*Nature-Inspired Processes and Structures: New Paradigms to Develop Highly Bioactive Devices… DOI: http://dx.doi.org/10.5772/intechopen.82740*

tailor-made scaffolds with the ability to fine-tune the tissue regeneration process. Four different biological prerequisites are necessary for BTE such as osteogenic cells, osteoinductive stimulus, osteoconductive matrix scaffolds, and mechanical environment which led to design scaffolds with appropriate macroporous structure, good degradability, and better osteoconductive properties [67]. A 3D structure is not enough to obtain a material with osteoinductive stimulus, but the chemical composition plays a decisive role. Both concepts (chemical composition and 3D architecture) are at the basis of biomimicry; hence, to obtain scaffolds with chemical composition very close to natural bone, a bioinspired synthesis method mimicking the natural biomineralization process was carried out [68].

In this respect, previous studies by Tampieri et al. exploiting the biomineralization process abovementioned developed biocomposites made of collagen and hydroxyapatite for bone and osteochondral regeneration [69–71].

Exactly as it happens in nature, collagen molecules promoted complex 3D arrangement and the heterogeneous nucleation of a low crystalline hydroxyapatite also due to the incorporation of foreign ions, usually present in human tissue, into the apatite phase. In details, biomineralization process was reproduced in the laboratory dropping an acid solution containing PO4 <sup>3</sup><sup>−</sup> ions mixed with collagen gel into an alkaline solution containing the Ca2+ ions exploiting a neutralization process. The pH of the suspension is increased up to neutral pH where two different mechanisms are simultaneously triggered; on the one hand, the collagen fibers reach the isoelectric point leading to their assembly into a 3D network; on the other hand, the mineral nucleation starts in correspondence to the carboxylic groups exposed by the collagen molecule that bind calcium ions [69–72]. One of the advantages of this material is the capability to entrap some foreign ions into HA lattice obtaining a hybrid material mimicking natural mineralized tissues. In particular, CO3 <sup>2</sup><sup>−</sup> ions can occupy two different sites of the apatite lattice. B-substitution occurs at the PO4 <sup>3</sup><sup>−</sup> site improving the osteoblasts adhesion and is typical of young and immature bones; conversely, carbonation in site A refers to partial substitution of OH<sup>−</sup>, which increases the stability of mineral phase, and in fact it is more typical of mature bone tissue. Mg2+ promotes the HA nucleation and bioavailability decreasing the crystallinity. Sr2+ is able to restore the bone turnover balance; this is important for the treatment of osteoporotic bone fractures [73, 74].

The aptitude of the apatite lattice to host several isovalent and heterovalent ion substitutions permits to synthesize apatite nanocrystals with multiple substitutions that can be used in different applications in regenerative medicine and nanomedicine. Furthermore, besides the incorporation of foreign ions, also the control mechanisms exerted by the organic phase allow to produce a more biomimetic apatite thanks to nearly amorphous crystal state and crystal orientation; in this way, cells well recognize hybrid composite without any inflammatory reaction and start to interact with it promoting the adhesion and proliferation on its fibers [75, 76]. Therefore, the use of bioinspired mineralization process is a tool able to confer unique properties to hydroxyapatite otherwise impossible to find in stoichiometric hydroxyapatite as well as in composites where hydroxyapatite was simply mixed with collagen [70].

Among bone defects, large chondral articular defects represent a major problem in orthopedic practice [77], and tissue engineering is providing promising results [78]. However, the results for the treatment of cartilage lesions are still controversial, and osteochondral lesions are even more severe relating to two different tissues featuring different self-healing abilities and cell lineages involved. 3D scaffold, usually, is able to well regenerate a single tissue, as cartilage tissue, and in case of osteochondral damage, additional autologous bone grafting is often necessary [79]. To overcome these limitations and to increase the advantages for osteochondral

