Change of TypeI and TypeII Collagen Biosynthesis by Growth Factors in Cultured Cells Isolated from Rabbit Intervertebral Disc

Lee, Jin Woo; Kim, Nam Hyun

Abstract

Growth factors influencing the function of chondrocytes are insulin-like growth factor I(IGF-I), basic fibroblast growth factor(bFGF), transforming growth factor-beta1(TGF-beta1), and epidermal growth factor(EGF). To find out the role of four kinds of growth factors in the biosynthesis of type I and II collagen represented as the phenotype of the disc cells, we cultured the disc cells isolated from rabbit intervertebral discs primarily and then checked cell proliferation, the expression of type I and II procollagen mRNA, and the immunohistochemical stains with type I and II collagen antibodies during in vitro culture in the maintenance medium containing low serum concentration with adding four kinds of growth factors. The results are as follows. FBS(10% Fetal bovine serum) group showed the highest cell proliferation potential. EGF and TGF groups showed remarkable cell proliferation, but there was no significant difference in IGF and FGF groups comparing to control group. A partial clone that encodes the rabbit type II procollagen C-propeptide region(RbCo12A1) was successfully isolated by reverse transcription-polymerase chain reaction using total RNA extracted from articular chondrocytes of rabbits. The identity of the cDNA clone was confirmed by DNA sequencing of the polymerase chain reaction products. A comparison of human al(II) cDNA sequence showed high sequence homology(83.6%). Type I procollagen mRNA expressed highly in EGF group. FGF, IGF, and TGF groups showed no significant expression comparing to control group. FBS group showed lower expression than control group. Type II procollagen expression was increased with passage of time, so at Day 10 it was the highest in all groups. Control group showed the highest expression among 6 experimental groups. The expression of type II procollagen in FGF and TGF groups was slightly lower than that of control. EGF and IGF groups showed markedly decreased expression comparing to control group. That in FBS group was the lowest, so it was three times lower than control group. In immunohistochemical stains with type I collagen, there was no difference among control, FBS, and EGF groups. FGF, IGF, and TGF groups showed increased positivity on stain comparing to control group, but the positivity didnt exceed 10%. For type II collagen, EGF and FGF groups showed decreased positivity, but there was no significant difference in FBS, IGF, and TGF groups comparing to control group. On the basis of this study, it may be concluded that TGF-pl showed the possibility of regeneration or delay the degeneration process of the intervertebral disc through the contribution to the stimulatory effects of cell proliferation and the synthesis of type II collagen. For the clinical use of this, more studies about the combination effects with FBS or other kinds of growth factors and finding out the ideal concentration about TGF-pl will be needed.