Molecular Mechanism Of Chondrogenesis Induced By Collagen-Based Hydrogels

Objective: Effect of collagen-based hydrogel on chondrogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSC) was investigated by investigating the expression of related signaling pathways. Further study demonstrated the impact of collagen-based hydrogel on chondrogenic differentiation in vivo by investigating the expression of related signaling pathways and molecular mechanisms.Methods:In vitro model of chondrogenic induction was constructed by rBMSC isolated and cultured in vitro to study the effect of collagen-based hydrogel on chondrogenic differentiation of rBMSC by investigating related signaling pathways.4 groups were designed:B group was the simple cell group, rBMSC three-dimensional (3D) cultured without chondroinductive factor TGF-β1 and collagen-based hydrogel; BT group was the growth factor group, rBMSC 3D cultured with TGF-β1 but without collagen-based hydrogel; BC was the collagen materials group, rBMSC 3D cultured with collagen-based hydrogel; BTC was the positive control group, rBMSC 3D cultured with collagen-based hydrogel and TGF-β1. After 14 days,28 days and 42 days of culture, the samples in all groups were taken out for analysis. DNA content and Hematoxylin-eosin (HE) were used to detect the cell proliferation and cell morphologyin experiments; Calcein-AM/PI and Phalloidin/hoechst33258 fluorescence staining were for observation of cell viability and distribution and dynamics of fibrous actin (F-actin); Total glycosaminoglycan (GAG) secretion was examed by safranin O staining and DMMB method. qRT-PCR was used to detect the gene expression of cartilage specific gene (Acan, Col Ⅰ, Col Ⅱ and Col Ⅹ) and signaling molecules involved in signaling pathways (TβR-Ⅱ, TβR-Ⅰ, Smad2, Smad3, Smad4, Wnt-5a, Ihh and PTHrP); The expression of collagen type I and type II collagen was evaluated by immunohistochemical detection; The protein levels of ERK1/2, p38, N-cadherin and fibronectin was detected by western blot method. Effect of collagen-based hydrogel on chondrogenic differentiation of rBMSC by the above methods to investigate the expression of related signaling pathways.The effect of collagen-based hydrogel on expression of related signaling pathways of chondrogenic differentiation in vivo by cartilage defect model of rabbit articular.5 groups were designed:The blank control group was implanted without cells and collagen-based hydrogel, B was the simple cell group implanted 3D cultured rBMSCs, BT as the growth factor group was implanted 3D cultured rBMSCs with adding TGF-β1, BC was the collagen materials group implanted 3D cultured rBMSCs with adding collagen-based hydrogel, BTC was the positive control group implanted 3D cultured rBMSCs with adding TGF-β1 and collagen-based hydrogel. After 4 weeks,12 weeks and 24 weeks of culture, the samples removed from experimental animals were taken out for analysis. The repair of articular cartilage defect was tested by macroscopic observation, macroscopic scores and histological scores. The elastic modulus of articular cartilage defect was detected by biomechanical properties testing; Cell morphology and secretion of extracellular matrix were detected respectively using HE and Masson staining; Total glycosaminoglycan (GAG) secretion was examed by safranin O staining; qRT-PCR was used to detect the gene expression of cartilage specific gene (Acan, Col Ⅰ, Col Ⅱ and Col Ⅹ) and signaling molecules involved in signaling pathways (TβR-Ⅱ, TβR-Ⅰ, Smad 2, Smad 3, Smad 4, Wnt-5a, Ihh and PTHrP); The expression of collagen type Ⅰ and type Ⅱ collagen was evaluated by immunohistochemical detection. The above all methods were used to investigate the repair of articular cartilage defect in vivo and the expression of related signaling pathways.Results:RBMSC isolated and cultured in vitro were used study the effect of collagen-based hydrogel on chondrogenic differentiation of rBMSC and the expression of related signaling pathways. By means of histolqgical tests (including HE staining, safranin O staining, calcein-AM/PI staining and phalloidin/hoechst33258 staining) and biochemical tests (including DNA content and GAG content detection), we investigated that the inductive results of BC group were superior or similar to the BT group, and the best inductive results were showed in BTC group, indicating that the collagen-based hydrogel material has the similar effect of chondrogenic induction compared with the growth factors. The secretion of proteoglycans and collagen type Ⅱ in BC group were superior or similar to the BT group by immunohistochemistry and qPCR detection, indicating that the collagen-based hydrogel material has the similar effect of chondrogenic induction compared with the growth factors. However, lower secretion of collagen type Ⅰ in BC group was showed with respect to the B and BT groups, suggesting that collagen-based hydrogel material in maintaining cartilage phenotype and preventing dedifferentiation was more effective. Collagen type Ⅹ failed to be detected in each group, indicating that there was a slight tendency osteogenic differentiation. The results of PCR showed that the gene expression of signaling pathways in the BT, BC and BTC groups were higher compared with the B group, the highest gene expression of signaling pathways was investigated in the BTC group in general, however, the gene expression of Ihh in each group were inferior to the B group, which indicated that collagen material could adjust the TGF-β,Wnt and Ihh-PTHrP signaling pathways to induce cartilage formation. The protein levels of ERK1/2, p38, N-cadherin and fibronectin were detected by western blot, the research indicated that the protein expression of ERK1/2, p38, N-cadherin and fibronectin were found in each group after gathering phase, differentiative stage and hypertrophy stage. Nonetheless, the protein up-regulation of p38, N-cadherin, fibronectin and the protein down-regulation of ERK1/2 were exhibited in BC group, which suggested that collagen material could enhance cell aggregation growth by promoting the secretion of adhesion protein (N-cadherin and fibronectin) and adjusting the MAPK (ERK1/2、p38) signaling pathways.Through the model of articular cartilage defect in vivo,we could observe that the effect of repair in articular cartilage defect in BT, BC and BTC group were better than that in B group by gross observation, biomechanical properties testing, histological tests (including HE staining, safranin O staining and Masson staining) and histologic grading, moreover, the effect of repair in articular cartilage defect in BC group were superior to the BT group. The best results of repair in BTC group and the worst results of repair in the control group could be observed. The secretion of proteoglycans and collagen type Ⅱ in BC group were superior or similar to the BT group using immunohistochemistry and PCR detection, suggesting that the BC group has the better effect of repair in articular cartilage defect compared to the BT group. However, lower secretion of collagen type Ⅰ in BC group was showed with respect to the B and BT groups, suggesting that collagen-based hydrogel material in maintaining cartilage phenotype and preventing dedifferentiation was more effective. Collagen type X failed to be detected in each group, indicating that there was a slight tendency osteogenic differentiation. PCR results showed that the gene expression of signaling pathways in the BT, BC and BTC groups were higher compared with the B group, the highest gene expression of signaling pathways was generally investigated in the BTC group, however, the gene expression of Ihh in each group were inferior to the B group, which indicated that collagen material could adjust the TGF-P, Wnt and Ihh-PTHrP signaling pathways to induce cartilage formation.Conclusion:The results hinted that the effect of collagen-based hydrogel on chondrogenic differentiation of rBMSC may be associated to TGF-β, Wnt, MAPK and Ihh-PTHrP signaling pathways. In addition, there was a analogous mechanism on chondrogenic induction between biological materials and TGF-β. Thus, biological materials was superior to growth factors on chondrogenic induction in vivo, indicating that biological materials can directly realize chondrogenic induction instead of the growth factors and comprehensive repair of articular cartilage defect by optimizing materials.