*Bio-Inspired Technology*

Ion-doped apatites obtained as bone cements offer interesting perspectives as a new class of injectable biomaterials that can found application as bioactive pastes for the regeneration of bone defects with complex geometry and not easily accessible by implantation of 3D solid scaffolds (e.g., femur head, tibial plateau, vertebral body, and maxilla). A very interesting perspective, further extending the possible application of bioactive pastes and cements, is the development of printable self-hardening biomaterials (**Figure 2**). Such pastes, to be prepared with rheologic properties enabling flowability, cohesion, and hardening in short times, to allow layer-by-layer deposition, can be processed by micro-extrusion to obtain solid scaffolds with enhanced bioactivity, thanks to the possibility to maintain biomimetic chemical composition, without the need of conclusive sintering process

*(a) Scheme of the development of self-hardening formulations as printable pastes, (b) typical nanostructure of hardened apatite cements, and (c) very tight interface between Sr-substituted apatite cement and bone in* 

**5. Hybrid scaffolds obtained by bioinspired assembling/mineralization** 

Hard tissues are biological constructs incorporating minerals into soft matrix to create a protective shield or a structural support such as the bone, teeth, and cartilage [7]. The non-mineralized region, called also soft tissue, can be connective, muscular, nervous, or epithelial. Especially examining bone tissue, it is a highly dynamic and vascularized tissue which has an ability to self-heal and remodel through a well-orchestrated process; the bone remodeling is a constant process, targeting to replace odd bone through resorption by means of osteoclasts and to produce new bone by means of osteoblast which usually completes in 4–6 months. However, the high regenerative capacity is lost when there is a large segmental defect, severe non-unions, or bone tumor resection [66]. To overcome these issues, the concept of bone tissue engineering (BTE) has been developed producing

**process for bone and osteochondral regeneration**

**40**

for consolidation.

**Figure 2.**

*rabbit test.*

regeneration, biomaterials provide the template for tissue development that can be adjusted in shape, size, and orientation according to defect features [80].

For this reason, several authors have highlighted the need to modulate a multilayered scaffold capable to reproduce different biological and functional environments of osteochondral region to promote regeneration [80, 81]. To create construct with more favorable integrative properties in the osteochondral site, bilayer or tri-layer composite is developed such as a polylactide-co-glycolide copolymer, the first scaffold reported for clinical use; however, it showed poor repair tissue quality at imaging, as well as unsatisfactory clinical outcomes [82, 83]. One of the difficult points in the osteochondral regeneration is the interface between material's layer and host tissue and between layers of host tissue; the cartilage repair should be followed by an adequate regeneration of the subchondral structure and by the effective union with surrounding host tissue [84]. Tampieri et al. designed a composite scaffold consisting of three different but integrated layers, corresponding to cartilage, calcified cartilage, and bone components [69]. It was developed to better mimic structure and composition of the whole osteochondral unit, showing promising clinical results even in challenging conditions, such as complex lesions or osteoarthritic knees [85, 86]. Exploiting biomineralization process, a different extent of mineralization was nucleated on collagen fibers developing a tri-layer with a gradient of hydroxyapatite ranging from a mineral content of 60–70% corresponding to subchondral bone and 30–40% corresponding to mineralized cartilage up to 0% corresponding to hyaline cartilage (**Figure 3**). Furthermore, in the top layer (hyaline cartilage), hyaluronic acid was added to create microstructural features improving the hydrophilic ability to reproduce columnar-like structure converging toward the external surface, where it formed horizontal flat ribbons, thus resembling the morphology of the *lamina splendens*.

Chemical-physical investigation highlighted that chemotactic information provided by collagen-induced unique features in the inorganic phase, promoting the nucleation of a biomimetic apatite very close to the biological one present in the bone [87]. In vivo evaluation demonstrates that it differentially supports cartilage and bone tissue formation in the different histological layers [88]. After 6 months from implantation of graded hybrid composites on femoral condyles of

### **Figure 3.**

*Representation of multilayered hybrid scaffold obtained by in-lab biomineralization and its application in osteochondral defect.*

