**3.4 Platelet-rich plasma (PRP)**

Platelets play a fundamental role in hemostasis and are a natural source of growth factors. More than 30 growth factors have been identified in PRP; among them, the following six growth factors play an important role in cartilage

**Figure 1.** 

*Scheme of preparation of P-PRP and L-PRP from whole blood using five steps.* 

regeneration. They are TGF-β1, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) [67, 68].

The concentration of platelet in PRP used for cartilage repair should be two to three times higher than that of baseline [69]. PRP can be prepared by the following five procedures (**Figure 1**). **Step 1:** blood (9 parts) is added into 3.8% sodium citrate solution (1 part) in a centrifuge tube and centrifuged at 500 g for 5 min to obtain three layers. **Step 2:** The supernatant at the top layer is transferred into a new tube, which is called as platelets-containing plasma, and the middle layer is transferred into another new tube, which is called leukocytes-containing plasma. **Step 3:** The platelets-containing plasma is centrifuged at 2000 g for 5 min to separate platelet-poor plasma (PPP) from the platelet pellet. **Step 4:** The platelet pellet is resuspended with appropriate amount of PPP to make pure PRP (P-PRP). **Step 5:**  The leukocytes-containing plasma is mixed with platelet pellet and resuspended with appropriate amount of PPP to make leukocytes-containing PRP (L-PRP). Both P-PRP and L-PRP can be used for cartilage tissue engineering [70].

## **4. Bioactive molecules used for cartilage tissue engineering**

Bioactive molecules used in cartilage tissue engineering include two kinds of materials: one is small molecular weight bioactive compound and the other one is high molecular weight materials including some nature biomaterials and synthetic polymers. Both of them play critical role in cartilage tissue engineering.

#### **4.1 Kartogenin (KGN)**

Kartogenin (KGN), a small heterocyclic molecule, has been discovered to enhance chondrogenic differentiation of human MSCs by regulating the *Current Tissue Engineering Approaches for Cartilage Regeneration DOI: http://dx.doi.org/10.5772/intechopen.84429* 

CBFbeta-RUNX1 transcriptional program [71, 72]. Animal studies have shown that KGN can promote rabbit meniscus regeneration [73] and wounded rat enthesis repair [70, 74]. *In vitro* and *ex vivo* experiments showed that KGN can reduce nucleus pulposus cell degeneration induced by interleukin-1beta (IL-1β) and tumor necrosis factor-alpha [75]. More recent studies indicated that KGN inhibited pain behavior, chondrocyte inflammation, and attenuated osteoarthritis progression in mice [76]; enhanced collagen organization and mechanical strength of the repaired enthesis of mouse rotator cuff [77]; and induced chondrogenic differentiation of dental pulp stem cells [78].

These findings invigorate research into small-molecule therapy and regenerative medicine for cartilage diseases. It also provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis (OA). KGN and other structurally related small molecules that can promote selective differentiation of MSCs into chondrocytes may prove to be extremely useful for improving the outcome of cell-based therapy by stimulating endogenous mechanisms for repair of damaged cartilage, thus enhancing the joint's intrinsic capacity for cartilage regeneration [79].

#### **4.2 Simvastatin**

Simvastatin is a kind of HMG-CoA reductase inhibitor, which is widely used therapeutically to reduce morbidity and mortality in patients with hyperlipidemic cardiovascular disease [80]. In addition to lowing low-density lipoprotein (LDL) cholesterol, statins have broad-range pleiotropic effects, including antiinflammatory effects, which could exert an effect on synovium and cartilage [81]. Animal studies found that simvastatin markedly inhibited not only developing but also established collagen-induced arthritis [82]. Simvastatin inhibited the IL-6 and TNF-α production of human chondrocytes and cartilage explants in a concentration-dependent manner. Higher concentrations of simvastatin decreased nitric oxide (NO) production in both of human chondrocytes and cartilage explants [83]. Statin treatment has also been shown to positively regulate components of the extracellular matrix in a rabbit OA model [84]. More studies have shown that local application of simvastatin enhanced tendon-bone interface healing in rabbits [85]. These studies have shown that the effect of simvastatin on articular chondrocytes may provide novel insight regarding the role of cholesterol homeostasis and signaling during cartilage development.

#### **4.3 Biomaterial scaffolds for cartilage tissue engineering**

Biomaterial scaffolds play an important role in cartilage tissue engineering, which act as a carrier to deliver the cells and bioactive molecules to the damaged tissue areas and also work as a template for tissue regeneration, to guide the growth of new tissue.

There are two groups of biomaterial scaffolds used for cartilage tissue engineering. They are synthetic polymers and natural polymers. Commonly used natural materials in cartilage repair are agarose, alginate, chitosan, collagen, fibrin, and hyaluronan.

