| Background:Tissue engineering is currently the world's hot field of cross-discipline in the area of medical and materials, and attached the world scholars's attention to rapid development. Currently in cell extraction, purification, proliferation and oriented induction so have seen a breakthrough. But in the world today the progress of tissue engineering scaffold development and cytology status can not meet. While the world invested heavily, but progress of tissue engineering is still slow. Present difficult problems mainly in the design and production of material microstructure. Since the early nineties of last century, Polymer foam processing technology innovation gradually. And can be processed to porous polymer foams with arbitrary shape and good biocompatibility. All of these has driven tissue engineering in the fields. However, this micro-structure can not fully meet the needs of cell and tissue growth. Its mechanical strength is not ideal, and its degradation products in the body prone to acid reaction. Is not yet reported using controllable micro-structure calcium phosphate porous bioceramics(Tricalcium phosphate and Hydroxyapatite) as a biological scaffold materials.Objective:This research used controlled pore structure ofβ-tricalcium phosphate porous ceramic material with good biocompatibility and high mechanical strength, and can better meet the needs of cartilage tissue engineering construct. This porous bioceramics with characteristics of controllability microstructure have high gas content, spherical pore distribution, the same size, the ball hole communication rate is 100%;spherical cavity wall smooth, the difference between male and female face is 0.2-1μm; each ball hole has 1 to 8 interconnection with the adjacent hole connected. This experiment used theβ-tricalcium phosphate porous ceramic composite cartilage cells in vitro for cartilage tissue engineering in order to find the right building bio-ceramic micro-structure of scaffold, and study for in vivo biological ceramic material. And this experiment established the articular cartilage defect model, and using in vitro bio-ceramic composite material repair cartilage defects in rabbit articular cartilage model, to get the bservation and assessment of restoration effects. In this way, we get experimental basis, so as to further clinical application in patients with articular cartilage defects.Method:Step 1:2 months old New Zealand rabbits were selected, embolism death by venous injection of air. We have carefully stripped rabbit ear cartilage and knee joints cartilage and digested isolated adult rabbit articular cartilage cells and auricle cartilage cells in vitro. By this method, we observed these two kinds of cartilage cell production rate, survival, adhesion rate, morphological changes, proliferation rate and we carried out staining of cartilage phenotype, measured the content of GAG and optimized for cartilage cells in vitro and in vivo tests. Step 2:We use rabbit auricle cartilage cells combined with different micro-structures bio-ceramic materials cultured in vitro, calculated the proliferation of cartilage cells in the scaffold the growth of different trends and optimized suitable microstructure for the growth of rabbit auricle cartilage cells inβ-TCP bioceramics scaffolds. Step 3:We sintered the appropriate micro-structure ofβ-TCP bioceramic scaffolds for cartilage tissue engineering. Step 4:We combined this scaffold with rabbit auricle cartilage cells, then implanted into rabbit articular cartilage defect model.Results:(1) Survival and adherence rates of these two kinds of cells are as follows:articular cartilage:digestion time 6.49+1.87h, cell production rate 5.79±1.7×105/100mg, survival rate 87.20±9.11%,24h adherence rate 70.23±8.17%. Auricle cartilage:digestion time 4.60±0.31h, cell production rate 5.57±0.54×105/100mg, survival rate 83.83±3.12%,24h adherence rate 72.16±7.44%. The P valus of these two cells'access rate, survival rate,24h adherence rate is 0.259,0.05,0.83, and these are not statistically significant is the figure. The digestion time of auricle chondrocytes was lower than articular cartilage cells, and the P value is 0.002. This may be because the auricle cartilage is more soft than the articular cartilage and the cell matrix surrounding is aslo more loose. (2) We used MTT to detect cell proliferation, cell growth curve. Passage cells of articular cartilage cells showing strong growth and proliferation, and passed into the logarithmic phase after After a brief plateau period. The 1st-4th generation cells have no significant changes in the growth curve. The 5th generation of cell growth ability started to diminish and the 6th cells proliferation was markedly reduced. Passage cells of auricle cartilage cells showing strong growth and proliferation, and passed into the logarithmic phase in the vaccination day. The 1st-4th generation cells have no significant changes in the growth curve. The 5th generation of cell growth ability started to diminish and the 6th cells proliferation was markedly reduced. Particularly the 2nd generation of cartilage cell proliferation is the strongest, and there is no incubation period in the growth curve. (3) Porous structure ofβ-TCP porous bioceramic composite materials can be better combined with chondrocytes for cartilage tissue engineering and cultivation, and can maintain the chondrocyte phenotype for a long time. The micro-structure of intercnnection diameter 100μm and pore diameter 530-600μm more adapted the auricle cartilage cell culture. Cartilage cells in the environment of porous micro-structure can be maintained the phenotype to 4th generation. (4) Usingβ-TCP bioceramics composite cartilage cells compared with the control group to confirme that Chondrocytes combined withβ-TCP bioceramics composite structure of tissue engineered cartilage can repair the articular cartilage defects model. Auricle cartilage cells and Articular cartilage cells combined with the micro-structure of intercnnection diameter 100μm and pore diameter 530-600μm can better maintain the cell phenotype and extracellular matrix update speed. We used D'Driscoll method to accordance the cartilage morphology score. These three groups of control group, articular cartilage cells group and auricle chondrocytes group score followed by increased. The score is control group 5.25±0.88, articular cartilage cells group 18.37±1.40, auricle chondrocytes group 19.12±1.45. Articular cartilage cells group and auricle cartilage cells group both has statistically significant difference with control group. There are no statistically significant difference between articular cartilage cells group and auricle cartilage cells group.Conclusion:1, Auricle cartilage cell dissociation method is simple and digestion time is less than articular cartilage cells. Researcher can get plenty of the seed cells of tissue engineering for a short-term.2, Auricle cartilage cells grew faster and there are no significant difference in the first two generations of auricle cartilage cells in the secretion of glycosaminoglycan than the original cells. It is the application of cartilage tissue engineering seed cells.3, Porous structure ofβ-TCP porous bioceramic composite materials can be better combined with chondrocytes for cartilage tissue engineering and cultivation, and can maintain the chondrocyte phenotype for a long time.4, Micro-structure has some impact on the maintenance of chondrocyte phenotype. The micro-structure of intercnnection diameter 100μm and pore diameter 530-600μm more adapted the auricle cartilage cell culture.5, cartilage cells combined withβ-TCP bioceramics constructed tissue-engineered cartilage can repair of articular cartilage defects. 6, Auricle cartilage cells and articular cartilage cells combined with the micro-structure of intercnnection diameter 100μm and pore diameter 530-600μm can better maintain the cell phenotype and extracellular matrix update speed.
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