The Effects Of IGF-Ⅰ On The Three Dimensional Cultured Rabbit Articular Chondrocytes

Objective1.To explore and resolve the problem of the descend rate of proliferation and de-differentiation during the culture of the chondrocytes.2.To study the effects of IGF-I on the tendency of growth and de-differentiation of the three-cultured chondrocytes, in order to gain large amount of good type chondrocytes.3.To establish a stable system of three-cultured chondrocytes, to provide a foundenmental research for self-grafted of chondrocytes and treatments of the jury of bone and joint in the clinics and constitution engineering.Methods:1.The source of the chondrocytes :the chondrocytes are come from the joints of shoulders,knees and coxas of four weeks newsland rabbits, digested by machine and enzyme,and then, make them into cell suspension. 2.The monoplay and three-culture of the chondrocytes: firstly, the chondrocytes were monoplay cultured, then, the second generation of chondrocytes were transplanted into alginate three-cultured system.3.The detect of the effects of IGF-I on the cellproliferation of the chondrocytes A)The cellproliferation of the three cultured chondrocytes with different concertrations of IGF-I were dected by MTT.B) To draw cell growth curve,in order to detect the difference of the cell number between the different groups.4.Evaluation of the chondrocytes the form of the cell were observed by microscope and H-E stain, collagen II was observed immunologically.Results:1.The chondrocytes digested by machine and enzyme were successfully cultured in monolayer and three dimensional culture system. The three dimensional culture model were successfully established 2.The three dimensional cultured chondrocytes proliferated into mass state, the cell number of the group with IGF-I is higher than that without IGF-I.3.The oD of chondrocytes with different concentrations IGF-I was dected by MTT, the results shew that the oD of the group with IGF-I of 50ng/ml was higer than any other groups,its' promoting effect is the highest.4. Compared with the pre-seeding state of condylar chondrocytes, the harvested cells from alginate medium showed an equivalent level of expression of type II collagen after eight weeks,H-E stain shew that the form of the chondrocyte was round. The chondrocytes cultured in alginate system maintained the excellent differentiated phenotype.Conclusions:1.Different concentrations of IGF-I can all stimulate the proliferation and the formation of the mass,the best concentration is 50ng/ml.2.The chondrocytes cultured in alginate system with IGF-I maintained the excellent differentiated phenotype and keeped normal form and function of the normal chondrocytes. 3.The three dimensional culture system was successfully established, a large amount of good type chondrocytes were gained in three dimensional culture system with IGF-I. This provide a probility for a great number of seed cells.4.The proliferation rate of the monolayer cultured chondrocytes is faster than three dimensional cultured chondrocytes in short period. but the monolayer culture system can't keep the excellent cell phenotype, so it can't be used as a long-term cell cultured system. We can use the monolayer culture system firstly, and then, after several generations culture of chondrocytes, we can transplant the monolayer cultured chondrocytes into three dimensional cultured system in order to gain a large amount of good phenotype chondrocytes in a short period.