The Experimental Study Of MSCs Seeded On Vancomycin Loaded Scaffolds For Treating Infected Radius Defect In Rabbits

Background and Objective:Infected bone defects are common caused by trauma or war high-energy injured fracture. The treat strategy aims at elimination of infection as the first and major objective and bone union as the next objective because of infection. The inflammation is characterized by a predominant presence of leukocytes and macrophages, which contribute to the destruction of bone tissues.Vascular channels are compressed and obliterated by the inflammatory process. The destruction of vascular channels leaves a portion of dead and infected bone (sequestrum) that is detached from the adjoining healthy bone and surrounded by avascular soft tissue. Owing to the impaired vascularity, antibiotics may not be delivered adequately to the lesion by the intravenous route. The local delivery of antibiotics is an effective option for treating infection. The most common of these is the incorporation of antibiotics into bone cement (polymethylmethacrylate (PMMA)) or a biodegradable carrier as slow release system .These materals can eat many types of infections,but They can't repair bone defects.The best release system can not only control bone infection, but also repair bone defects.Bone tissue engineering technigue is the best selection to repair bone defects.Scafflds are the important element of tissue engineering.Antibiotic loaded scafflds can not only release local antibiotic but also be attached by seeding cells.In this experiment,MSCs were isolated and inducted from rabbits in vitro and seeded on the vancomycin loaded collagen DBM scafflods to control bone infection and repair bone defects. and provide a potential treatment procedure for infected bone defects.Methods:1.The original generation of MSCs were isolated from bone marrow of rabbits by density gradient centrifugation and adherent culture.Culture-expanded were idetified with osteogenic, chondrogenic and adipogenic differentiation. 2.MTT assay, alkaline phosphatase (ALP) activity and reverse transcription polymerase chain reaction (RT-PCR) were used to observe the influence of different concentrations of vancomycin (0μg/ml,100μg/ml,200μg/ml,400μg/ml,700μg/ml,1000μg/ml)and delayed release vancomycin on the proliferation and osteoinductive differentiation in vitro.3.The vancomycin loaded collagen DBM scafflods were made and the morphology, release characteristics of vancomycin and bacteriostasis ability were observed.4.Mesenchymal stem cells seeded on the vancomycin loaded scaffolds .The MSC/scaffold constructs were cultured in vitro and the biocompatibility was detected using MTT assay, alkaline phosphatase (ALP) activity and scanning electronic microscopy (SEM). The cellar oncogenicity of the MSCs on the the vancomycin loaded scaffolds was observed.5.To investigate the in vivo anti-infection and osteogenesis of the MSCs/ vancomycin loaded scaffolds, the biomaterals were implanted in rabbit infected radii bone defect and studied by macroscopic , C-reactive protein (CRP), erythrocyte sedimentation rate(ESR), X-ray radiographs, the histologic images and biomechanics.Results:1.Four days of MSCs of original generation,the fibroblast-like cell colony was observed and MSCs have fulfilled the bottom of the monolayer cultural bottle in 9-12 days,while the passage cells having done in 4-7 days.After the osteogenic induction, most of cells were polygonal and clustered. ALP staining was strong positive. Calcareous infarct could be detected by tetracycline dying in cell clump and stained red by alizarin red. After the adipogenic induction,most of cells were stained positive by oil red. After the chondrogenic induction,most of cells were stained positive by alcian blue.2.The results of MTT assay, alkaline phosphatase (ALP) activity and reverse transcription-polymerase chain reaction (RT-PCR) of osteogenic genes showed that different concentrations of vancomycin (0μg/ml,100μg/ml,200μg/ml,400μg/ml,700μg/ml,1000μg/ml)and delayed release vancomycin have no effect on MSCs proliferation and osteogenic differentiation3.The release of vancomycin from the vancomycin loaded scaffolds was good . The release of vancomycin and bacteriostasis ability can be sustained for 32 days.4.The morphology of MSCs attached onto the scaffolds was evaluated using SEM shows that cells have dramatically reproduced and aggregated with each other to form stratified cell layers, accompanying with filamentous fibers formed on the surface. Statistical analysis indicated significant difference in the cell number between MSCs seeded on the vancomycin loaded collagen DBM scaffolds and empty DBM scaffolds through MTT assay. the level of ALP activity increased substantially on he vancomycin loaded collagen DBM scaffolds. Statistical analysis indicated that there was significant difference between all groups (p<0.05).5.The caryotype of MSCs that were seeded on the vancomycin loaded collagen DBM scaffolds and cultured on them for 10 days was nomal,the clone formation experiment showed negative and the tumorgenesis experiment of nude mouse showed also negative.6.The MSCs/ vancomycin loaded scaffolds and vancomycin loaded scaffolds control infection and facilitate new bone formation. Moreover, the introduction of MSCs to vancomycin loaded dramatically enhanced the efficiency of new bone formation.The vancomycin PDLLA/β-TCP could control infection but could not repair bone defects.The empty collagen DBM scaffolds could not control infection.Conclusions:1.The MSCs of third generation obtained from dissociating red marrow by density gradient centrifugation and adherent culture have matched the MSCs features in aspects of appearance.The MSCs can successfully be inductived to osteoblast, chondrocyte and adipocyte.The MSCs of third generation have high purity and considerable quantity and are good seeding cells in bone tissue engineering.2.Different concentrations of vancomycin (≤1000μg/ml)and delayed release vancomycin have no effect on MSCs proliferation and osteogenic differentiation .3.The vancomycin loaded collagen DBM scaffolds have good ability of release of vancomycin and bacteriostasis .They are good local delivery system. the vancomycin loaded scaffolds are biocompatible and have no negative effects on the MSCs in vitro.The MSCs on the vancomycin loaded collagen DBM scaffolds can grow dramatically and keep osteogenesis.The MSCs have no tumorgenesis.4. The MSCs/ vancomycin loaded scaffolds and vancomycin loaded scaffolds control infection and facilitate new bone formation. The vancomycin loaded scaffolds have the potential to be applied in orthopedic to treat infected bone defect.