Masuda Koichi, Sah Robert L, Hejna Michael J, Thonar Eugene J-M A
Department of Orthopedic Surgery and Biochemistry, Rush Medical College, 1653 W. Congress Parkway, Chicago, IL 60612, USA.
J Orthop Res. 2003 Jan;21(1):139-48. doi: 10.1016/S0736-0266(02)00109-2.
Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.
大多数组织工程软骨的尝试都涉及将培养的细胞接种到生物或合成支架中。我们开发了一种新颖的两步培养方法，该方法使得成熟的成年牛软骨细胞在无需合成基质的情况下，能够在体外形成软骨样组织。第一步包括在维持细胞圆形形态及其分子表型（通过II型胶原蛋白和聚集蛋白聚糖的产生来评估）的条件下培养软骨细胞。这一步是通过将分离的软骨细胞培养在藻酸盐珠中实现的，直到细胞重新建立富含蛋白聚糖的细胞相关基质（CM）。第二步包括在从珠子中回收后，将带有CM的细胞在具有多孔膜的组织培养插入物上进行培养。在本研究中，将年轻成年牛关节软骨细胞在含有10%或20%胎牛血清（FBS）的藻酸盐珠中培养。培养7天后，通过在钙螯合剂柠檬酸钠缓冲液中孵育珠子20分钟来溶解藻酸盐珠。经过短暂离心后，回收带有CM的细胞，将其重悬于含有10%或20% FBS的培养基中，并接种到组织培养插入物上。在插入物上培养1周后，带有CM的单个细胞逐渐整合形成一团软骨组织。用20% FBS培养导致组织形成最佳。这些易于从插入物上回收的组织随后进行生化和组织学分析。生化结果表明软骨细胞在组织中保持表型稳定。新生组织具有相对较高的蛋白聚糖/胶原蛋白比率。组织学检查显示该组织含有被甲苯胺蓝强烈染色的软骨样基质。这种无支架系统似乎是在体外研究可移植软骨组织发育的理想选择。
