Bone Marrow Stromal Cells Modified By Bone Morphogenetic Protein-2 Gene Combined Nano-hydroxyapatite/Collagen Composite Scaffold For A Rabbit Lumbar-Fusion Experiment

PrefaceSpinal fusion has been one of the most popular procedures performed in spine surgery,however,some clinical investigations have shown a considerable rate of failure to achieve a solid bony union.The most common clinical approach for preventing nonunions has been the use of internal fixation,which provides the initial stability and mechanical environment for bony fusion.Despite the improved mechanical strength of the recent spinal instrumentations,spinal bony fusion is the objective.Traditional spinal fusion procedures use autogenous bone as a graft to provide the osteoinductive and osteoconductive components necessary for the formation of new bone at the operative site.Although autogenous bone graft is though to be a gold standard in the achievement of solid spinal fusion,there is frequently an inadequate supply of autogenous bone graft for performing multilevel spinal arthrodesis.In addition,the morbidity of autogenous bone graft harvest is reported to be as high as 30%,with the most frequent complications including chronic donor site pain,nerve injury,infection,fracture,hematoma,and increased operative time.The healing of a spine fusion is a multifactorial process,the donor site morbidity,limited supply,and imperfect success rate of autogenous bone graft has alerted researchers to search for new suitable alternatives.The ideal autograft alternative is a material that may be used entirely in place of autogenous bone graft to achieve the same or a better fusion success rate.The rationale underlying the use of a matrix/stem cells/growth factor combination as an alternative to autogenous bone is that the osteoconductive and osteoinductive components of such a combination will attract,support,and simulate host osteogenic cells to form new bone tissue.Currently,there has been a considerable clinical interest in the use of matrix/stem cells/growth factor combination for bone regeneration and osseous graft supplementation therapy.Accordingly,the current study introduced collagen based composite (nano-hydroxyapatite collagen,nHAC) as a scaffold to carry adenovirus-mediated bone morphogenetic protein-2(BMP-2) transfected rabbit bone marrow stromal cells (BMSCs) in vitro,and evaluated its effect on the relative success of gene therapy by means of a rabbit spinal-fusion experiment.Materials and Methods1,Adenovirus-mediated bone morphogenetic protein-2(BMP-2) transfected rabbit bone marrow stromal cells(BMSCs) in vitro(1) Production of the Adenoviral VectorTwo different adenoviral constructs were prepared for this study: Adenovirus-Lacz construct(Ad-Lacz) and adenovirus BMP-2 construct(Ad-BMP-2). 293 cells were incubated in Dulbecco's Modified Eagle Medium(DMEM) containing 10%fetal bovine serumand.Ad-BMP-2 were purified and detected by PCR.Titers were determined by standard plaque assay.(2) Ad-BMP-2 transfected rabbit BMSCs in vitroRabbit BMSCs were isolated and cultured by the method of complete bone borrow culture.BMSCs were identified by inducting multipotent differentiation.BMSCs differentiated into osteoblasts were assessed by Von Kossa staining to observe the mineralized nodules and standard alkaline phosphatase(ALP) assay.Adipocytes differentiation of BMSCs was assessed by Alizarin Red-S staining.Ad-BMP-2 transfected rabbit BMSCs.mRNA and protein expression of BMSCs was detected by BMP-2 hybridization in situ and immunohistochemical technique.Ad-Lacz transfected rabbit BMSCs in the same way.X-gal staining was used to detect the transfection rate and Western blot analysis was used to determine if BMSCs infected with Ad-BMP-2 produced BMP-2 protein.(3) The effect of BMP-2 gene transfection on BMSCs proliferation and differentiationThe change of BMSCs cell cycle after transfection was observed by flow cytometry.Alkaline phosphatase activity and staining,immunofluorescence analysis of osteocalcin and collagen I were used to analyze the effect of BMP-2 gene transfection on BMSCs proliferation and differentiation.2,BMSCs modified by BMP-2 gene was planted into the scaffold of nano-hydroxyapatite collagen(nHAC) to construct tissue engineering bone in vitroBMSCs transfected by Ad-BMP-2 were collected after they were trypsinized with trypsin.Then the BMSCs were planted into the scaffold of nHAC equably and were cultured at 37℃in a humidified atmosphere with 5%CO₂.The adhesion and growth of cells on the nHAC scaffold were observed by scanning electronic microscopy(SEM).Energy spectrometer was used to detect the secretion of calcium around cells.Western blot analysis was used to determine the expression BMP-2 protein.3,BMSCs by Ad-BMP-2 Gene transfer combined nHAC scaffold for a rabbit Lumbar-Fusion Experiment(1) Surgical ProcedureSixty mature New Zealand white rabbits(weight 2.0-2.5kg) were divided into five groups:BMSCs transfected by Ad-BMP-2 with nHAC group(group A),BMSCs not transfected by Ad-BMP-2 with nHAC group(group B),nHAC group(group C), autologous bone group(group D) and control group(group E).Rabbits were anesthetized by abdominal injection of 1.5 ml/kg of 3%sodium pentobabital.After being placed in the prone position in sterile fashion,an anterior approach by posterior peritoneal was made,followed by developing to expose the vertebral body and intervertebral space of L4-5 or L5-6.After removing the nucleus pulposus,cartilage and annulus fibrosus and endplates,graft materials were placed and fixed.Penicillin was intramuscularly injected on postoperative day 3 for antibiosis.(2) Radiographic EvaluationThe L5-L6 spines from each group animals were examined by lateral plain radiographs sequentially at 4,8,and 12 weeks after surgery.Fusion and location of new bone formation were observed.