**43**

*Nature-Inspired Processes and Structures: New Paradigms to Develop Highly Bioactive Devices…*

sheep, a new hyaline-like tissue is formed, and a good integration of scaffolds with host cartilage is observed; furthermore, a strong proteoglycan staining, columnar rearrangement of chondrocytes, and an underlying well-structured subchondral trabecular bone are shown. Besides, hybrid scaffold was completely resorbed, and no remarkable difference was revealed with or without seeding of chondrocyte cells, highlighting as chemical-physical features of hybrid composite allow the recruitment of bone marrow stem cells directly from the underlying subchondral

In conclusion, the ability of the scaffold to induce orderly osteochondral tissue

**6. Biomorphic transformation of natural structures: a new way to obtain biomimetic scaffolds for regeneration of load-bearing segmental** 

Among the bone diseases, those affecting portions of long bone subjected to mechanical loads are the ones which most seriously impact on the quality of life of sufferers. The incidence of such pathologies is particularly relevant among the aged people (osteoporosis); anyway, more recently the number of relatively young patients affected by bone diseases has increased mainly owing to modern lifestyles (e.g., intense sport activity, obesity, etc.). In this case, pain and disability also impact on the psychological well-being, leading to anxiety, depression, fear for the future, and altered perception of the social role. Such feeling is nowadays shared by the aged people also, because of the increased expectation of an active life and well-being even among the elderly. For this reason, the abovementioned numbers in terms of socioeconomic costs and number of hospitalized people are likely to

Due to the inability of the current manufacturing technologies to form mechanically strong porous inorganic structures with a hierarchic pore organization and complex morphological details in the submicron scale, the healing of load-bearing bone segments still relies on bioinert dense implants based on alumina, titanium, etc. A significant change in engineering and ceramic processing is needed, thus greatly expanding the existing tools enabling the development of porous and massive ceramic bodies with designed smart functions. The current manufacturing approach in ceramic development is based on powder synthesis, forming, and thermal consolidation (sintering); the idea is to surpass the existing approach, by developing new "one-step synthesis/consolidation processes" to obtain new 3D ceramics with properties and functions not achievable with the current manufacturing approach. In particular, this is relevant when the ceramic phases with desired functional properties have low thermodynamic stability such as nanosized and atomic position, so that the existing ceramic process, particularly sintering, destroys labile phases increasing their stability but deleting its smart functional properties. Particularly, the sintering process, which is fundamental to consolidate ceramic bodies, impairs the maintenance of ceramic phases characterized by low crystallinity, nanosize, and nonstoichiometric composition. These features, relying on low thermodynamic stability, are very often the source of functions that cannot

repair without the introduction of cells makes it attractive for several reasons: (i) from a practical and commercial standpoint, because it could be used as an off-theshelf graft in a one-step surgical procedure; (ii) from a surgical standpoint, it could be inserted under minimally invasive conditions due to its flexibility; and (iii) from a biological standpoint, because the problems related to the cell culture would be

*DOI: http://dx.doi.org/10.5772/intechopen.82740*

bone [88].

eliminated [89].

**bones**

increase in the next future.

be expressed by a stable, sintered ceramic phase [20].

*Nature-Inspired Processes and Structures: New Paradigms to Develop Highly Bioactive Devices… DOI: http://dx.doi.org/10.5772/intechopen.82740*

sheep, a new hyaline-like tissue is formed, and a good integration of scaffolds with host cartilage is observed; furthermore, a strong proteoglycan staining, columnar rearrangement of chondrocytes, and an underlying well-structured subchondral trabecular bone are shown. Besides, hybrid scaffold was completely resorbed, and no remarkable difference was revealed with or without seeding of chondrocyte cells, highlighting as chemical-physical features of hybrid composite allow the recruitment of bone marrow stem cells directly from the underlying subchondral bone [88].

In conclusion, the ability of the scaffold to induce orderly osteochondral tissue repair without the introduction of cells makes it attractive for several reasons: (i) from a practical and commercial standpoint, because it could be used as an off-theshelf graft in a one-step surgical procedure; (ii) from a surgical standpoint, it could be inserted under minimally invasive conditions due to its flexibility; and (iii) from a biological standpoint, because the problems related to the cell culture would be eliminated [89].
