The Effect Of Tendon Stem Cells Transduced With Sox9 On Tendon Bone Healing

Objective1.To compare the capacity of tendon stem cells(TSCs)and bone marrow stem cells(BMSCs)to promote tendon-bone healing in order to screen for better seed cells that promote tendon-bone healing.2.Sox9 was transduced into TSCs to build the Sox+/Scx+ cells,after transplantation,the tendon-bone healing effect was observed.Methods1.TDSCs and BMSCs were isolated as previously described in our team and we identified them by FCM and Immunofluorescence.The proliferation rates of TSCs and BMSCs were compared using the CCK-8 method.RNA of third-generation TSCs and BMSCs was extracted,and the expression of osteogenesis-,tenogenesis-,and chondrogenesis-related genes was determined using q RT-PCR.For in vivo animal studies,24 Sprague Dawley rats were divided into three groups: the TSC group,the BMSC group,and the cell-free control group.Stem cells and fibrin gel were mixed together and injected into the interface between the Achilles tendon and the bone in rats.Rats were sacrificed at weeks 4 and 8.The presence of heterotopic ossification at the tendon-bone junction was detected using microCT.The repair of the tendon-bone interface was detected using Alcian blue and Picrosirius red staining.2.We chose a suitable MOI according to the pre-experiment to have the transfection.Three groups were divided: TSC group,Lv group and Sox9 group.RT-PCR,WB and IF were used to compare the expression level of Sox9 in each group;The proliferation rates were compared using the CCK-8 method.We observed the transfection effect on differentiation by culture cells in a higher density,WB test and immunofluorescence technique.In vivo studies,96 Sprague Dawley rats were divided into four groups: the cell-free control group,the TSC group,the Lv group and Sox9 group.Cells and fibrin gel were mixed together and injected into the interface between the Achilles tendon and the bone in rats.Rats were sacrificed at weeks 4 and 8.Micro CT and in vivo fluorescence imaging technique were used to evaluate biological safety of lentivirus transfection.The repair of the tendon-bone interface was detected using Alcian blue and Picrosirius red staining.Biomechanical properties were compared by a mechanical test system.Result1.TSCs and BMSCs look similar.Both TSCs and BMSCs exhibited positive staining for the MSC marker CD90 but negative staining for the hematopoietic cell marker CD34 and the endothelial cell marker CD31.The proliferation rate of TSCs was significantly higher than that of BMSCs.The expression level of Scx in TSCs was significantly higher than that in BMSCs,whereas the expression levels of osteogenesis-related genes(Alp L and Runx2)were higher in BMSCs.The expression levels of Sox9,an important transcription factor in chondrogenesis,was not significantly different between these two cell types.At week 8,specimens from rats in these three groups exhibited heterotopic ossification.The heterotopic ossification level in the TSC group was lower than those in the other two groups,but there was no significant difference among the three groups.Staining of tissue sections from the tendon-bone junction using Alcian blue showed that at week 4 and 8,more fibrocartilage structures were formed in the TSC group than in the BMSC and the control groups,and these differences were statistically significant.Tissue sections of specimens from weeks 4 and 8 were stained with Picrosirius red,and the repair of collagen at the interface between the tendon and the bone was observed under a polarizing microscope.The results showed that the new type Ⅱ collagen in the TSC group was significantly more mature than that in the BMSC group and the control group.2.GFP was observed by confocal microscope 72 hours after transfection.The results of RT-PCR,Western-blot and immunofluorescence showed that the expression of the Sox9 rise;CCK-8 results suggested that there was no difference among the groups at each time point.We cultured the cells in a higher density and found that the cells in Sox9 group and Differentiation Induction group aggregated since day7.WB and immunofluorescence showed that in Sox9 group Col Ⅱ,a cartilage associated protein,expresses increasingly.The degree of heterotopic ossification in Sox9 group is significantly higher than that in control group.We see a small amount of fluorescence signal in the heart of Lv group,while the fluorescence signal in the rat liver,heart and kidney of Sox9 group is stronger.In the week 2,there is no significantly difference in fibrocartilage formation between the Sox9 group and the control group,while,in the week 4,the difference is significant.The maturity of collagen in Sox9 group in tendon-bone interface in the week 2 and 4 are significantly higher than that in the control group,also,the maximum load and the tensile strength of the Sox9 group are significantly higher than those in the control group in the week 2 and 4.Conclusions1.The cells we isolated from Achilles tendon and bone marrow were identified as stem cells.2.TSCs proliferate faster than BMSCs in vitro and this may lead to a better tendon-bone healing.3.In vivo,the healing effect on tendon-bone interface of TSCs is better than that of BMSCs.4.The lentivirus mediated Sox9 overexpression vector is successfully transfected into TSCs.5.After transfection,cells in Sox9 group express more Col Ⅱ protein and have a tendency to chondrogenesis.6.Cells in Sox9 group have a stronger power to reconstruct the tendon-bone interface and a better biomechnical strength,however,the possibility of heter otopic ossification and transferring to important organs increase as well.