(3) Biomechanical TestingBiomechanical testing to evaluate the solidity of the L5-L6 fusion site was performed by a three-point flexion-bending test.The biological character of fusion segment was compared and accounted.(4) Gross and Histological AnalysisThe specimens of the spinal segments of all the animals were harvested at 4,8,12 weeks postsurgery.They were inspected grossly first,and then sent for light microscopic examination.In the laboratory,they were stained with hematoxylin and eosin(HE stain),and observed under a light microscope to examine for bony fusion. HE-stained specimens were analyzed by computational photogrammetry and the pixels of new form bone were calculated by image-analysis software.Immunohistochemical stain analysis was used to observe the expression of BMP-2.(5) Statistical AnalysisSPSS11.5 for windows was used for statistical analysis.Comparisons in each group were made using one-way analysis of variance.Significance for all tests was defined as P＜0.05.Results1,Part 1 (1) Determination of Ad-BMP-2DNA-PCR analysis showed that 1.2 kb gene segment was observed in Ad-BMP-2 tranfected group,whereas in Ad-Lacz transfected group or 293 cells not transfected group there were none.(2) BMSCs cultureAfter primary culture was sustained for 24-72 hours,the round cells displayed a polygonal morphology.At day 7-10 of the culture,the rate of proliferation accelerated to form the colony with approximately 80%confluency and at day 14-21,the cells grew circinately and fused to sheets.The cultured cells appeared to grow more rapidly after passages were done.(3) Determination of BMSCsThe cultured cells proliferated with cobblestone-like appearance after mineralized fluid was added into the medium,more dispersed compact round conglomeration could be observed among the cells.Von Kossa staining showed black mineralized nodule among the cells and standard alkaline phosphatase(ALP) assay displayed the positive brown-black stain in plasma.After adipocytes induction,orange-red stained fat globelet was observed by Alizarin Red-S staining.The cultured cells were identified as BMSCs because of the multipotent differentiation to blastocytes and adipocytes.(4) BMP-2 expression of BMSCs transfected by Ad-BMP-2Positive X-Gal staining was observed in histologic sections with more than 90% of tranfection rate.In the hybridization in situ and immunocytochemical analysis,we observed positive signal in the plasma of transfected BMSCs.In the Western blot analysis,positive signal lane of BMP-2 was observed in transfected group,whereas in the control group no signal was seen.It was proved that Ad-BMP-2 transfected BMSCs efficiently with high expression of mRNA and protein of BMP-2 gene.(5) The effect of BMP-2 gene transfection on BMSCs proliferation and differentiationFlow cytometry showed there was no difference of cell proportion of G1 phase and S phase between tansfected BMSCs group and the control group,which indicated that the synthesization of cell DNA and proliferation was not effected by transfection. Alkaline phosphatase activity of transfected cells was significantly higher than that in the control group.Positive green flurescent substance in plasma by immunofluorescence analysis of osteocalcin and positive expression of collagenâ… by immunocytochemical analysis indicated that transfected BMSCs differentiated to osteoblasts.2,Part 2BMSCs transfected by Ad-BMP-2 adhered to the surface and hole of the nHAC scaffold and grow well by scanning electronic microscopy(SEM) observation.Energy spectrometer detection showed that higher calcium content than that in the control.Positive expression BMP-2 protein were detected by Western blot analysis in Ad-BMP-2 transfected BMSCs group while negative in Ad-Lacz transfected BMSCs group and non-transfected BMSCs group,indicating that BMSCs transfected by Ad-BMP-2 was not effected by the nHAC.3,Part 3Radiographic evaluation,gross and histological analysis showed that all specimens were graded as nonunion in group E,complete fusion in group A,B,D and partial fusion(75%) in group C at 12 weeks.Computational photogrammetry demonstrated that the area of new formation bone in group A was obviously higher than that in group B,C.There was no difference on the area of new formation bone between group A and E,which indicated that bone fusion of group A is the same as the autografts.Biomechanical testing was performed to compare the stiffness of the specimens.The results showed that they were not significantly different between group A and E.Immunohistochemical analysis of BMP-2 showed strong positive reaction in group A and brown-yellow granule distributed in mesenchymal cells,chondrocytes, osteoblasts and bone cells,while weak positive reaction was observed in group B and C. It indicated that BMSCs transfected by Ad-BMP-2 could express BMP-2 persist and stably.Conclusions1,Adenovirus-mediated BMP-2 gene could be transfect to rabbit bone marrow stromal cells with high level transfection rate and expression.2,The proliferation of BMSCs transfected by Ad-BMP-2 was not effected by transfection,BMSCs could express gene products and differentiate to blastocytes.3,nHAC scaffold could facilitate bony ingrowth and biocompatible,which was in favor of the adhesion,growth and proliferation of trans-gene cells.Trans-gene cells could express BMP-2 and induct the differentiation to blastocytes of BMSCs.4,BMSCs transfected by Ad-BMP-2 could express extrinsic gene and promote the differentiation to blastocytes of planted BMCSs and bony fusion in rabbit.5,BMSCs transfected by adenovirus-mediated BMP-2 Gene combined collagen based composite(nHAC) scaffold was a new substitute of autografts with bioactivity of blastocytes induction.