biomechanical loads in the early stages of new bone formation.
