Inductive Efiects Of Exogenous Hyaluronic Acid On Difierentiation Of Bone Marrow Derived Mesenchymal Stem Cells

1. ISOLATION, CULTURE AND PROLIFERATION OF BONE MARROW DERIVED MESENCHYMAL STEM CELLS (MSCS) IN VITROObjective:To explore the approaches to isolate, culture and proliferate the bone marrow derived mesenchymal stem cells (BMSCs) of rabbits.Methods:5.Oml of bone marrow was aspirated from one-month-old rabbit. Mscs were isolated using percoll-density centrifugation and/or repurified by cell attachment. Light microscopy was used to study the morphologic features. Cell surface markers were identified by flow cytometry. antigens. The growth curve of Mscs was obtained based on the cells proliferation of the1st,3rd.5th,7th generations.Results:Mesenchymal stem cells were purified by density gradient centrifugation and/or cell attachment. They growing outward in a swirl ing pattern with the same shape in the3rd and5th generations. Mscs proliferated more rapidly than those of7th generation or latter. Cell surface markers of adherent cell were detected as CD44(+),CD90(+), while CD34(-).Conclusion:Purified Mscs were obtained from rabbit bone marrow cells via this protocol. A strong proliferation ability was shown especially after Mscs were cultured in vitro. Bone marrow derived mesenchymal stem cells were verified as suitable feed cells for further research. 2. THE INFLUENCE OF EXOGENOUS HYALURONIC ACID ON PROLIFERATION AND DIFFERENTIATION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLSObjective:To Investigate the role of the intra-articular environment to the Mscs by studying the influence of exogenous hyaluronic acid (hyaluronic acid, HA) on proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (Mesenchymal stem cells, Mscs), and to basis the clinical applications for Mscs-based tissue-engineered cartilage.Methods:The rabbit Mscs were isolated and cultured in the method of whole bone marrow and adherent culture. The4th passage cells were used for the experiment. The experimental group cells were induced by HA solution (with0.25mg/ml HA+HG-DMEM supplemented with10%FBS), with TGF-β3induced group as a positive control. The negative control group was joined the regular medium. The features of chondrocytes were identified by toluidine blue staining, immunohistochemistry and RT-PCR to detect the expression of collagen II after7,14,21d's induction respectively.Results:After induced by HA, the speed of cell proliferation slowed down. The cell morphology changed from long spindle to polygonal, oval. The extracellular matrix showed metachromasia with toluidine blue and positive with type Ⅱ collagen immunohistochemical staining. RT-PCR detection also showed the expression of type Ⅱ collagen mRNA. All the above showed the characteristics of cartilage cell differentiation. But their expression was weaker than the positive control group.Conclusion:The exogenous HA can induce the rabbit Mscs differentiate to chondrocytes. But its capacity is weaker than TGF-β3. The results indicated that the intra-articular environment played a positive role in chondrogenic differentiation of Mscs and supported that HA could be used as a matrix for cartilage tissue engineering. 3. THE EFFECTS OF EXOGENOUS HYALURONIC ACID IN DIFFERENT CONCENTRATION ON CHONDROGENIC DIFFERENTIATION OF RABBIT BONE MARROW DERIVED MESENCHYMAL STEM CELLSObjective:To investigate the effects of exogenous hyaluronic acid (HA) in different concentration on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (Mscs).Methods:The un induced Mscs in passage3were divided into four groups with different supplement:Group A:basic medium+HAO.1mg/ml;Group B:basic medium+HAO.2mg/ml;Group C:basic medium only (negative control);Group D:basic medium+TGF-β310ng/ml(positive control). The morphologic features and proliferation of Mscs were observed with light microscope. The growth curves were made based on the data obtained above. The chondrogenic differentiation was identified by immunohistochemistry and RT-PCR to detect the expression of collagen Ⅱrespectively.Results:The speed of cell proliferation decelerated after HA was added. The cell morphology changed from long spindle to polygonal, oval. The extracellular matrix showed metachromasia with toluidine blue and positive with type Ⅱ collagen immunohistochemical staining.The mRNA expression of type Ⅱ collagen were determined by RT-PCR as an index of chondrogenic differentiation of MSCs. There is no statistic difference between expression of type Ⅱ collagen of MSCs cultured with HA in different concentration. Though both of them were lower than positive control.Conclusion:The exogenous HA induces chondrogenic differentiation of MSCs regardless of concentration. HA will be known as not only an essential component of extracellular matrix but a chondrogenic inducer. Lower concentration of HA cannot decelerate The chondrogenesis of MSCs, which encourage tissue engineering applications of MSC in chondrocyte defects, as the natural environment in the joint is favorable for chondrogenic differentiation. 