Objective To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage.Methods Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type Ⅱcollagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct wascross-linked by use of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensilestrength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performedto evaluate the degradability of the scaffold in vitro. Results A bulk scaffold with desired configuration was obtained. The mechanical test showed that the crosslinking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100250 μm. In vitro lysozyme degradation indicated that crosslinked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. Conclusion Chitosan and typeⅡcollagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite socaffold meets requirements for tissue engineered scaffold and may serve as an alternative cellcarrier for tissue engineering cartilage.
Objective To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle. Methods Acellular cartilage matrix was made from human auricle cartilage. The MSCs were isolated from the nucleated cells fraction of mice marrow by centrifuge.The MSCs were embedded in acellular cartilage matrix. After 10 day’s combined culture, the specimens were observed with optical and electrical microscope.Results The MSCs could well proliferate in the acellular cartilage matrix. The cells were not well-distributed in acellular cartilage matrix. There were more cells in the peripheral part of the matrix than in the central part of the matrix. Most of the cells were in cartilaginous lacunae. There were 1 or 2 cells in every cartilaginous lacunae.Conclusion The MSCs can be used as seed cells of tissue engineering and can well proliferate in the acellular cartilage matrix and become tissue engineering cartilage.
Objective To observe the efficiency and biological characteristics in regenerating in vitro tissue-engineered cartilage from epiphyseal chondrocyte-scaffold complex. MethodsThe first passage epiphyseal chondrocytes were collected and mixed with the biological gel-matrix, the chondrocyte-gel fluid wasdropped into the scaffold to form a complex. The complexes were in vitro cultivated. The changes of complexes in morphology and synthesis of collagens type ⅡandtypeⅠ and aggrecan were observed under the gross and the inverted and light microscopes. The sulfate GAG content in complexes was measured by the the modified dimethylmethylene blue method. Results During cultivation, thecomplexes could keep its original shape with the stable homogeneous three-dimensional distribution of chondrocytes，gradually became milk white and translucence with their rigidity increasing. In the 1st week, the chondrocytic lacunae formed in the complexes. After 2 weeks, the complex was gradually reorganized into the mature engineered cartilage with rich collagen typeⅡand aggrecan and typical cartilage histological structure, but with negative immunological staining of collagen typeⅠ. In the 4th week, the engineered cartilage resembled the nature epiphyseal plate in the characteristic of histological structure, and had over 34% of the sulfate GAG content of the natural epiphyseal plate. Conclusion Theepiphyseal chondrocyte-scaffold complex can be reorganized into typical cartilage with the epiphyseallike histological structure, and be fit for repairing the epiphyseal defect. The tissue engineered cartilage cultivated for 1-2 weeks may be a good choice for repairing epiphyseal defect.
OBJECTIVE To investigate the possibility of repairing the cartilage cartilage defect with homogeneous chondrocytes combined with Pluronic. METHODS: Homogeneous cartilage chondrocytes of adult New Zealand rabbits were harvested and cultured in vitro, which were marked by 3H-TdR and mixed with Pluronic. The medial or lateral condyle defects were made (phi 4 mm, extending down to the calcified zone) in 20 rabbits. In the experimental group, the right defects were repaired by homogeneous chondrocytes combined with Pluronic; in the control group, the left defects were repaired by Pluronic only or were left un-repaired. The animals were sacrificed in the 4th, 8th and 16th weeks after operation respectively. The repair results were observed and the cell source of repair tissue was distinguished. RESULTS: In the experimental group, the cartilage defects were repaired by the cartilage-like tissue after 8 weeks of operation; the defects were completely filled with mature cartilage tissue, which integrated smoothly with articular cartilage 16 weeks later. In the control group, only a small amount fibrous tissues were seen on the surface of defects. Autoradiographic assessment showed that the repair cells came from the implants, but not from self-chondrocytes. CONCLUSION: It is a good way to repair articular cartilage defects with homograft of tissue engineering cartilage. It is a convenient method to mark with 3H-TdR to discriminate the resource of the repair cells.
OBJECTIVE To investigate possibility of cartilage cultured in centrifuge tube as graft materials. METHODS: Articular chondrocytes isolated from a 3-week-old rabbit formed cartilage after cultivation for 2 weeks. Articular cartilage of humeral head, growth plate of proximal tibia and meniscus were collected from a 6-week-old rabbit. The ultrastructure of chondrocytes and extracellular matrix in the three kinds of cartilages and cultured cartilage were observed by transmission electronic microscopy. RESULTS: Cartilage cultured in centrifuge tube possessed unique ultrastructure and was similar to articular cartilage and growth plate, but it was markedly different from meniscus. The four kinds of cartilages were characteristic of respectively different chondrocytes and extracellular matrix. Cultured cartilage showed typical apoptosis of chondrocytes and "dark chondrocytes" appeared in growth plate. Condrocyte apoptosis was not seen in articular cartilage and meniscus. CONCLUSION: Cartilage cultured in centrifuge tube has unique ultrastructure and may be used as graft materials for articular cartilage and growth plate.
Objective To sum up the research advances of the seed cell and the culture system using in tissue engineering cartilage. Methods The recent original articles about the seed cell and the culture system in tissue engineering cartilage were extensively reviewed. Results At present, autologous or homologous cells is still major seed cell and the three dimensional culture system is also major system for tissue engineering cartilage. Conclusion The source of seed cell for tissue engineering cartilage. Conclusion The source of seed cell for tissue engineering cartilage should be further explored, and the culture system need to be improved and developed.
Objective To investigate the possibility of sheep joint cartilage defect repair with tissue engineered cartilage constructed by using porous bioceramics as scaffold and TGF-β induced autologous bone marrow derived mesenchymal stem cells(MSCs) as seed cell. Methods In the experimental group(n=12), autologous MSCs were isolated and expanded in vitro and then implanted into the pre molded porous β-TCP; the cell β-TCP complex was implanted into sheep right humeral cartilage defect. The defects in β-TCP (n= 12) group were repaired by B-TCP only, while defects in the control group (n= 4) were left un-repaired. Samples were extracted 12 and 24 weeks after operation for histological, histochemical and immunohistochemical analysis. Results In the experimental group, cartilage-like tissue formation could be seen on the surface of the implants. Microscopic analysis demonstrated obvious degradation of B-TCP and extensive new cartilage formation 12 weeks after operation, containing rich extracellularmatrix. The cells were stained positively with type II collagen. The bioceramics had almo st completely been degraded and abundant cartilage formation could be seen in the whole defects 24 weeks later. In the B-TCP group, marginal cartilage ingrowth could be seen 12 weeks after operation and the number of chondrocytes increasedmarkedly after 24wee s. However, no cartilage can be found in the middle of the material. In the control group, only a small quantity of new cartilage formation could be seenalong the margin of defects. Conclusion It is feasible to generate tissue engineered cartilage with porous B-TCP and auto logousM SCs for cartilage defect repair.