The Effect Of Vascular Endothelial Growth Factor Combined With Cells-Scaffold Compound For The Repair Of Rat Femoral Bone Defects

Bone defect on clinically is very common.The therapy of bone defect is one of the tough problems in department of orthopaedics.There are two different treatments:expectant treatment and surgical treatment.Expectant treatment includes changing external fixation,fixing the fracture absolutely and extending the fixation time.Surgical treatment includes fracture position cleaning,internal fixation,implant biomaterial.The commonly used materials of the therapy are autogenous bone materials, variant bone materials and artificial bone materials.Along with the development of the study of implant materials,tissue-engineering become an investigative hot spot at present.Tissue-engineering utilizes the philosophy and technique of life science and engineering.It is through scheming, constructing,cultivating and conserving living cells to study and research organism surrogate,to recover and rebuild the constitution of human tissue and organ,to maintain or improve the functions of tissue and organ.Now it is focuses on three aspects:seed cells,implant materials and construct modes.Objective:This paper is to evaluate the effect of vascular endothelial growth factor (VEGF) combined with the compound of marrow mesenchymal stem cells (MSCs) and nano-hydroxyapatite/collagen(nHAC) bone on the treatment of rat femoral bone defects. Methods:Rat MSCs were isolated and purified in vitro,and the rat MSCs of 3rd-generation were seeded onto prepared nano-hydroxyapatite/collagen (nHAC).Their adhesion situation was analyzed by scanning electron microscope(SEM) after 5,10 days.Animal models of femoral bone defect were established in 30 Sprague-Dawley rats.They were divided into 3 groups randomly:In group A,as the control group,defect was filled with nHAC;in group B with nHAC/MSCs;in group C with VEGF/MSCs/nHAC.Animals are executed in batch postoperative 2,4,8,12 weeks.General conditions, macroscopic evaluation,X-ray,the bone density measurement and histomorphology examination of bone defect repairing sample and statistical analysis were performed in order to compare the effects of repairing bone defects of these groups.Environment scanning electron microscope(ESEM) observation was made 12 weeks after operation.The animal disposal was consistent with the ethical requirements.Results:1.Rat bone marrow mesenchymal stem cells(MSCs) grow adherent.The speed of generation is fast.The growth curve is fit for Logistic growth curve.2.Rat bone marrow mesenchymal stem cells(MSCs) and nano-hydroxyapatite/collagen(nHAC) are cultivated in vitro,the scanning electron microscope(SEM) is used to observe at 5^th,10^th day.It can be seen that MSCs cell population adhered onto nHAC at 10^th day (38.52±7.21) are obviously more than at 5^th day(21.28±4.70),P＜0.01.3.All rats awaked in 3 hours after the operation.All the general state of health of Sprague-Dawley rats was fine.The postoperative recovery and food intake are normal.There is no inflammation present in the wound. The concrescence is fine.4.Macroscopic evaluation:The bone defection of group C was completely repaired at postoperative 12 weeks.The prosthetic condition of bone defection was obviously better than group A,B those were partly recovered.5.Radiographic and radiographic scoring:In group C,there were few callus formations in the radial defects in 2 weeks;evident bridges grafting was observed in the defects in the 8th week;the defects were filled with newly formed bone completely 12 weeks.In group A,B,there were more callus formations in 12 weeks,but no bridges grafting was observed and there was osteosclerosis in the two ends.Bone nonunion can be seen in 2 samples of group A by the end of 12 weeks.Radiographic scoring of group A in 2,4,8,12 weeks are 1.51±0.16,1.87±0.20,2.16±0.33, 3.86±0.30.Group B are 2.30±0.18,2.87±0.16,3.25±0.48,5.60±0.29. Group C are:2.68±0.17,4.15±0.37,5.43±0.31 7.63±0.35.There were significant differences in new bone formation between every two groups in 2,4,8 and 12 weeks after operation(P＜0.05) 6.The bone density measurement:the bone density of group A postoperative 2,4,8,12 weeks are 0.448±0.111,0.872±0.361, 1.192±0.109,1.233±0.056 respectively;group B are 0.773±0.075, 1.227±0.078,1.496±0.394,1.736±0.169;group C are 0.862±0.013, 1.476±0.076,1.820±0.070,2.187±0.188.Differences every two groups in 2,4,8 and 12 weeks after operation are significant(P＜0.01).7.Histomorphology:There were hyperemialymphocytes and lots of spindle cells in every group at 2^nd week after operation.At 4^th week after operation there are lessen inflammatory reaction and new capillaries can be seen in the implant materials.New osteoid formation was obvious in group C.At 8^th week after operation,the implant scaffold degradation was incompletely.We found osteoblasts in group B and woven bone tissues in group C.At 12^th week after operation,the implant scaffold disappeared totally.The bone regeneration of group A was less than other groups and there were few hyperemia and necrotic tissue in some samples.Mature osteocytes and new discus bone can be seen in group B,mixing construction of discus bone and lamellar bone can be seen at bone defects of group C.8.The Environment scanning electron microscope(ESEM) results showed that there were rigidity bone fibers and bone lacunas in group A;bone trabecula structure with voids in group B;regular bone trabecula construction and Havers' system in group C at 12^th weeks after operation. Conclusion:According to the experimental results,we can make the conclusion:the conjunctive extent of the rat bone marrow-derived mesenchymal stem cells (MSCs) and nano-hydroxyapatite/collagen(nHAC) cultivated together in vitro is high.According to it,we can construct tissue-engineering bone.The bone defect repair ability of tissue-engineering bone which constructed with VEGF and cells-scaffold compound of MSCs and nHAC is stronger compared with the compound of MSCs and nHAC or nHAC alone.It can well repair segmental bone defects.