4. THE EFFECTS OF EXOGENOUS HYALURONIC ACID WITH DIFFERENT MOLECULAR WEIGHT ON CHONDROGENIC DIFFERENTIATION OF RABBIT BONE MARROW DERIVED MESENCHYMAL STEM CELLSObjective:To study the effects of exogenous hyaluronic acid (HA) with different molecular weight on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (Mscs).Methods:The un induced Mscs in passage3were divided into five groups with different suplement:Group A:basic medium+HAO.1mg/ml(average molecular weight1000kD);Group B:basic medium+HA HAO.1mg/ml(average molecular weight1800kD);Group C:basic medium+HA HAO.1mg/ml (average molecular weight2000kD); Group D:basic medium only (negative control);Group E:basic medium+TGF-β310ng/ml (positive control). The morphologic features and proliferation of Mscs were observed with light microscope. The growth curve were made based on the data obtained above. The chondrogenic differentiation were identified by toluidine blue staining as well as immunohistochemistry and RT-PCR to detect the expression of collagen Ⅱrespectively.Results:The speed of cell proliferation decelerated after HA was added. The cell morphology changed from long spindle to polygonal, oval. The extracellular matrix showed metachromasia with toluidine blue and positive with type II collagen immunohistochemical staining. The mRNA expression of type II collagen were determined by RT-PCR as an index of chondrogenic differentiation of MSCs. There is no statistic difference between expression of type II collagen of MSCs in Group B and Group C. However, each experimental group shows difference with control group. No expression of type II collagen were observed in negative groups.Conclusion:The exogenous HA with different molecular weight can induce chondrogenic differentiation of MSCs with different ability. Hyaluronic acid with higher average molecular weight enhance The chondrogenesis of MSCs effectively.But all of HA shows weaker ability of inducement than TGF-β3. The improvement of molecular weight of hyaluronic acid in exocellular environment helps chondrogenesis of MSCs. 5. EFFECT OF AUTOLOGOUS BONE MARROW DERIVED MESENCHYMAL STEM CELLS INDUCED BY EXOGENOUS HYALURONIC ACID ON REPAIRING KNEE JOINT CARTILAGE DEFECT OF RABBITSObjective:To explore the effects of the autologous bone marrow-derived mesenchymal stem cells (MSCs) indudced by exogenous hyaluronic acid on repairing of the full-thickness articular cartilage defect, and to investigate whether the mesenchymal stem cells can or cannot keep the cartilage phenotype which were induced by exogenous hyaluronic acid in vitro and promote cartilage repairing.Methods:Twenty-four four-month-old Japanese rabbits were made the model of the cylindrical full-thickness articular cartilage defect (5 mm width and4mm depth) by hand drill. The rabbits were divided into four group by the different filler in the injured articular cavities:Group A(n=6) calcium alginate gel mixed with MSCs which were induced by exogenous hyaluronic acid; Group B(n=6) calcium alginate gel mixed with un-induced MSCs; Group C(n=6) calcium alginate gel only and Group D(n=6) untreated. The macromorphology and histologic scores were made respectively after5weeks,8weeks and12weeks of operations. Chondrocytes morphology were observed by HE staining and the expression of collagen IIwas detected by RT-PCR.Results:Cartilage defects in Group A and Group B were repaired better than other groups after8weeks of operation. The repairing tissue combined with normal cartilage tightly than control. There was statistic difference in histological scores between MSCs groups with negative or blank controls while Group A and Group B are in the same. The repaired cartilage also showed better histological results in Group A and Group B after12weeks of operation.It connected with normal tissue smoothly and tightly.No obviously border were observed as well. Group A showed the best statistically repairing result than any others. The histological observation showed that there were similarly hyaline cartilage chondrocytes in group A while fibrous cartilage chondrocytes in Group B. Group C and Grouop D were repaired with fibrous tissue only. The expression of collagen II was only detected in Group A and Group B. It was better in Group A than Group B.Conclusion:MSCs induced by exogenous HA in vitro keeps chondrocytical shape and function well in rabbit. It shows cartilage repairing capability in vivo. Un-induced MSCs differentiate into chondrocytes spontaneously in joint cavity with a weaker repairing capability than induced MSCs. All the repairing effects of MSCs improves as time going.