Nano-hydroxyapatite-chitosan Composite Scaffolds Combined With Peripheral Blood Mesenchymal Stem Cells Enhance Bone Regeneration In Rat Tibia Bone Defect Model

BackgroundVarious reasons,including congenital deformities,complex trauma,degenerative disease,and tumor resection may result in critical-sized bone defects,and spontaneous healing can't completely occur,and on the contrary the critical-sized bone defects often require large amounts of bone for reconstruction.Clinically available sources of bone for skeletal reconstruction are limited by several factors including accessibility and amount of available autologous grafts,immunological rejection of allograft,and durability of synthetic substitutes.All these drawbacks have fueled the search for an alternative approach to repair critical-sized defects.So far,bone tissue engineering has been a common method for repairing large bone defects and obtained great progress in recent years.It is a significant strategy that usually involves translating mesenchymal stem cells into three-dimensional porous scaffolds to facilitate new bone formation by osteogenic induction.Mesenchymal stem cells may serve as ideal seed cells in bone tissue engineering for several advantages including expansion in vitro,multipotential differentiation especially easily differentiating into osteoblasts and no immunological rejection.Owing to their strong proliferation and osteogenesis capacity,bone marrow mesenchymal stem cells(BMMSCs)have been commonly recognized as reliable cell source for repairing large bone defects.However,autogenous BMMSCs require an invasive procedure to harvest.This makes patients suffering,tissue damage and local infection inevitably.Furthermore,BMSCs have lower self-renewal and proliferative ability due to patient aging and diseases such as osteoporosis and arthritis.Comparatively speaking,peripheral blood mesenchymal stem cells(PBMSCs)are harvested by minimally invasive and relatively simple procedure,with more effective cost economic cost is low,thus may become an ideal substitute of BMMSCs.It has reached a consensus that single material is difficult to meet the needs of the bone tissue engineering scaffolds while composite material can overcome the shortcomings for each other.Nano-hydroxyapatite-chitosan(nHA-CS)composites can not only overcome brittleness and the poor fatigue resistance of the nano hydroxyapatite material,but also solve the problem of the natural polymer materials,such as poor mechanical properties,easy to dissolve.With high porosity,good mechanical properties,biodegradability,biocompatibility and osteoconduction,nHA-CS composites are widely used in bone tissue engineering due to the ability of facilitating cells adhesion and proliferation.In this experiment,we combinate nHA-CS with PBMSCs as a composite to fill into the tibia segmental defects in rats and evaluate the therapeutic effect of PBMSCs-nHA-CS composite on bone defect restoration in rats.Our findings will provide a theoretical basis for future treatment of long bone defect.PurposeThis research aimed to explore the effect of nano-hydroxyapatite-chitosan(nHA-CS)osteo-induced peripheral blood mesenchymal stem cells(PBMSCs)composites for repairing the bone defect area.MethodsIsolating and cultivating PBMSCs from rats by using density gradient centrifugation and adherent growth method,and in parallel amplification in vitro,thereafter detect the surface markers and osteogenetic differentiation potential of PBMSCs.After fabricate nHA-CS scaffolds through coprecipitation method,we use scanning electron microscopy(SEM)to observe their three-dimensional structure,the determination of the porosity.In addition,the degradation rate in vitro is also evaluated.Totally 42 rats were randomly divided into three groups,including the control group(no scaffolds),nHA-CS only and PBMSCs-nHA-CS group.The nHA-CS osteo-induced PBMSCs composites(nHA-CS+cells group)and the nHA-CS scaffolds(nHA-CS group)were implanted into a 5 mm bone defect of the left tibia in rats.At 8 and 12 weeks post-implantation,the defect healing were assessed by X-ray,microcomputerized tomography(micro-CT)and HE staining.All statistical analysis was accomplished by SPSS 20.0 software.Specific data is showed as mean±standard deviation(X±SD),while data among groups would use ANOVA analysis(One-way ANOVA),comparision among groups LSD method(least significant difference),if heterogeneity of variance,Dunnett'S T3 test method would be used;if the P-value<0.05,we can consider the difference is statistically significant.ResultsPBMSCs presented long fusiform morphology,and had the capacity of amplification after cultivation in vitro.Flow cytometry analysis revealed that the surface markers expression of PBMSCs were in accordance with MSCs.It was confirmed that by PBMSCs could differentiate into osteoblasts after osteogenesis induced in vitro because of positive expression of ALP and calcium nodule formation.The nHA-CS scaffolds exhibited three-dimensional porous network structure with bore diameter ranging 100?m from 200?m and high porosity that connected to each other.X-ray and Micro-CT analyses revealed that much greater bone formation was obtained for the PBMSCs-nHA-CS group compared to the nHA-CS and control group at each time point.The percentages of bone mineral density and bone formation volume in the PBMSCs-nHA-CS group was significantly greater than those nHA-CS and control group(P<0.05).Similarly,Histological examination indicated that more new bone was detected in the PBMSCs-nHA-CS group compared to the other two groups at each time point(P<0.05).ConclusionsThese results suggested that better repairing effect was obtained in the PBMSCs-nHA-CS than that in the nHA-CS group and control group.In addition,the nHA-CS scaffolds co-cultured with PBMSCs have fairly strong osteogenic capability and may serve as a promising substitute for regenerating bone defects.