摘要
背景:软骨细胞体外培养时常发生失分化,合成糖胺多糖的能力下降,延缓软骨细胞的失分化速度是组织工程学需要解决的重要课题。目的:观察不同接种密度下,软骨细胞合成糖胺多糖的能力。设计、时间及地点:对比观察细胞学实验,于2007-01/05在山西医科大学第二医院骨科实验室完成。材料:1月龄新西兰兔5只。方法:采用0.4%Pronase酶和0.025%Ⅱ型胶原酶消化分离双膝关节关节软骨细胞,来源于同一只兔的软骨细胞分为两部分,一部分以2×104/cm2接种,传代时仍以相同密度接种。另一部分在细胞贴壁后,人工降低细胞密度至2×103/cm2培养。倒置显微镜下观察细胞形态和增殖情况。原代和传1代细胞于细胞融合后换液。主要观察指标:换液后12,24,36,48,60h以改良Alcianblue染色沉淀法测定糖胺多糖质量浓度。结果:原代高密度培养组关节软骨细胞为多边形,轮廓清晰,三四天即可见集落形成,集落周边细胞较中心瘦长,为长多边形,传1代细胞形态无明显变化。低密度培养细胞早期散在分布,7d左右形成集落,细胞形态与高密度培养无明显差异。原代低密度培养软骨细胞长到融合所需时间较原代高密度培养细胞所需时间长。原代低密度培养组上清液中糖胺多糖质量浓度显著低于原代及传1代高密度培养组软骨细胞(P<0.001,P<0.05),且时间越长,质量浓度相差越大。结论:与低密度培养相比,平面高密度培养可提高软骨细胞合成糖胺多糖的能力,明显减缓软骨细胞的失分化速度,提示高密度培养更有利于软骨细胞维持表型,是软骨平面培养的较好方式。
BACKGROUND： Chondrocytes may dedifferentiate when they are cultured in vitro, and the capacity of synthetizing glycosaminoglycan （GAG） is also reduced, how to delay the dedifferentiation of chondrocytes is a crucial topic in the field of tissue engineering. OBJECTIVE： To observe the performance of chondrocytes synthetizing GAG at different inoculum densities. DESIGN, TIME AND SETTING： A controlled cellular experiment was performed at the laboratory of Department of Orthopaedics in the Second Hospital of Shanxi Medical University between January 2007 and May 2007. MATERIALS： Five New Zealand rabbits of one month old were used in this study. METHODS： Articular chondrocytes were isolated from both knees and digested using 0.4% pronase enzyme and 0.025% Ⅱ type collagenase. The chondrocytes harvested from the same rabbit were divided into two sets, one was seeded at a constant density of 2× 10^4/cm^2 in primary and subculture, the other was cultured at a reduced density of 2× 10^3/cm^2 following cellular adhesion. Cellular morphology and proliferation were observed under inverted microscope. The culture media were renewed after the primary cells and passage 1 cells were confluent. MAIN OUTCOME MEASURES： GAG concentration was determined using the modified precipitation method with Alcian blue at 12, 24, 36, 48 and 60 hours following the renewal of culture media. RESULTS： Articular chondrocytes in the primary high-density culture group were polygonal with clear boundaries, they have shown to form colony at 3-4 days. Cells around colonies were more slender than those in the center of colonies, shaping as long polygon. There was no obvious change observed in the morphology of passage 1 cells. In the low-density culture group, cells scattered at early stage and formed colonies at 7 days, cellular morphology showed no significant differences in comparison with high-density culture group. The time of primary cells becoming confluent in the low-density culture group was prolonged compared with high-density culture group. The GAG concentration in supernatants in the primary cells of low-density culture group was significantly lower than that in primary cells and passage 1 cells of high-density culture group （P 〈 0.001, P 〈 0.05）. The GAG concentration showed a greater difference along with the prolonging of culture time. CONCLUSION： High-density culture is better then low-density culture to enhance the performance of chondrocytes synthetizing GAG and to retard the velocity of chondrocytes dedifferentiation, which suggests high-density culture contributes to maintain the chondrocytes phenotype and can be considered as a good way of plate culture.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第33期6589-6592,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
the National Natural Science Foundation of China, No. 30872616~~
