Combination Of Rabbit BMSCs Cell Sheet And Nano Biomimetic Scaffold For Functional Tubular Bone Tissue Engineering

BackgroundBone tissue engineering research hold great potential for treating bone defect caused by trauma(burns),inflammation and tumor.The main objective of the study was to tissue engineer the functional bones which have the mechanical force and function of normal bones.In recent years,the vast progress of the cell seeding technology and scaffold materials research have made functional bone tissue engineering possible.Currently,the construction of bone tissue engineering still follows the basic model that single cell suspension were seeded into scafolds directly differentiate into bone in vivo.Moreover,the application of engineered bone in clinical encountered numerous technical problems.These problems consisted of the large-scale amplification of seed cells in vitro,the selcetion of the scaffold materials, the effective and uniform combination of the seeded cells and the scaffold materials,the central support large piece of tissue engineering bone,the optimal cultivation techniques,degradation of the scaffold, vascularization of the tissue engineering bones in vivo,the construction of the tissue engineered bones of different forms,etc.The reconstitution of functional bones with suitable mechanical force in vitro is the key points of the bone tissue enginerring research.ObjectiveThe rabbit(from 2 weeks to 8weeks) BMSCs(bone marrow stromal cells) were used as seeded cells in this study,and the electric nano-biomimetic material PCL-CO(polycaprolactoone-collagenâ… ) was used as scaffold materials.The cell sheet technique will transformed the BMSCs membrane and PCL-CO to tubular functional bones in the rotary bio-reactor.The tubular functional bones were implanted subcutaneously in the nude mice.Diffrent analysis from the mechanical testing,histological section,immunohistochemistry to molecular biology were applied in study.The research results would play an important role in bone defects repair and functional tissue engineering area. Methods1.Rabbit BMSCs were purified and amplified with the density gradient centrifugation and adherent culture method.Cells were well characterized by cell growth,cell morphology and multi-induction potential and so on.Passage of 2-3 cells were directly differneitated into osteoblasts,adipocytes and chondrocytes.2.Characterization of the electric spinning nanofiber PCL-CO's basic structure,biocompatibility through cell viability testing,electron microscopy,laser scanning confocal microscope,etc.3.The compound of the cell sheet of the rabbit BMSCs and the electrospin PCL-CO nanofibers were contructed,differentiated and dynamically cultivated in rotary tissue reactor for 4 and 8 weeks. Utilization of histological section,HE stain,alizarin stain, safranine-O/fast green stain and immunohistochemistry to detect the expression of collagen typeâ… ,collagen typeâ…¡,OCN and OPN. Samples were underwent biomechanics analysis as well.4.Implantation the in vitro engineered tubular bones subcutaneously in the nude mice and observation of regional tissue reaction.After 4 and 8 weeks implantation,samples were analysised by histological section and immunohistochemistry as well as biomechanics test.Results1.Protocol of the combination of the density gradient centrifugation and adherent culture method could be applied to isolate and purify rabbit BMSCs,which presented the ability of three-stage differentiation under appropriate induction conditions.Cells showed specific physiological, biochemical reactions as well as the specific protein and histological characteristics.2.Electric spinning nanofiber PCL-CO possed satisfactory physical characteristic and cell compatibility.3.The compound of the cell sheet of BMSCs and PCL-CO membrane formed the tissue with suitable mechanical strength and the expression of bone-specific proteins under the osteogenesis induction in bioreactor.4.The artificial tissue grew into vascularized tubular bones with the mechanical strength more than 200MPa in nude mice.The tissue contained cancellous bone structure and chondrocytes,indicating endochondral ossification.Conclusions1.The rabbit BMSCs isolated by the method of the combination of the density gradient centrifugation and adherent culture method posseed stable cell trait,and it was easy to be purified and amplified.It should be the ideal seed cells since it could be differentiated into osteoblast, cartilage cells,fat cells.2.PCL-CO had no negative effects on the proliferative activity of rabbit BMSCs and it presented good biocompatility.It may be one of ideal tissue engineering materials for bone.3.Under dynamic culture in a rotary bioreactor through cell-sheet technique,the co-constructed complex of BMSCs sheet and PCL-CO membrane formed growing tubular bone-like tissue.4.The enginerred bones implanted subcutaneously in the nude mice formed highly vascularized tubular bones by the way of cartilaginous osteogenesis,containing both cartilage and cancellous bone structure.5.The model of function bone engineering with the rabbit BMSs cell sheet and nano-PCL-CO subcutaneous tissue engineering bone provided an experimental basis for clinical bone defeat repair and prefabricating composite flaps(skin flap/tissue engineering bone flaps).