**5. Tissue engineering based on multiphase designs for cartilage regeneration**

Tissue engineering can be defined as the creation or induction of the formation of a specific tissue, in a specific location, through the manipulation and selection of cells, matrices, and biological stimuli. It is an interdisciplinary field that applies principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function [40].

Currently, tissue engineering combines the contribution of cells, undifferentiated or not, which are placed into a scaffold where growth factors can be added to accelerate cell proliferation and differentiation so that after being transplanted to a damaged structure and reaching its regeneration.

#### **5.1 Strategies for cartilage tissue engineering**

In the cartilage tissue engineering, the constant development of new designs combining biomaterials, with different cellular sources and modifying the cell culture methodology within the scaffolding systems is driven by the need, still not satisfied, to have a gold standard for functional and long-term repair of chondral and osteochondral defects.

As a cellular source for the formation of cartilage, chondrocytes, or alternatively, mesenchymal stem cells can be used. In the case of mesenchymal stem cells, there are a series of known factors that induce their differentiation toward the chondral phenotype, among which are the use of a culture medium without serum, enriched with dexamethasone, ascorbate, TGFβ, and BMPs, being the method of three-dimensional cultivation at high density one of the most used for this purpose [41, 42].

For the implantation of cells in the cartilage defect, they should be embedded in the thickness of scaffolding. These cellularized graft needs to be maintained for some period of time in culture in order to allow the cells to secrete enough ECM to functionally replace the normal cartilage and facilitate its complete integration. A newly developed osteochondral construction with inferior mechanical properties can also contribute to mechanical imbalance near the defect region until its mechanical properties have matured [43]. Mayr et al. demonstrated that the cartilage component of the osteochondral graft had only half the rigidity of the surrounding cartilage 6 months after implantation [44]. The longer the osteochondral graft takes to mature into mechanical properties, the longer the surrounding cartilage will be exposed to an excessive load, which may contribute to degenerative processes. Therefore, it is necessary to select scaffolds that allow building structures related to the biological behavior of cells into an adequate environment. In any case, for the production of cartilage, it is important to achieve the maximum production of extracellular matrix because the mechanical behavior of the implanted artificial tissue is favored, especially when using less resistant scaffolds than the normal articular cartilage.

The application of cells in scaffolds, as tissue engineering does, makes cartilage regeneration strategies complex but allows the process to be orchestrated efficiently. The critical point in these strategies is the expansion of the cells in culture in order to generate a suitable production of ECM *in vitro* and with a supportive impact on the mechanical properties *in vivo* [45].

Another challenge to overcome regarding this strategy is to achieve a competent integration of the graft after implantation. The integration of the implanted tissue with the organ requires remodeling, degradation, and at the end, formation of new tissue. The remodeling of the implanted tissue is essential for its functionality [45].

In the last decades, the strategies have been designed without considering the cartilage as a complex tissue with a functionally stratified three-dimensional structure. Today, efforts are focused on achieving a benchmark in the cartilage formation process with the development of a multiphasic implant, not only because it recapitulates the nature of native tissue, but also it takes advantage of the healing capability of bone to promote the implant integration with the surrounding tissue and then bone healing and cartilage formation. The architecture of the scaffold and the presence of migratory cells within or immediately around the graft in the bone phase of the osteochondral tissue then play a key role in the integration and therefore tissue repair.

#### **5.2 Multiphasic scaffolding**

 During the last decade, there have been many new developments in various aspects of scaffolding manufacturing. Computer-aided designs and fabrication technologies are used to fabricate custom scaffolds for irregularly shaped defects [46, 47].

 The materials used for scaffolds and matrices are increasingly intelligent and more versatile, and can be modified to incorporate bioactive peptides [48]. Although scaffold fabrication technologies are advancing at a rapid pace, no engineering strategy used to date can completely recapitulate the biochemical and physical characteristics of native osteochondral tissue. Although it is generally useful to simplify the approach of *in vivo* repair from an engineering point of view, for a successful *in vivo* result, the biological complexities that take place within the joint must also be taken into account in the design.