 Agarose is a galactose polymer, which is suitable for cell encapsulation, especially for chondrocytes. When the ADSCs were cultured in agarose, they were differentiated into chondrocytes as evidenced by upregulation of the production of glycosaminoglycan (GAG) [86]. Moreover, dynamically loaded cell-seeded agarose hydrogel provided better graft tissues in a repair model of full thickness defects in rabbit joint cartilage [87]. PRP combined with agarose as a bioactive scaffold has shown to enhance cartilage repair [88].

Another extensively studied natural scaffold used for cartilage tissue engineering is alginate, which is a polysaccharide extracted from brown algae. Generally, alginate is hydrophilic and water-soluble, thickening in neutral conditions, which is of great importance for *in situ* hydrogel formation [89]. The good gelling properties of alginate-based scaffolds allowed them to be used as an injectable scaffold for the damaged cartilage repair. Human dental pulp stem cells were cultured in 3% alginate hydrogel and implanted in a rabbit damaged cartilage area. Three months after surgery, significant cartilage regeneration was observed [90]. More studies have been done by mixing the cells or/and growth factors with alginate solution to form gel microspheres in an isotonic CaCl2 solution (**Figure 2**). The findings have shown that the cells are distributed homogeneously inside the gel microspheres. Those cell-containing alginate beads can be used as chondrogenesis-promoting scaffolds for cartilage regeneration [91, 92].

Chitosan is another natural polysaccharide extracted from crustacean shells, particularly from shrimps and crabs. Chitosan contains glucosamine and hyaluronic acid (HA), which are basic components of the native cartilage. Therefore, chitosan is widely used for cartilage tissue engineering. The recent studies have shown that chitosan-hyaluronic acid hydrogel promoted wounded cartilage healing in a rabbit model [93, 94].

Collagen is a main component of the extracellular matrix (ECM) of chondrocytes. Collagen gel has been widely used as substrates for articular cartilage substitutes [95, 96]. Injectable type II collagen gel has been used to treat full-thickness articular cartilage defects [97]. Clinical study has demonstrated that collagen gel can be used to replace cartilage and subchondral bone [98].

Fibrin hydrogels used for articular cartilage repair has been well documented by a review paper [99]. It has been reported that chondrocytes survived in the fibrin gel and enhanced their synthetic activity as evidenced by the increase of the production of GAG and collagen type II [100]. Human fibrin hydrogels have been approved by the Food and Drug Administration (FDA) for cartilage tissue engineering [101].

 Hyaluronan is a main component of native cartilage. Similarly to the other native biomaterial scaffold, hyaluronan is the most widely used scaffold for cartilage tissue engineering. The studies have shown that hyaluronan upregulated collagen II expression and downregulated collagen I expression in human MSCs when they were cultured in hyaluronan gel [102].

Although bioactive natural scaffolds have very good biocompatibilities, their mechanical properties still need to be improved. In addition to natural bioactive scaffolds, synthetic materials provide good mechanical properties suitable for cartilage tissue engineering. These synthetic polymers are either used alone or combined with natural biomaterials for cartilage research.

#### **Figure 2.**

*The intermolecular network of alginate molecules is formed in calcium chloride solution. Alginate can be dissolved with sodium chloride (left image), but cross-linked each other in calcium ions-containing solution to form hydrogel (right image).* 

#### *Current Tissue Engineering Approaches for Cartilage Regeneration DOI: http://dx.doi.org/10.5772/intechopen.84429*

The most famous synthetic polymers for cartilage regeneration are polylactic acid (PLA), polyglycolic acid (PGA), and their copolymer polylactic-co-glycolic acid (PLGA). These polymers have a beneficial range of mechanical characteristics and high biocompatibility. Owing to the fact that PLA-PGA polymers have been successfully used in the clinics including sutures, screws, and pins [103–105], they are also used for articular cartilage defect repair in rabbits [106] and meniscal lesion repair in dogs [107]. Currently, two PLA-based scaffolds have been clinically used for cartilage repair: one is BioSeed ®-C and the other one is TRUFIT CB™. The PLA-based polymer scaffolds have shown significant improvement in patient outcomes for the treatment of post-traumatic OA and focal degenerative cartilage defects [108, 109].

Polyethylene glycol (PEG), a nontoxic synthetic polymer, is widely used with other natural materials to enhance their mechanical strength for cartilage tissue engineering. The studies have indicated that PEG-based hydrogel can promote chondrogenic differentiation of MSCs *in vitro* and *in vivo* [110, 111]. Injectable hydrogels used for cartilage tissue engineering have been well summarized by several review papers [112]. PEG-HA scaffold-treated patients achieved significantly higher levels of tissue fill in cartilage defects [113].
