| Osteochondral defect cases are increasing caused by degenerative joint disease and trauma, and there is a limited ability to repair itself. It can not completely restore the function of cartilage tissue by present treatments for osteochondral defect and trauma, the rapid development of tissue engineering technology has brought new hope for repairing the cartilage defects with large areas. It is important for tissue engineering to build cell-scaffold complex as well as in vitro environment of constructing tissue engineering. Static culture is a traditional cultured way of tissue-^engineered construction, but it will be obviously limited of the diffusion of oxygen and nutrients, the provision of nutrients and the discharge of metabolic waste will be blocked, the proliferation and differentiation of cells in the scaffold will be definitely affected when tissue exceeds a certain thickness. With the deeper study, the dynamic culture of bioreactor has been chosen by much more researchers. In this study, the acellular cancellous bone scaffold was regarded as bone biomimetic scaffold, the chitosan and gelatin were mixed to prepare chitosan/gelatin porous scaffold as cartilage biomimetic scaffold by freeze drying foaming method with the characteristics of chitosan cross-linked anionic polymers. After that, human adipose-derived stem cells (hADSCs) were inoculated into the two scaffolds with chondroinductive and osteogenic media to construct tissue engineering cartilage and bone, respectively. After being cultured two weeks, the two parts were stitched together and cultured in a spinner flask to make for the osteochondral construction dynamically.The preparation and physical properties of acellular cancellous bone and chitosan/gelatin hydrogel scaffolds:After the bones were cleaned, degreased with methanol/chloroform, the cells were taken off with sodium deoxycholate and protein was subsequently removed by H2O2, the acellular cancellous bone scaffolds were prepared.2%chitosan acetic acid solution and gelatin solution were mixed in the mass ratio of3:1, then they were cross-linked in carbodiimide/N-hydroxyl-succinamide-/morpholine ethane sulfonate (EDC/NHS/MES) for6h after freeze drying, the scaffolds were treated with Na2HPO4for2h to neutralize acetic acid and prepared finally after freeze drying once again. The samples size were cut into the shape with size of5mm×5mm×5mm, their surface appearances of bone and hydrogel were observed under a Scanning Electron Microscopy (SEM), their modulus of elasticity were measured with Universal Testing Machine (UTM), the porosity of hydrogel scaffold was detected by a pycnometer method and measured swelling ratio, the composition of cancellous bones were analyzed by infrared detection. The results showed that the average pore size of cancellous bones were284.5±83.62uμm, the elasticity modulus were41.27±15.63MPa. IR detection showed that the scaffolds remained hydroxyapatite and other inorganic components and a small amount of protein which was same with natural elements of human bone, so cancellous bone had good biocompatibility. The average pore size of hydrogels were118.25±19.51μm, the porosity, swelling rate and elasticity modulus of scaffolds were82.60±2.34%,361.28±0.47%, and61.2±0.16KPa, respectively. Two kinds of scaffolds had good biological compatibility and physical properties and they were suitable as support material for tissue-engin osteochondral tissue engineering.Isolation, cultivation and osteogenic, chondrogenic differentiation of ADSCs:ADSCs were isolated using trypsin digestion, and then they were continued to culture and passaged. hADSCs at passage5were cultured in osteogenic and chondrogenic induction medium, osteogenic and chondrogenic differentiation ability were detected. The results showed that ADSCs grew with shuttle shaped and attached, proliferated well, could be passaged in5days. ALP expressed positive after7days of induction, the results of von-Kossa staining showed that induced cells secreted large amounts of calcium nodule after28days induction. It could be observed a lot of metachromatic matrix in chondrocyte cytoplasm and surrounding mucilage by toluidine blue and saffron after O staining after7,14days chondrogenic induction in vitro.Construction cell-cancellous bone construct, cell growth and matrixin secretion in scaffolds:hADSCs at passage7were inoculated into cancellous bone scaffolds at density of106cells/mL. All scaffolds were added40μL with100μL micro-pipette. After the constructs were cultured at37℃and5.0%CO2in a humidified incubator for2h, then they were added enough osteogenic induction medium. Half constructs were transferred into spinner flask with40rpm as the dynamic experimental group I, the other constructs and simple cancellous bone were put in two T-flasks at static condition as the control group II and blank group III, respectively. Osteogenic differentiation ability was detected qualitatively and quantitatively with alkaline phosphatase (ALP) kits, cell growth and-survival were investigated qualitatively using Dead/Live staining, the cell metabolism of glucose was analyzed through detecting kits, and cell adhesion and extracellular matrix secretion were observed by SEM. The results showed that ALP expression of cells in cancellous bone was much obvious, Dead/Live results showed cell distribution and growth was much better, SEM showed that matrix secretion from cells was much more at dynamic spinner flask environment. Engineered bone could be rapidly constructed at dynamic culture.Construction cell-hydrogel construct, cell growth and matrixin secretion in scaffolds:It was same to build cell-hydrogel construct with tissue engineered bone.20constructs were cultured at static condition as the control group II, the other20constructs were transferred into spinner flask as the dynamic experimental group I. Chondrogenic differentiation ability of ADSCs within hydrogel scaffolds was investigated with Toluidine Blue and Safranine O staining, the cell metabolism of glucose and lactic acid was analyzed through detecting kits, the cell distribution, adhesion and extracellular matrix secretion within scaffolds were observed by Dead/Live staining and scanning electron microscopy (SEM), respectively. The results showed it could express more proteoglycans in dynamic spinner flask, the cell distribution within scaffolds in spinner flask was more uniform and the scaffolds could be filled mostly by extracellular matrix.Construction of osteochondral constructs and their culture at dynamic and static condition:hADSCs at passage7were inoculated into cancellous bone and hydrogel scaffolds at density of107cells/mL following the method of constructing tissue engineering bone and cartilage. After being added enough osteogenic and chondrogenic medium for two weeks, respectively, the two parts were sewed together to construct tissue engineered osteochondral complex. Half constructs were transferred into spinner flask with40rpm as the dynamic experimental group I, the other constructs were put in T-flask at static condition as the control group II for correlation detection within a certain period of time, respectively. The two part of the interface connection was observed by inverted microscope, the distribution and permeability of cells in scaffold each layer of static and dynamic group were investigated using laser scanning confocal microscope, cell adhesion and extracellular matrix secretion in composite interface, cartilage and bone scaffolds were observed by SEM. The results showed that two groups of complex connected closely, Calcein staining showed that cell survival and distribution were much better and uniform in dynamic group. SEM results showed that cells and matrix secretion were similar at the interface and engineering bone of two groups, there were more cell clusters stretching on the hole wall and more matrix secreting in engineering cartilage in dynamic group. |
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