The osteochondral tissue has a heterogeneous multilayer structure composed of uncalcified cartilage (superficial, middle, and deep zone), calcified cartilage and subchondral bone.

Essentially, a multiphasic scaffold should be biocompatible able to guide the structuring of new chondral and osseous tissue, taking into account the presence and biological functionality of the interface region between them (tidemark) to achieve the mechanical properties of articular cartilage. The widespread approach uses multicomponent systems, and the exquisite melding of natural and synthetic biomaterials where the assembly strategy is fundamental since it determines the topography and the structural arrangement in which the extracellular matrix is organized, a random or a well-ordered orientation of the fibers within the chondral phase in particular.

 It can be postulated that the typical lack of orientation of the collagen fiber in the repaired cartilage also has a role in the prevention of a strong integration at the level of the cartilage. The surface area of the cartilage in the normal cartilage is horizontally aligned, parallel to the direction of the joint. However, within the repaired cartilage, this provision is often lacking; therefore, the border adjacent to the native

#### *Therapeutic Potential of Articular Cartilage Regeneration using Tissue Engineering Based… DOI: http://dx.doi.org/10.5772/intechopen.84697*

cartilage tissue and modified by genetic engineering is susceptible to rupture. The vertical orientation of collagens near the subchondral bone has been attributed to the anchoring of cartilage tissue against large strains [49]. The lack of orientation of the collagen in a dynamic loading environment of the joint probably has a role in the *in vivo* failure of the implanted constructions and the decreased integration.

 Additionally, it is well documented that the rigidity of the cartilage depends on the depth, and that the superficial layer of the cartilage deforms much more than the deeper layers [50]. In this respect, when the cartilaginous component of the osteochondral scaffolds lacks the deformation patterns that vary in depth, it is likely that the levels of compression deformation mismatched between the cartilage and the implant cause a higher shear stress in the interfacial region, causing a break. Tissue engineering cartilage grafts with variable depth compressive properties have also been proposed in the past [51], and can be incorporated into future osteochondral designs.

Multiphasic scaffolds can be designed considering two or three different phases (biphasic or three phase, respectively), each of them with an architecture and composition of particular biomaterials. Since the cartilage and the subchondral bone, part of the osteochondral unit, have different biological and mechanical requirements, the first approaches in the design of multiphase implants were based on the use of two different biomaterials in order to reach a tissue-specific scaffold design; moreover, the use of different combinations of biomaterials for each phase has been reported.

Polylactic-acid (PLA)-coated polyglycolic acid (PGA) scaffold molded by the computer-aided design and manufacturing (CAD/CAM) technology proved to be ideal scaffolds for cartilage regeneration, where the presence of PLA provides adequate rigidity for the chondral phase, which is attached to polycaprolactone/ hydroxyapatite (PCL/HA), an osteoconductive material, where HA generates a favorable topographical surface and biomimetic microenvironment in terms of bone tissue. The regenerated cartilage and subchondral bone showed a wellstructured transition zone between the two phases, which has resulted in a better integration with the host and with mechanical properties capable of supporting the solicitation of the chondral tissue [52].

The importance of generating in the scaffold a tissue-specific microenvironment that allows the undifferentiated migrant cells of the host to find a niche for the adequate differentiation toward the chondrocytic lineage is evident.

Yun-Jeong et al. have shown that not only the microenvironment generated by the composition of the biomaterials impact on a better imitation of the osteochondral unit, but also the scaffolding structure has an important influence, being an aligned structure the most adequate in comparison with a randomly structured scaffold [55]. A stratified design of aligned channels in a biphasic scaffold using collagen type I and biphasic calcium phosphate (BCP) to mimic the cartilage and bone tissue, respectively, was manufactured by using an exquisite unidirectional freeze-casting process. Collagen is flexible, and it has specific molecular domains able to induce and support cell bioactivity [53]. Likewise, BCP provides significant osteoconductivity, bioactivity, and mechanical features [54]. However, privileging on the composition, the generation of a biphasic scaffold with a longitudinal roughness of the inner channels that serves as a guide for the correct adhesion of the cells; it results in a topography that truly emulates the osteochondral unit and shows *in vivo* a superior regeneration of the osteochondral tissue compared to the random structure [55].

