Characteristic Identification Of Amniotic Stem Cell Derived Condylar Chondrocytes

Objective: To use Transwell co-culture system to induce human amnion mesenchymal stem cells(HAMSCs) to differentiate into condylar cartilage cells, establish cartilage cell seed bank, and explore a new way for the treatment of temporomandibular joint osteoarthrosis.Methods: 1. The mechanical separation and two-step enzyme digestion method was used to separate mandibular condylar chondrocytes(MCCs) of newborn rabbits. The differential adhesion method was used to purify the cells. Morphological features of cells were observed by microscope. MCCs were immunohistochemically stained and identified with HE, toluidine blue and Type Ⅱ collagen. 2. MCCs and HAMSCs were respectively placed in the upper and lower chamber of Transwell culture dish with the ratio of 4:1, and induced and cultured for 7d, 14 d and 21 d. The cell morphology and cells after immunohistochemical staining, identification and induction with HE, toluidine blue and Type Ⅱ collagen were observed under inverted microscope. The average positive staining area was calculated.Results: 1. The primary generation of MCC cells was short-spindle-shaped and polygonal. They grew in the shape of paving stones and gradually gathered together into clusters. The cell growth cycle was about 10 d. The second generation of MCC cells was polygonal and triangular. The cytoplasm was stained purple, and the cell nucleus was stained blue. They were oval or round. One or two nucleolus could be seen. The cytoplasm was uniformly stained blue by toluidine blue, bluish violet metachromatic granules could be seen, and cell nucleus were stained blue. In the immunohistochemical staining with Type Ⅱ collagen, brown metachromatic granules of cytoplasm could be seen. The cell nucleuses were stained dark blue after counter staining. The growth cycle was shortened to 5d. 2. In Transwell co-culture system, the induced group was cultured for 7d. Cells in the lower chamber still had the shape of long spindle. After the immunohistochemical staining with toluidine blue and Type Ⅱ collagen metachromatic granules were sometimes seen. After 14 d of co-culture, Cells in the lower chamber had morphological diversity. After immunocytochemical staining with toluidine blue and Type Ⅱ collagen, metachromatic granules in the cells gradually increased. After 21 d of co-culture, Cells in the lower chamber were polygonal and similar to that of MCCs. Toluidine blue staining and Type Ⅱ collagen immunocytochemical staining was positive. No metachromatic granules were detected in the cells of the control group. 3. The average positive staining area of Type Ⅱcollagen was 35.8% for MCCs group. The average positive staining area of cells in the lower chamber in the induced group gradually increased with the extension of co-culture time. It reached 33.6% on Day 21, and had no significant difference with that of MCCs group(P>0.05). The average positive staining area of average positive staining was only 1.4%, which had significant difference with that of MCCs group and the induced group(P<0.05).Conclusion: 1. After enzyme digestion method was used for two times, high-quality MCCs could be obtained. 2. Transwell co-culture system can induce HAMSCs to differentiate into MCCs. 3. When Transwell co-culture system is used to induce HAMSCs to differentiate into chondrocytes, the best co-culture time is 21 d.