NOTCH ligand signaling plays a major role in the chondrogenic differentiation of cells
[100][162]. NOTCH-2 modulates the activities of NOTCH-3 and -1, hence influence the growth and development, and homeostasis of chondrocytes and articular cartilage
[101][102][103][163,164,165]. NOTCH-3 represses the proliferation of terminally differentiated chondrocytes within the cartilaginous tissues
[104][166]. In a 3D-cultured chondrogenesis, there is a downregulation of NOTCH ligands and receptors
[105][106][167,168]. While MSC undergoes terminal chondrocyte differentiation, NOTCH-3 receptors were upregulated and were highly expressed
[100][107][162,169].
6. Engineered Chondrogenesis by D-MSCs
Due to the intrinsic limited potential of cartilage tissue to heal, cartilage tissue engineering gave a robust breakthrough in the field of regenerative and translational medicine. The field of tissue engineering provides the biological substitute of limited available tissues to restore and maintain the naïve homeostasis and to improve the biomechanical strength and function of the tissues. The integral components of tissue engineering are stem/stromal cells, scaffolds, and bio-micromolecules
[108][109][110][178,179,180]. The successfully engineered tissue relies on tissue ergonomics that include harvest and expansion of appropriate cells, the addition of optimum levels of growth factors and cytokines, and provision of 3D scaffold and extracellular matrices until the healing gets completed.
Growth factors: The cardinal growth factors responsible for cartilage engineering are TGF-β1 and -β3, BMP-2 and -7, IGF-1, and bFGF. TGF-β1 induces and maintains chondrogenesis of MSC through chondrostimulatory signaling by p38, ERK-1, JNK, N-cadherin expression, and suppresses the IL-1 catabolism
[111][181]. TGF-β1 controls Wnt-mediated signaling and β-catenin TCF pathway in early MSC chondrogenesis
[112][182]. TGF-β3, when co-cultured with bovine MSCs in a chitosan scaffold, stimulates the growth of hyaline cartilage and integrates into the host cartilage
[113][183]. BMPs are involved in Hedgehog and TGF- β signaling in regulating chondrogenesis both in vitro and in vivo
[114][115][116][184,185,186]. BMP-2 inhibits IL-1 effects and enhances cartilage cell production by inducing chondrogenic factors, whereas BMP-7 enhances ECM production
[117][187]. IGF-1 downregulates MMP-13 by upregulating collagen 2 expression and GAG synthesis
[118][188]. IGF-1 induces in vitro and in vivo chondrogenesis in a dose- and time-dependent manner
[119][189]. The evidence stated that bFGF-18 in a concentration-dependent manner stimulates and enhances chondrogenesis in the osteochondral lesion of rabbit knees
[120][121][190,191].
Bioactive molecules: Kartogenin (KGN) enhances MSC chondrogenesis by upregulation of CBFβ-RUNX1 transcription
[122][192]. Evidence states that kartogenin promotes tendon and meniscus regeneration
[123][124][193,194]. KGN inhibits pain stimulus, attenuates chondral degeneration and inflammation, supplements the biomechanical strength of repaired bones and tendons in-vivo animals, and robust chondrogenic differentiation of DP-MSCs
[125][195]. Simvastatin, a hypolipidaemic molecule, enhances positive effects on synovium and cartilage tissues, thereby reducing inflammation, degeneration and halts arthritis progression
[126][196]. A higher concentration of statins decreased the production of nitric oxide in chondrocytes and cartilage explants
[127][197].
Bioactive scaffolds: Scaffolds, an integral part of tissue engineering, are of natural [collagen, fibrin, hyaluronan, alginate, agarose, and chitosan] and synthetic [polylactic acid (PLA), polyglycolic acid (PGA), and copolymer polylactic-co-glycolic acid (PLGA)] polymers. The ideal scaffold should be biocompatible, optimum porosity, biodegradable, elastic natured, mechanical strength, easy fabrication, non-toxic, long-term effectiveness, and support cell attachment and proliferation
[128][129][198,199]. Platelet-rich plasma seeded with agarose enhance cartilage and tendon regeneration
[130][200]. MSCs cocultured with collagen or agarose enhance chondrocyte differentiation along with increased production of ECM and GAGs
[131][201]. Alginate, an injectable scaffold, is used in regenerating focal chondral defects and in autologous chondrocyte implantation
[132][133][202,203]. DP-MSCs accelerate chondrogenesis when cultured with growth factors and alginate beads
[134][204]. The synergistic effects of chitosan and hyaluronic acid hydrogel enhance the healing of cartilage defects in rabbits
[135][205]. Synthetic polymer scaffolds are used in the repair of osteochondral defects in rabbits
[136][206] and meniscal lesions in dogs
[137][207].
Chondrocytes release factor XIIIA, whose upregulation leads to hypertrophic chondrocyte differentiation in OA chondrocytes. In the murine OA cartilage model, there is an interplay between FXIIIA and α1 subunit of α1β1 integrin and tissue glutaminase 2 (TG2) mobilization, which leads to remodeling of the cartilage matrix. In absence of TG2, FXIIIA fails to undergo chondrocyte hypertrophy
[138][208]. The conjunction of plasma membrane-bound TG2 and FXIIIA with a raised expression of FXIIIA upregulates the p38 MAP kinase signaling pathway in chondrocytes of OA cartilage in situ
[139][209]. In turn, p38 signaling significantly increases SOX-9, which inhibits both in vitro and in vivo chondrocyte maturation to hypertrophy by DP-MSC-induced chondrogenesis
[140][210].
Cordycepin is a potent antioxidant molecule with anti-tumorigenic and anti-inflammatory properties
[141][211]. During MSC-induced chondrogenesis, cordycepin upregulates type 2 collagen, SOX-9, and TGF-β1 and -β3 expression, whereas downregulates type 10 collagen and Runx-2
[100][162]. Cordycepin has the potential to construct engineered cartilage by the inhibition of chondrocyte hypertrophy through PI3K/Bapx1 and the westin signaling pathway
[142][212]. Hence, cordycepin plays a major role in cartilage and chondrocyte metabolism.
DP-MSCs loaded onto nanostructured PEG-GELMA-HA hydrogel form 3D spheroids, which further differentiate into chondrocytes in vitro
[143][61]. Scaffold-assisted chondrogenesis upregulates procollagen type 2 and 10, aggrecan, alkaline phosphatase, and SOX-9 genes and downregulates Nanog, Slug, Snail, and Twist genes
[144][145][172,213]. IHC analysis exhibit type 2 collagen deposition in DP-MSCs co-cultured with PEG-GELMA-HA hydrogel scaffold. These findings direct the usage of DP-MSCs to construct engineered cartilage in focal cartilage and osteochondral defects.
Expanded chondrocytes from MSCs co-cultured with PGA-fibrin scaffolds revealed considerable expression of type 1 and 2 collagen and further resulted in the formation of hyaline cartilage. Upon optimal addition of platelet-rich plasma to cartilage tissue, the formation of hyaline cartilage was robust with higher expression of collagen type 2
[146][214].
Loading of human dedifferentiated chondrocytes into collagen sponge, in the presence of hypoxia and BMP-2, resulted in chondrogenesis, which is transfected onto siRNAs targeting collagen type 1 and HtrA1 serine protease, which are raised in OA cartilage. Such a mechanism led to the improvement of chondrocyte phenotype differentiation. Transplantation of in vitro cultured cells into nude mouse model in vivo resulted in neochondrogenesis with hyaline matrix formation
[147][215].