Engineered Tendon With Hypoxic Pre-culturing Adipose-derived Mesenchymal Stem Cells And Small Intestinal Submucosa To Repair Achilles Tendon Defect Of Rats

Objective: In vitro studies have confirmed that hypoxia preconditioning adipose-derived mesenchymal stem cells(ADMSCs)can enhance its ability to survive and to tendon cell differentiation potential.The current research we planed to investigate the effect of using the engineered composite of ADMSCs-seeded small intestinal submucosa(SIS)to repair Achilles tendon defct in rats model and pre-culturing ADMSCs under hypoxia increases the effect of using the engineered composite to repair tendon defct.We hoped that this study would provide a new theory basis for treatment of tendon defect.Methods: ADMSCs were isolated from the adipose tissue the Sprague-Dawley rats.ADMSCs were purified and subcultured.Markers of the third passage ADMSCs were detected by the flow cytometer.The third passage ADMSCs were cultured under normoxic(20 % O2)or hypoxic(2 % O2)conditions for 7 days.ADMSCs which were cultured under normoxic(20 % O2)or hypoxic(2 % O2)conditions were labeled with CM-DiI.Then the cells labeled with CM-DiI were seeded the SIS.ADMSCs-seeded SIS was cultured for 48 h under normoxic(20 % O2).Select 50 male SD rats(250-300 g)as the experimental animal model.From the SD rats randomly selected 30 is divided into A,B,C three groups,and each group had 10 rats.The remaining 20 rats' left Achilles tendons were defined as a group D and on the right side of the Achilles tendo were defined as a group E.Group A was defined as the control group.B,C,D,E,four groups of rats' lateral band and postero-lateral band of the bilateral Achilles tendon were cut about 0.5 cm.Group B was defined as the tendon autograft group.Group C was defined as the SIS group.Group D was defined as the normoxic ADMSCs combined with SIS.Group E was defined as the hypoxic ADMSCs combined with SIS.The tendon autograft or the SIS(ADMSCs seeded on SIS)was sutured on the proximal and distal of the tendon defect with the modified Kessler' method.Put plaster cast on the legs of the all rats.After Four weeks,the tendon Graft would be observed macroscopically.In addition,the effect measures included biomechanical testing,histological analysis,and CM-DiI labeling/tendon markers immunohistochemistry.Results:(1)After Four weeks,HE staining of the sample: Group A showed that a small amount of long spindle cells could be found and the tendon collagen fibers are arranged density wavy toward a direction;Group B showed that a large number of fibroblasts could be found and the extracellular matrix sequence is not neat;Group C,group D and group E showed some spindle cells on the SIS in one direction along the extracellular matrix arranged regularly,and the percentage of long spindle cells in the group E were more than in group C and group D,but the long spindle cells number of group D is higher than that of group C.Besides C,D,E in the three groups,group E had the most extracellular matrix,but the extracellular matrix in group D is more than ingroup C.(2)After Four weeks,Masson staining of the sample: Group A showed that collagen fibers were blue,cytoplasm was red,nucleus was black and a small amount of spindle cells arranged along the direction of collagen fiber;Group B showed that a large amount of disordered arrangement,spindle cells could be found,and a large number of collagen fibers were arranged no rules deposited Outside the spindle cells;Group C,group D and group E showed that some spindle cells were aligned along the collagen fibers.In group C,D and E,group E had the densest collagen fibers,also their order were the best.But the group D deposition of collagen fibers and the order was better than that of group C.(3)After Four weeks,immunohistochemistry of the Group D and group E:There were a lot of Tnmd protein and Mkx protein expressed in the long spindle cells and oval cells both of the Group D and group E.The number of positive cells which expressed Mkx protein in group E was significantly higher than that of group D(P<0.05).(4)After Four weeks,ADMSCs tracer and immunofluorescence Mkx chemical staining of the Group D and group E :Group D and group E have a lot of ADMSCs,and the proportion of positive ADMSCs which expressed Mkx protein in group E was significantly higher than that of group D(P<0.05).(5)After Four weeks,biomechanical test: The ultimate force load in the group E was significantly greater than that in the group C or group D(32.34 ±2.71 N vs 20.33±1.47 N or 27.78±2.11 N,respectively),but the ultimate force load in the group C(20.33±1.47 N)was less than that of the group D(27.78±2.11N).The ultimate force load in thegroup E(32.34 ±2.71 N)was close to but still significantly less than that of the group B(37.62±1.54 N).Conclusion:(1)The outcome of using the engineered composite of ADMSCs-seeded SIS to repair Achilles tendon defct was better than that of using the SIS alone.(2)Hypoxia could promote ADMSCs compound SIS treatment effect for the treatment of tendon defect.(3)ADMSCs attached to the SIS were possible to differentiate into tendon cells,beside hypoxia might ehhance the effect of differentiation.