**6. Influence of vascularization on scaffold design for osteochondral regeneration**

Vascularization is the bottleneck in tissue engineering. Creating constructs in the laboratory that lack of the proper vessels network will fail after implantation as the cells will not get enough oxygen and nutrients and will die. This fact is even more significant for osteochondral regeneration. Bone is a highly vascularized tissue while cartilage is avascular. When vascular networks invade cartilage surface from the subchondral zone, it might lead to an ossification of the cartilage from the deep and intermediate zone implying a joint damage and increasing pain. The design of the optimal scaffold to control angiogenesis, promoting vessel growth from preexisting ones, on the bone side and inhibiting it on the cartilage side is relevant for osteochondral regeneration.

One strategy to improve bone formation is to use growth factors (GFs) that can activate angiogenesis within the scaffold. There are several GFs involved in angiogenesis, such as vascular endothelial growth factor (VEGF), platelet-derived GF (PDGF), bone morphogenic proteins (BMPs), fibroblasts growth factors (FGFs), and tumor growth factor beta (TGFβ) [60]. Uploading VEGF is widely used, as the VEGF activates endothelial precursor cell (EPC) migration and proliferation activating the angiogenic process, and subsequently promotes the recruitment and survival of bone forming cells improving bone regeneration. However, the presence of high levels of VEGF is one of the factors related to OA progression, inducing cartilage degeneration and pain [61].

Therefore, the scaffold design for osteochondral regeneration must fulfill different properties that are shared by the two tissues, such as cell adhesion and proliferation and a high production of ECM; but others must deal with angiogenic promotion for bone or angiogenic inhibition for cartilage. Furthermore, the already observed side effects of supraphysiological doses of bone-related GFs heterotopic bone growth, pseudoarthrosis, local inflammation, and immune response [62] must be controlled by means of delivery vehicles that will ensure the bioactivity of these molecules and the remaining in the target location over the therapeutic timeframe. This can be done by covalent attachment to the scaffold, noncovalent binding, or with the nanoparticle carriers.

 Kempen et al. developed a system for the sequential release of VEGF with BMP-2. BMP-2-loaded PLGA microspheres in a poly(propylene fumarate) (PPF) scaffold combined with a VEGF-loaded gelatin hydrogel in a rat subcutaneous model demonstrated both improved vessel and bone formation when compared to scaffolds that did not contain VEGF [63].

 García-Fernández et al. used antiangiogenic polymer based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and a methacrylic derivative of 5-amino-2-naphthalenesulfonic acid (MANSA) [64]. The use of this synthetic polymer completely inhibited angiogenesis by the interaction of the sulfonic acid groups with the bFGF and VEGF modulating their activity in the processes of endothelial cell migration and proliferation. Thus, the fabrication of a biphasic scaffold by combining two different polymers that can control angiogenesis can be an efficient approach.

An innovative approach that has been tested recently is the use of microRNAs(miRNAs) to modulate cell activity for regenerative medicine applications. miRNA is a single-stranded RNA, with a length between 21 and 25 nucleotides that can regulate gene expression, usually by destabilizing mRNAs or by suppressing translation. The roles of these miRNAs on bone diseases (such as osteoporosis, osteoarthritis, and rheumatoid arthritis) have been recently highlighted. Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures [65, 66]. miR-26a was reported to regulate angiogenesis by targeting BMP/SMAD1 signaling in endothelial cells [67].

 The use of these molecules as miRNA regulators can be done by using synthetic molecules, which either mimic or repress the function of endogenous miRNAs. The mimicking molecules will enhance the suppression of the target protein synthesis by degrading the miRNA or inhibiting the protein translation. On the other hand, miRNAs inhibitors (antagomirs) preventing the activity inside the cells will lead to a rise of mRNA and protein expression. This approach can be used to upload scaffolds with either agonist or antagonist molecules to induce or avoid vascularization.

Many scaffolds have been designed to fulfill the function of miRNA delivery, mainly hydrogels, nanofibers, and porous or spongy scaffolds. Besides, the normal desired properties such as biocompatibility, easy fabrication, easy sterilization, proper mechanical properties, and adequate porosity for vessels growth, the material must retain the miRNA complexes while facilitating their sufficient exposure to the infiltrating cells without affecting its mechanical properties [68].

