Chondrogenic Differentiation Of ASCs Cultured On The Collagen Scaffold Treated With GDF-5 For Repairing Intervertebral Disc Degeneration

PurposeThe intervertebral disc is a fibrocartilage-like organ with few cells and poor self-renewal ability. Extracellular matrix mainly including aggrecan and collagen constructed the cells scaffold. As the intervertebral disc degeneration occured, the nucleus pulposus water content fell, the type II collagen and aggrecan content decreased, chondrocyte apoptosis and the cartilage endplate calcification occured, intervertebral disc degeneration aggravated gradually. Therefore, searching for seed cells, scaffold and cytokine and forming complex to repair early intervertebral disc degeneration have been an attractive subject in spinal research.Adipose-derived stromal/stem cells (ASCs) can differentiate into different phenotypes reflecting their potential to regenerate various skeletal tissues including adipocyte, osteoblast, myocyte and chondrocyte. Collagen type I with the characters of no antigenicity, no toxic metabolite, no impact on cell proliferation and good biocompatibility has been commonly used in tissue engneering. Growth and differentiation factor-5 (GDF-5) were demonstrated to promote the aggrecan and collagen type II synthesis of intervertebral disc cell in vivo and in vitro.These properties formed the basis of our study to establish an experiment for using ASCs to undergo chondrogenic differentiation in vitro under the influence of GDF-5.In this study, we hypothesized and conducted that GDF-5 promoted the differentiation of ASCs into chondrogenic lineages in vitro. We observed biological effects of GDF-5 in the presence and absence of different concentrations on chondrogenic differentiation of ASCs cultured on collagen scaffolds and established the experiment foundation of the repairing intervertebral disc degeneration.Methods1. Study of the isolation culture and biological characteristics of rabbit ASCs.a. Adipose of Japan white rabbit was density gradient centrifuged, collagenase digestion and cultured in plastic culture flask.b. Immunohistochemistry and immunofluorescence analysis of cell surface markers CD₄₄, CD_49d and CD₁₀₆ in ASCs.c. The cells were examined by invert microscope and the growth curve was drawn according MTT.2. Effects of GDF-5 on chondrogenesis of the ASCs.a. The morphological change of GDF-5 (range, 0 to 100 ng/ml) on chondrogenesis of the ASCs at 7th, 14th and 21 st day.b. Alcian blue staining, toluidine blue staining and collagen type II immunohistochemistry staining were applied for the induced ASCs at 14th day under the microscope.c. Gene expression of collagen type II and aggrecan on treatment with the different concentration of GDF-5 at all the time points was detected by RT-PCR.3. Chondrogenic differentiation of rabbit ASCs treated with GDF-5 cultured on the type I collagen scaffoldsa. The ultrastructural architecture of the scaffolds and cellular attachment were revealed by scanning electron microscopy or light microscopy.b. The gene expression of aggrecan and collagen type II, secreted by the ASCs cultured on the collagen scaffolds was detected by RT-PCR.Results1. Success of the isolation culture of rabbit adipose derived stem cells.a. After density gradient centrifugation, the obtained cells mostly presented spherical shape. As the cells were adherent to the flask, they had similar long-spindle morphology and tightly arrayed to grow, showed no morphological changes in passage culture.b. Immunohistochemistry and immunofluorescence analysis of cell surface markers in MSCs showed that the positive expression results of CD₄₄, CD_49d and the negative expression results of CD₁₀₆. The negative results of marker of waveshape protein excluded the interference of fibrobast.c. Growth curve showed proliferation ability of ASCs was similar at passage 3 and passage 6.2. Effects of GDF-5 on chondrogenesis of the ASCs.a. We investigated the dose-dependent effects of GDF-5 (range, 0 to 100 ng/ml) on chondrogenesis of the ASCs at 7th day. The gene expression of collagen type II and aggrecan highly increased upon treatment with 100ng/ml GDF-5 at all time points and attained to the peak at 14th day.b. Alcian blue staining positive strongly was observed in the part of cells aggregation. Toluidine blue staining and collagen type II were positive in the cytoplasm at 14th day.3. Chondrogenic differentiation of rabbit ASCs treated with GDF-5 cultured on the type I collagen scaffoldsa. The ultrastructural architecture of the cell scaffolds and cellular attachment were revealed by scanning electron microscopy or light microscope. The extracellular matrix was observed on the surface of the cells adhering to the scaffolds at 14th day.b. The gene expression of aggrecan and collagen type II, secreted by the ASCs cultured on the collagen scaffolds increased significantly.Conclusion1. The combination of density gradient centrifugation and adherent culture is an effective method to isolate ASCs from rabbit adipose. ASCs have the characters of stable growth and high proliferation ability.2. After the ASCs in monolayer culture and cultured on collagen type I scaffold were treated with GDF-5, the expression content of aggrecan and collagen type II increased obviously and these induced cells possessed the part biological function of chondrocyte. The good biological compatibility between collagen type I scaffold and ASCs was demonstrated simultaneously.