The Effects And Mechanisms Of Collagen TypeⅠScaffold Architecture On Chondrocyte Differentiation

Tissue Engineering and Regenerative Medicine (TERM) provide a whole new way toaddress cartilage repair. Mesenchymal stem cells (MSCs) can overcome the limited supply ofchondrocytes and were widely used in the application of cartilage TERM research. The cellniches and extracellular matrix components contacting with cells have an important impact oncell differentiation. Therefore, in order to guide the design and development of materials forcartilage defects, it is significant to study the function and impact of material on celldifferentiation.Collagen type I have attracted much more attention as cartilage TERM material becauseof its excellent biocompatibility and bioactivity. But different researches showed different andeven conflict results on whether collagen type I had the ability to induce MSCs chondrocytedifferentiation. The main reasons for this discrepancy can be attributed to:1) the difference ofcell source and the different differentiation ability of cells caused by inappropriate cellpassages and culture conditions;2) the difference of source, purity and scaffold architecture ofcollagen. Cell line like ATDC5can avoid these instability commonly observed on MSCs.Based on ATDC5model cells, this study established an evaluation system to assess the impactof material on cell chondrocyte differentiation, compare the effects of different collagen type Iscaffold architectures on cells chondrocyte differentiation and explore the effect of collagenon activation of FAK and MAPK signaling pathways.By analyzing the stability of16common reference genes in chondrocyte differentiationof ATDC5cells through geNorm algorithm, together with comparing the expression levels ofthese reference genes, this study filtered out Ppia and Hprt as the ideal reference genes ofqPCR experiments during chondrocyte differentiation. Also, we further confirmed that Ppiaand Hprt worked well for chondrocyte differentiation of mouse MSCs. Then this studyoptimized various parameters in the experiments for evaluating effects of materials on ATDC5chondrocyte differentiation. The result of gene expression, cell proliferation, staining andquantitative methods confirmed that ATDC5quickly entered the chondrocyte differentiationprocess and expressed cartilage-related genes when induced by serum-free medium containing TGF-β3. Though insulin is commonly used in ATDC5cells chondrogenicdifferentiation, TGF-β3medium is more suitable for evaluation of materials used in cellchondrogenic differentiation. This study also filtered out the optimal concentration of TGF-β3was10ng/ml. When induced by this concentration, ATDC5had the highest cartilage-relatedgene expression level and the lowest ossification-related gene expression level.Collagen type I scaffolds with three different morphological structures were produced:1)Non-recombinant fiber sponge scaffolds with connectivity porous were prepared bylyophilization. Scanning electron microscope showed that the aperture of scaffolds wasapproximately300μm, and the hole wall was non-recombinant fiber structure.2) Gel with100-200nm diameter recombinant fiber networks prepared by NaOH neutralization.3)Recombinant fiber-coated cell culture dishes made by in vitro recombinant and in-suitdemineralization process. Sirius Red staining and3D images showed the recombinantfiber-coated film was uniform and smooth. Sirius Red staining and3D images showed therecombinant fiber-coated film was uniform and smooth. Collagen recombinant fibers were100-200nm in diameter, about68nm of periodically D-band, about3.46nm of gap depth,closer to the morphology of in vivo collagen fiber.On the basis of established evaluation system, this research studied the effects ofdifferent type I collagen architecture on early cell chondrogenic differentiation. The results ofcell morphology, proliferation, and gene expression exhibited that the specific role of collagenin cartilage differentiation depended on its morphology and structure. Collagen gelrecombinant fiber networks facilitated cell shape to change spherical, which was more benefitfor chondrocyte differentiation than collagen film and sponge. Compared with ATDC5cellmicromass without collagen, the presence of collagen might provide some extracellularmatrix signaling to promote cell chondrogenic differentiation.Next, the effects of different type I collagen architecture on ossification differentiationwere studied. OM Containing β-glycerol phosphate, vitamin C and dexamethasone couldinduce ATDC5ossification, and can guarantee that all of the cells can be in contact with eachother and the material by inhibiting cell proliferation. The results showed type I collagen filmhad neither promotion nor induction on ATDC5ossification. However, porous sponge-type Icollagen scaffolds not only promoted but also induce ATDC5ossification. Finally, this study investigated the effects of type I collagen gel on chondrocytedifferentiation of hBMSCs and confirmed the results of ATDC5cells. The results showed typeI collagen could promote hBMSCs chondrogenic differentiation in conjunction with TGF-β3,and the outcome using0.25mg/ml of collagen gel was better than2.5mg/ml. This studydemonstrated that the presence of type I collagen maintained the stability of FAK expressionand activated MAPK signaling pathways by western blotting analysis.