### **7. Biomaterials for multiphasic scaffolding**

Biomaterial scaffold properties are fundamental to guide and recreate the native environment. The biomaterials for osteochondral applications in first insight must be biocompatible and should be intrinsically osteoinductive, osteoconductive, chondroinductive, or chondroconductive, and not less insignificant, and must possess a degradation rate that allows the formation of new tissue.

As previously stated, an ideal scaffold for the treatment from a multiphase point of view must have a chondrogenic matrix that is flexible, resistant and with pores small enough to mimic the hyaline cartilaginous matrix and an osteogenic matrix that should be mechanically competent similar to cancellous bone and bioactive, which has larger pores that mimic the microenvironment of the subchondral bone.

Achieving an articular cartilage design capable of mimicking its anisotropic mechanical behavior, still represents one of the greatest challenges in the cartilage tissues engineering [69]. In addition, the ideal biomaterial for cartilage should allow the cartilage composition to be recreated in terms of the liquid and solid phases of the connective tissue, reproduce its zonal organization, and facilitate the integration of the neoformed tissue with the adjacent native tissue.

Functionally, we can classify biomaterials into: protein-based polymers, such as fibrin, gelatin, collagen, and silk fibroin [70–74]. Biopolymers based on carbohydrates such as alginate, chitosan, agarose and polyethylene glycol [75–78], and synthetic polymers such as polylactic acid, polyglycolic acid, polycaprolactone and polylactic-coglycolicacid (PLGA) are the most common [79–81].

#### **7.1 Carbohydrate-based polymers**

These kinds of biomaterials are comprised of cross-linked polymers that swallow a great amount of water, which empathizes with the features of cartilage ECM, thus favoring the maintenance of spherical morphology within the scaffold [76]. Furthermore, synthetic materials and growth factors can be added in order to enhance chondrogenesis.

A material with adequate characteristics for cartilage engineering is chitosan, a polycationic polysaccharide that can be degraded enzymatically by the lysozyme *Therapeutic Potential of Articular Cartilage Regeneration using Tissue Engineering Based… DOI: http://dx.doi.org/10.5772/intechopen.84697* 

 present in the MEC of human cartilage. Chitosan has a chemical similarity with GAGs, which gives it the ability to interact with them [82]; through various *in vitro* studies, it has been demonstrated that scaffolds based on chitosan especially in combination with other biomaterials such as collagen II [108], hyaluronic acid [83], or fibroin [84] promote chondrogenic activity and support the production of aggrecan and type II collagen, thus improving cartilage repair [108].

#### **7.2 Protein-based polymers**

Among the materials of a protein nature is gelatin, which is formed from denatured collagen and can bind to growth factors, proteins, and peptides and is also capable of promoting efficient cell adhesion. On the other hand, there is the collagen that constitutes the main structural component of the ECM, and its use as a scaffolding material allows the cells to retain their phenotypes [85].

 Collagen is a naturally occurring protein found in various fibrous tissues such as bone and cartilage. Collagen-based scaffolds have been used for cartilage tissue engineering applications as biomaterials due to its biocompatibility and biodegradability. Type I collagen gels seeded with bone marrow-derived MSCs have shown the formation of cartilage and subchondral bone after implantation in a full-thickness osteochondral defects macaque model. After 24 weeks, the defect had been covered with cartilage-rich reparative tissue, suitable integration with the surrounding cartilage tissue, and restoration of trabecular subchondral bone [86].

 As part of this group of biomaterials is the silkworm fibroin, which is a natural biopolymer, with properties such as biocompatibility and biodegradability that allow it to be currently used for the development of a wide variety of biomedical devices and new regeneration technologies [87]. Fibroin is the main constituent (72–81%) of silkworm cocoons *Bombyx mori* [88], is a hydrophobic glycoprotein containing a large amount of hydrogen bonds, its composition and molecular orientation allows the formation of a semicrystalline structure formed by a highly ordered phase of antiparallel β-sheets that give it strength and tenacity, separated by less ordered β-sheet spacers that in turn contribute to the flexibility and elasticity of the fiber [89].

