| Background:Tendon/ligament injuries are common in both the workplace and in sportactivities. Tendon is relatively cell-poor with insufficient capacity forself-repair/healing; frequently result in some complications such as scar tissueformation or calcification, which have spurred a demand for the development oftissue-engineering strategies for tissue replacement. Of particular interest in recentyears is the use of Bone Marrow derived Mesenchymal Stem Cells (BMSCs) toregenerate functional tendons. However, ectopic bone formation aftertransplantation of BMSCs has been reported in some cases. There was alsoevidence of tumor induction by undifferentiated BMSCs in some specificcircumstances. So we suggest that growth factors induce the tenogenicdifferentiation of BMSCs in vitro, and then construct stem cells tendon alternativebiomaterials for tendon repair/regeneration.Objective:1. To isolate and characterize BMSCs.2. To test the tenogenic effect of different growth factors on the tenogenicdifferentiation of BMSCs in vitro and define the optimal condition fortenogenic differentiation of BMSCs in vitro.3. To construct stem cells tendon alternative biomaterials in vitro by thetenogenic process of BMSCs.4. To promote tendon regeneration by stem cells tendon alternative biomaterialsconstructed by tenogenic process of BMSCs in vitro in a rat acute patellartendon injury model.Methods:1. Density gradient centrifugation method isolated BMSCs from greenfluorescent protein transgenic rat. The isolated BMSCs were characterized by flowcytometry and osteogenesis, chondrogenesis, adipogenesis differentiationassay.2. BMSCs at passage3were cultured with different concentrations of Growthand Differentiation Factor6(GDF-6), Growth and Differentiation Factor7(GDF-7) and Connective Tissue Growth Factor (CTGF). After cultured for2weeks in vitro, the mRNA expression of scleraxis and tendomodulin weretested by quantification real time PCR, and the optimal concentration of eachgrowth factor was defined. The protein expression of tendomodulin, tenascinC and type I collagen were tested by immunocytochemical staining, and theprotein expression of tendomodulin were tested by western blotting, thendefined the optimal condition for the tenogenic differentiation of BMSCs.3. BMSCs at passage3were cultured under the optimal condition for2weeks.The stem cells tendon alternative biomaterial was constructed in vitro and thentransplanted into nude mouse subcutaneously. After12weeks, the biomaterialsamples were collected for histology.4. Animal model: SD rat patella tendon window injury model. The BMSCs, stemcells tendon alternative biomaterials were transplanted respectively intotendon injury site and the control group were not transplanted any cells orbiomaterials. After2,4and6weeks post operation, the patella tendon sampleswere collected for histology. After4weeks post operation, the patella tendonsamples were collected for biomechanical test.Results:1. The flowcytometry results showed that isolated BMSCs were positive forCD90,CD44, negative for CD31,CD34. The multi-differentiation potentialassay showed that isolated BMSCs had osteogenesis, chondrogenesis andadipogenesis abilities.2. The quantification real time PCR, immunocytochemical staining and westernblotting results showed that CTGF (25ng/ml) and GDF-6(20ng/ml)significantly increased mRNA expression of tenomodulin,scleraxis(p<0.05),and also the protein expression of tenomodulin,tenascin C and type Icollagen.3. Histology results showed that the stem cells tendon alternative biomaterialsformed tendon like tissue in the nude mouse. The collagen fibers were red staining and dense, the cells were alignment with collagen fibers.4. Histology results showed that the healing outcome in the stem cells tendonalternative biomaterials group was better than other groups. The cellularitywas decreased, extracellular matrix production was increased and the collagenfibers were more parallel. Biomechanical test results showed that the ultimatestress and young's modulus in the stem cells tendon alternative biomaterialsgroup were significantly higher than that of BMSCs and control groups(p<0.05).Conclusions:1. The isolated BMSCs have the multi-differentiation potentials and express thespecific markers of mesenchymal stem cells.2. Growth and Differentiation Factor6(GDF-6), Growth and DifferentiationFactor7(GDF-7) and Connective Tissue Growth Factor (CTGF) can promotethe tenogenic differentiation of BMSCs in vitro, furthermore, CTGF(25ng/ml)and GDF-6(20ng/ml)have better effect on the tenogenicdifferentiation of BMSCs in vitro.3. The BMSCs can form a cell sheet after induced by CTGF(25ng/ml)in vitro,and then use it to construct the stem cells tendon alternative biomaterials invitro. This biomaterial can form tendon like tissue in nude mouse model.4. The stem cells tendon alternative biomaterials constructed by tenogenicprocess of BMSCs in vitro can promote tendon repair in a rat acute tendoninjury model. |
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