The Study Of Tissue-engineering Bone Constructed By Surface Mordified Nano-hydroxyapatite /PLGA Composites

ObjectTo assess the feasibility and reliability of bone rabbit marrow-derived mesenchymal stem cells combined with PLGA/g-HA Scaffolds in construction tissue-engineering by vitro and vivo experiments, and make support for future clinical application.Methods(1) Isolation,culture condition and identification of Rabbit BMSCs:bone marrow mesenchymal stem cells(BMSCs) were obtained from 1d-aged White New Zealand rabbits' tibial and demoral bones. After harvesting the suspension of bone marrow, we isolated BMSCs by centrifugation over Percoll separated medium and cells were cultured at 37℃in a 5%CO2 air atmosphere. After two passages,the cells were cultured in osteoblast and adipogenic media and take respective staining identification.Till the three cell passages, cells were detached using trypsin and reseeded in 96-well culture plate.The density of BMSCs was regulated to 4×103cells/well.At 1d,3d,5d,7d,9d after plating, growth curve was drawed by microplate spectrophotometer.(2) The research of the biocompatibility of PLGA/g-HA composite material:The proliferation rate of BMSCs cultured under leaching liquor with different concentrations(10%,30%,50% and 80%) were detected by MTT assay. Meanwhile, the cell adhesion and morphology were observed with scanning electron microscopy. PLGA/g-HA scaffolds were cut into 10 mm×5 mm×5 mm in size, and extracts of the material in DMEM for 48 hours. Then obtain leaching liquor with four concentrations,10%,30%,50% and 80%, followed by reserving at 4℃after filtration. PLGA/g-HA scaffolds were resized for square in 20 mm×20 mm×0.5 mm, treated with ethanol for sterilization and reserved after UV lighting for 24 hours.BMSCs were seeded onto the surface of the prewetted scalffolds sized 20 mm×20 mm×0.5 mm. At 4h,8h,12h,24h, the amount of adherent cells were counted by trypsin digestion method. At 3d, the morphology of the adherent cells were observed by scanning electronic microscopy. (3) The experiment of using tissue-engineering bone to repair bone defect:Fifteen adult male rabbits (2.5-3.Okg) were initially included in this part for preparation of bone defect model in radius. Chose the three cell passages cells and adjusted the density of BMSCs to 1×106/ml.Then inoculated 100μl cell suspension on PLGA/g-HA and PLGA materials. Before implanting, we cultured in vitro for three days. Group management and intervention:according to different scaffolds, there were totally five groups, A; BMSCs seeded on PLGA/g-HA; B:BMSCs seeded on PLGA scaffold; C:PLGA/g-HA scaffold Only; D:PLGA scaffold Only; E:blank control group; Setting time points:post operation,4w, 8w,12w.Results(1) BMSCs fundamental state:Following primary harvest, BMSCs were in round shape and the structure of celluar nuclear was undentificable. Till 24h, the adherent cells started to stretch and became polygonal or spindly.At cultured 2-5d, some cells gathered as colony-forming unit fibroblast (CFU-F) and extension radially. The growth curve was appeared in S shape.From the curve we could tell that at the first 2 days BMSCs were in adaptive phase, then entered in logarithmic phase at 3rd day, at 7th day went into platform for growth. Soon afterwards, the growth rate started to decrease and maintain present status.(2) Corresponding staining identifications:BMSCs were induced to osteoblast demonstrated positive staining in Dye alizarin red, alkaline phosphatase and Von Kossa staining. Dye alizarin red:alizarin red staining was used to evaluate cells mineralization and mineralization nodes were showed in red; Alkaline phosphatase (ALP):With activity of osteogenic, the nuclear will appreaed in lavender and reddish brown particles in cytoplasm; Von Kossa staining:The color of calcium phosphate mineral were expressed obviously over times. Adipogenic differentiation of BMSCs demonstrated positive in Oil Red-O staining. Oil Red-O staining:On the first 7d, orange lipid droplets could be observed. The numerous intracellular lipid droplets pushed nuclear to one side.(3) Cells proliferation in PLGA/g-HA leaching liquor:From the measured data, there was no statistical significance between 10% group and negative group,30% group (P>0.05). There were statistical difference between 50%,80% group and negative group (P<0.01). However, all the cytotoxic grade were in Grade 1 fulfill our national standard. (4) Cell adhesion and morphology on the surface of PLGA/g-HA:BMSCs could stretch pseudopodium gradually and adherent on the surface of PLGA/g-HA firmly. And with prolonging cultur time, the amount of the adherent cells were showed increasing trend.(5) General conditions of animals:All the experiment animals were survived and awoke after 1h post-surgery. Most ones behaviored normal as pre-operation. There were two animals limped at 2-4d after operation and recovered in 5 days. The incisions were no obvious infection or inflamed phenomena and got primary healing.(6) X-ray assay:Post operation, all the fracture line were obviously clear. At 4w, there were plenty callus and the fracture line turned obscure, except E group. The amount of callus is higher than other fours in A group. Furthermore, all scaffolds were stable without any shifting. At 8w,the length of bone defect in the five groups were shorter than 1.5cm.And the fracture healing degree continued to carry different rates. At 12w, the newly formed bony callus were full of defect area in A group.C group rank second to A. The worst condition happened in E group.Conclusions(1) BMSCs can proliferate well in vitro which isolated by Percoll separated medium, growth in stable condition and maintain the powerful differentiation potential. It proved that BMSCs were candidate seed cell for bone engineering, because of its accessible harvest and proliferation.(2) PLGA/g-HA composite material had well biocompatibility and cellular adhersion that fulfilled the need of biomaterial in tissue-engineering bone. In our vitro study, BMSCs can proliferation in the surface of PLGA/g-HA composite material.(3) PLGA/g-HA scaffold appeared better osteoconduction than PLGA scaffold. In addition, the groups which combined with BMSCs demonstrated advantages in repairing bone defect. In future biomaterial area of bone repairing, PLGA/g-HA scaffold may be worth of being generalized.