Therefore, not only the pore size and porosity should be taken into account for a correct design, but also the alignment of the channels within the scaffold influencing cell migration and the proper pattern fibers of the ECM.

Likewise, multiphase can be assembled on the basis of a single biomaterial. It is possible to modify the physical properties such as roughness, pore size, and interconnectivity particularizing according to the phase, selecting a specific type of porogen and particle size, as well as through the use of different solvents and the polymerization process.

Biphasic scaffold with a cartilage phase consisting of a silk scaffold attached to a bone phase consisting of a strontium-hardystonite-gahnite (SHG)-silk scaffold has been designed by Jiao Li et al. [56]. The preparation implies a coating process of SHG ceramic scaffolds with a single silk layer using an aqueous silk fibroin solution then attached to the mixture of silk using methanol as an alternative solvent prior to silk scaffold formation in order to induce β-sheet formation in fibroin and the structure of the interface. The conformation of this design showed not only the ability to promote the differentiation of human mesenchymal stem cells toward the chondrogenic or osteogenic lineage, but also in addition, by having a well-stratified biphasic structure, the loading behavior validated the compression properties.

By the same token, a single biomaterial can be used and can generate distinct microenvironment using different molecules to biofunctionalize in a tissue-specific manner. Certainly, no biomaterial is intrinsically capable of satisfying all the requirements for the manufacture of complex and stratified tissues, so the biofunctionalization of these is presented as an alternative procedure to adapt the properties of the biomaterials to the needs of the chondral or bone tissue.

A biphasic, but monolithic scaffolds based on alginate, a highly biocompatible natural biomaterial able to support the growth of diverse cell lineages is designed by Schütz et al. through its strategy; scaffolds are fabricated by a diffusion-controlled system that allows the directed ionotropic gelation [57]. The final structure leads to the formation of channel-like, parallel aligned pores. In order to generate a chondral environment, alginate is biofunctionalized with hyaluronic acid, while for the bone phase, hydroxyapatite is used.

 This simple procedure generates two well-defined layers characterized by different microstructure and mechanical properties, which provide a suitable environment for cells to form the respective tissue. Although an interface between the chondral and bone areas of the implant is not structured, a stable connection between them is clearly demonstrated, which positively impacts the mechanical properties in the final design. According to the influence of biofunctionalization, it was demonstrated by gene expression analysis that the embedded stem cells differentiated into the chondrogenic lineage when they were cultivated in chondrogenic medium; additionally, under the stimulation of the hyaluronic acid present in this phase, the chondrocyte phenotype remained stable.

Biofunctionalization, especially for monolithic scaffolds, is a useful alternative to provide chondro- and osteoinductive properties. Aragonite is a biomaterial from coral exoskeletons, similar to human bone including its 3D structure and pore interconnections as well as its crystalline form of calcium carbonate (CaCO3) [58]. That features confers improved osteoconductive ability, suitable for bone regeneration.

 Interestingly, specific coral species differ in size and interconnectivity of coral pores, which expands the range of applications for different tissues. In order to induce chondrogenesis in a monolithic system of aragonite, the use of hyaluronic acid has been described by Korn et al. [59]. We have already discussed before, the relevance of channel generation aligned in parallel to guide the adhesion of the cells in the chondral phase and the subsequent structuring of the ECM. In this design, in addition to the biofunctionalization with hyaluronic acid, the mechanical modification of drilled channels is also added. The combination of the two strategies showed in a model of joint damage in goat the best results compared to aragonite alone, and in the absence of parallel *Therapeutic Potential of Articular Cartilage Regeneration using Tissue Engineering Based… DOI: http://dx.doi.org/10.5772/intechopen.84697* 

channels; it means a cartilaginous repair tissue with hyaline cartilage as shown by the marked expression of proteoglycans, as well as of collagen type II and absence of collagen type I.
