Enhancement of the stem cell engraftment and differentiation for cartilage regeneration using transglutaminase-4

Abstract

Purpose: Although mesenchymal stem cells (MSCs) transplantation with a hydrogel type scaffold is a promising strategy for repairing damaged articular cartilage, MSC-based cartilage tissue engineering has numerous limitations, including poor implanted cell engraftment, phenotypic alteration, soft mechanical properties, in vivo degradation and loss of strength. Transglutaminase (TG) has been used to crosslink proteins to generate biocompatible tissue scaffolds in cell engineering. However, there are no published reports regarding the use of TG in improving the engraftment and differentiation of stem cell. This study aimed to investigate the efficacy of transplantation of synovium-derived mesenchymal stem cells (SDSCs) encapsulated in a hyaluronic acid/ collagen/ fibrinogen (HA/COL/FG) composite gel by supplementing recombinant human transglutaminase-4 (rhTG-4) in treating osteochondral defects.

Materials and Methods: Human SDSCs were prepared from the synovial tissues of knee from human donors who underwent total knee arthroplasty. HA/COL/FG composite gel was generated with a mixture ratio of HA: COL: FG = 0.5: 0.5: 2.0. RhTG-4 was prepared using baculovirus expression system. The adhesion assay was carried out by directly counting the number of attached cells to the fibronectin coating plate at different concentrations of rhTG-4. The distribution of adherent cells was analyzed by crystal violet staining. To visualize cell adhesion and cytoskeletal proteins, immunofluorescence assay was performed and cystamine was supplemented to inhibit the rhTG-4 activity. SDSCs cultured in the HA/COL/FG composite gel were assayed for cell proliferation and viability at different rhTG-4 concentrations. The proliferation assay was carried out by direct counting the number of cells. The Live/Dead Viability kit was used for the assessment of the viability. To evaluate the extra cellular matrix (ECM) hardness, the cells were incubated to generate micro mass, according to the concentrations of rhTG-4 and the hardness was evaluated using a nanoindentation instrument. RhTG-4 activity in the hydrogel was estimated by measuring incorporation of biotinylated pentylamine to N, N-dimethylcasein.

In vitro chondrogenesis of SDSCs encapsulated HA/COL/FG composite gel supplementing rhTG-4 was verified. The expression of chondrogenesis-related genes (type I COL, type II COL type X COL, aggrecan, and SOX-9) was analyzed using reverse transcription- polymerase chain reaction (RT-PCR). To investigate the effect of integrin β1 on the TG-4-induced actin remodeling, the intracellular signal transduction pathway and the control of chondrogensis-related genes expression, integrin β1 siRNA was transfected to inhibit integrin β1 expression. To evaluate the in vivo cartilage regeneration, fifty-six knee joints of rabbit osteochondral defect models were made and divided into 3 groups: control group, in which the defect was left untreated; Gel/SDSC group, in which the defect was filled with the HA/COL/FG composite gel loaded with human SDSCs; Gel/SDSC+TG-4 composite gels group, in which the defect was filled with the supplement of rhTG-4 in HA/COL/FG composite gels loaded with human SDSCs. After 12 weeks, tissue specimens were assessed by macroscopic and histological evaluation, and DiI-labeled SDSCs distribution in the center of defect was evaluated.

Results: RhTG-4 treatment improved the attachment of SDSCs with increased adhesion to fibronectin and in a concentration-dependent manner. The immunofluorescence assay revealed that pretreatment with cystamine markedly downregulated rhTG-4-induced integrin β1 activation and dynamic actin remodeling, demonstrating the possibility of modulating the phenotype. SDSCs proliferation was significantly increased in rhTG-4 group. Nanoindentation analysis revealed that rhTG-4 stimulation increased the hardness of 3D micro masses. Activity of rhTG-4 in hydrogel was maintained for 2 h.

In the group supplemented with rhTG-4, the relative expression levels of type II COL and aggrecan mRNA were significantly increased as determined by RT-PCR. Immunofluorescence analysis revealed that pretreatment with integrin β1 siRNA markedly decreased rhTG4-induced actin remodeling. Intracellular signal transduction pathway and chondrogenesis-related genes including aggrecan and type II collagen were activated in an integrin β1 siRNA-dependent manner. In vivo study, Gel/SDSC+TG-4 group showed more repaired defect lesion compared to the other groups and yielded reconstructed tissue resembling native hyaline cartilage. The total macroscopic and histological scores were significantly higher and DiI-labeled SDSCs distribution was markedly increased at the center of defect in Gel/SDSC+TG-4 group.

Conclusion: RhTG-4 supplementation in the HA/COL/FG composite gel mediates cartilage regeneration by enhancing the engraftment and differentiation of SDSCs.