Mandibular Condylar Cartilage And Femoral Head Cartilage Study Of Proliferation And Differentiation In Vitro

Condylar cartilage and growth plate cartilage of femoral head are two different types of cartilage, abnormal cartilage occurred during the growth period will cause the mandible and long bone growth disorders. One unique characteristic of the condylar cartilage is that the cells in the proliferative layer have multilineage potential. Under a physiological conditions, progenitor cells differentiate into chondrocytes. however, under non-functional conditions, such as in vitro organ culture or under excessive tensile loading, progenitor cells undergoing chondrogenic differentiation are replaced by intramembranous bone. Hyaluronic acid can repair articular cartilage layers, maintaining no calcification of cartilage thickness and function of protection of the cartilage damage. TGF-β can promote chondrocyte proliferation, cell differentiation, extracellular matrix synthesis.This project will study the characteristic of proliferation and differentiation on condylar cartilage and femoral head cartilage in vitro. And study effect of the hyaluronic acid, TGF-β1 on proliferation and differentiation of cartilage, provides some theoretical supports about clinical studies on differentiation of mandibular condylar cartilage and related illnesses.Methods:Neonatal mice were decapitated and the explants(mandibular condyles and femoral heads) were processed for tissue culture in vitro. The gross observation, HE staining, Alizarin Red Staining, Alkaline phosphatase staining, Col X immunohistochemistry and Ki67 immunohistochemistry were conducted with the two type of cartilage.Results:1. Mandibular condylar cartilage proliferative cell layer and mature cell layer undergo hypertrophic differentiation, spontaneous calcification of matrix and endochondral ossification in vitro.After in vitro culture 4 weeks, a higer density light radiopaque area appeared in the condyle cartilage. Until the end of 8 weeks, the higer density light radiopaque area occupied most of the condylar cartilage and the condylar cartilage tissue grossly osteoblastic changes. However, the surface area of condyle cartilage didn’t change during after in vitro culture 4 weeks(P>0.05). The hematoxylin-eosin staining showed mandibular condylar chondrocyte in higer density light radiopaque area happened hypertrophic differentiation, Alizarin red staining confirmed the occurrence of calcified matrix.The alkaline phosphtase staining and TypeⅩcollagen immunohistochemical study further showed the condylar chondrocyte became hypertrophy and is going through the process of endochondral bone formation. ki67 immunohistochemistry showed chondrocytes still have proliferation activity after in vitro culture 8 weeks, confirmed the feasibility of in vitro methods.2. The surface area of femoral head cartilage significantly increased during cultivating in vitro, but chondrocyte didn’t became hypertrophy.After in vitro culture 8 weeks, femoral head cartilage didn’t appeared higer density light radiopaque area, but cartilage area measurements showed the surface area of femoral head cartilage significantly increased during cultivating(P<0.05). The hematoxylin-eosin staining showed femoral head cartilage cell volume increased markedly. Alizarin Red Staining showed there is no calcification occurred in the femoral cartilage matrix. Apart from the hypertrophy zone in cartilage, there is non-alkaline phosphatase and type x collagen expression. Ki67 Immunohistochemistry showed chondrocytes proliferation activity after in vitro culture 8 weeks.3. The surface area of condylar cartilage and femoral head cartilage significantly increased during cultivating in HA group, but had a inhibitory effect on condylar chondrocyte hypertrophy differentiation.After in vitro culture 8 weeks, condylar cartilage and femoral cartilage in the Hyaluronan group didn’t appeared higer density light radiopaque area.The surface area of condylar cartilage and femoral head cartilage significantly increased during cultivating(P<0.05). The hematoxylin-eosin staining showed condylar cartilage proliferative zone and fibrous layer became thinner, mature layer cells increase in size. Alizarin Red Staining showed there is no calcification occurred in condylar cartilage. Apart from the hypertrophy zone in cartilage, there is non-alkaline phosphatase and type x collagen expression. The hematoxylin-eosin staining showed femoral head cartilage cell volume increased significantly after in vitro culture 8 weeks. Apart from the hypertrophy zone in cartilage, Alkaline phosphtase staining and TypeⅩcollagen immunohistochemical study showed there is no expressed in other cartilage layer. Ki67 Immunohistochemistry showed both of chondrocytes still have proliferation activity after in vitro culture 8 weeks.4. The surface area of femoral head cartilage significantly increased during cultivating in TGF-β1 group, but hypertrophic differentiation of condylar cartilage occurred in advance.After in vitro culture 2 weeks, the higer density light radiopaque area in condylar cartilage in the TGF-β1 group was occurring earlier. After culture 8 weeks, the surface area of condylar cartilage didn’t significantly increased(P<0.05), but compared with blank group, the surface area of femoral cartilage significantly increased after culture 8 the weeks(P<0.05). The HE staining, Alizarin red staining, alkaline phosphatase staining and type x collagen immunohistochemical confirmed condylar chondrocyte happened hypertrophic differentiation and is going through the process of endochondral bone formation.Conclusion:1. Condylar cartilage and femoral head cartilage have different growth characteristics in vitro. condylar chondrocytes going through hypertrophic differentiation, spontaneous calcification of matrix and endochondral ossification in vitro.2. The characteristics of condylar cartilage proliferation differentiation maybe a useful research tool for the study of condylar cartilage development related disease mechanism.3. hyaluronic acid markedly promote the proliferation of condylar cartilage and femoral head cartilage in vitro, but had an inhibitory effect on chondrocyte differentiation.4. TGF-β1 plays an important role in femoral head cartilage proliferation and mandibular condylar chondrocyte hypertrophy differentiation in the early days.