Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Toshihiro Izumi, Toshiyuki Tominaga, Junichi Shida1, Futoshi Onishi & Moritoshi Itoman Department of Orthopaedic Surgery, Kitasato University School of Medicine,
1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan

http://www.springerlink.com/content/g523121861680452/fulltext.pdf

Cartilage graft is a common technique applied in repairing chondral or osteochondral defects. One way of performing cartilage graft is by autologous chondrocytes transplantation following cell culture. However, one of the potential problems of this method is that chondrocytes may change their phenotype during monolayer culture. To solve this problem, the experiment presented in this research paper hypothesized that chondrocytes cultured over agarose (suspension culture) as a source of graft material can retain the expression of type II collagen and glycosaminoglycans, and the expression of these two products for chondrocytes cultured in a plastic glass (2D culture) is also measured as a control.

105 cells/cm2 of rat chondrocytes are isolated from coast cartilage and are dispersed in Ham’s F-12 medium supplemented with 0.2 mg/ml bovine serum albumin, 50 mg/ml ascorbic acid, antibiotics–antimycotic, 10% fetal bovine serum ,and 25mM HEPES, pH 7.4. The isolated cells were collected, and seeded on six well plate either uncoated or precoated with 1.5% agarose. Chondrocytes cultured in uncoated dishes represented the monolayer culture, while chondrocytes in coated dishes represented suspension culture. Cultures were then maintained in a humidified atmosphere consisting of 95% air and 5% CO2 at 37 oC. Cells were cultured for two weeks, and medium was changed twice a week. After 14 days in culture, the expression of glycosaminoglycans is examined by a histochemical test in which the chondrocytes are stained with safranin O (a biological stain used in histology). On the other hand, the expression of type II collagen is examined first by an acid guanidinehiocyanate–phenol–chloroform method for RNA extraction and following by northern analysis. The cultured chondrocytes are then transplanted to a rat with osteochondral defects.

The result of this study shows that chondrocyte phenotype was maintained in suspension culture over agarose, as assessed by northern analysis and histochemistry compared with chondrocyte monolayer culture. Moreover, this study shows that suspension culture chondrocyte grafts in femoral osteochondral defect also resulted in hyaline cartilage formation compared with controls in this study. Surface of the condylar defect was smooth, and the matrix produced in the defect contained glycosaminoglycans.

The reasons I choose this paper are because it is related to our project and may provide some clues to accomplish the objects in our experiment. Instead of concerning the mechanical properties of the chondrocytes / agrose cell culture, measuring the expression of collagen II of our chondrocytes after one week of culture is also the other big challenge in this project. This problem can get complicated as we do not know which portion of the collagen is from the calf serum and which portion is from the cells. Therefore, solely running the sample and a negative control in western blot cannot tell us whether the cells are expressing collagen II or not. The application of northern blot used in this paper suggests a possible way to deal with this problem since the mRNA of collagen II would only come from the chondrocytes. Nevertheless, we still have to come up with some ways to quantify the protein.