Because of these unique intrinsic properties and their versatility, silk alone is used as a biomaterial for biotechnological processes and as well as in tissue engineering [56, 90, 109]; however, it can also be combined with other polymers; the combination of fibroin/hyaluronic acid is reported, which favors the cultivation of mesenchymal stem cells [91]. In this context, silk fibroin has interesting applications in the engineering of hard and soft tissues and has diverse characteristics among which is included the ability to support the proliferation and differentiation of various cell types, making it an attractive therapeutic candidate in cartilage regenerative medicine (**Table 1**) [56, 109].

 Silk fibroin has been used in several medical applications, and it can be used as fiber [92], electrospun fibers [93], films [94], or hydrogels [95]. The versatility of fibroin as a biomaterial makes it suitable for any type of application in tissue engineering, and applications that demonstrate greater maturity and close to its final application are in the field of regeneration of bone, cartilage, and ligaments.

In this regard, a very interesting application is the reconstruction of the cruciate ligament of the knee through the elaboration of a cord of silk fibers that later are sown with mesenchymal cells of the bone marrow that differentiate to ligament tissue, offering a mechanical resistance much superior to that of other organic materials and a great biocompatibility. This application is already in commercial phase in the United States, by a company specialized in the development of biomaterials based on silk fibroin (Serica) [96].

Regarding the regeneration of cartilage, fibroin has been used for the manufacture of biphasic implants in combination with bioactive ceramics or 70S bioactive glass, which



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

> **Table 1.**  *Multiphasic designs for osteochondral repair.*

has allowed the obtaining of scaffolds with stratified properties capable of satisfying the complex and diverse regenerative requirements of the osteochondral tissue [97].

### **7.3 Synthetic polymer-based biomaterials**

Several biodegradable and biocompatible polymers of synthetic origin have been developed for biomedical applications. The aliphatic polyesters, that is, poly (α-hydroxy esters), represent polymers that have great potential for their application in tissue regeneration. In this group are listed: poly (lactic acid) (PLA), poly (glycolic acid) (PGA), and poly (ε-caprolactone) (PCL) [12, 13]. There are three enantiomers of PLA, L-lactide, D-lactide, and mesolactide [98]. Of these, the most used are poly (L-lactide) (PLLA) and poly (D-lactide) (PDLA) [99]. Both PLLA and PDLA have a tensile strength and elongation at break (1–8%) [100, 101], its nature of slow crystallization predisposes that these materials are typically hard and brittle. *In vivo* studies have shown that highly crystalline PLLA degrades completely in 5 years, while mostly amorphous PDLLA loses strength in less than 2 months and is degraded in 1 year [102].

The material properties, degradation rates, and tissue compatibility of PLA can be modified by copolymerization with other monomers, resulting in copolymers such as poly (lactic acid-co-caprolactone) (PLGA), poly (lactic acid-co-caprolactone) (PLCL), poly (lactic acid-co-ethylene glycol) (PLEG), and poly (lactic acid-co-glutamic acid) (PLGM); this makes them biomaterials with highly adaptable properties for broad biomedical applications (**Table 1**) [108, 110, 113–115].

The most common synthetic material used for cartilage tissue engineering has been nonwoven PGA and PLA mesh. PGA has demonstrated good chondrogenesis both *in vitro* and *in vivo* [103]. A combination of a cell-free poly (L-lactic-coglycolic acid) scaffold and in situ bone marrow stem cells has been used for focal fullthickness cartilage defects in a rabbit model, demonstrating suitable integration of the implant and hyaline-like cartilage regeneration in 24 weeks [104].

These polymers have been approved by FDA: a PGA, PLA, and also polydioxanone-based copolymer; BioSeed1, BioTissue Technologies, Freiburg, Germany has been used for hyaline cartilage regeneration in human trials. This scaffold is cellularized with autologous articular chondrocytes showing improved clinical scores in human trials [105].
