"Two-step" Bone Tissue Engineering Strategy For Rat Bone Defect Treatment

Objective Present study aim to explore the best bone tissue engineering(BTE)strategy.On one hand,we want to optimize the cells transplanting strategy to improve the survival of seeding cells after transplantation,and we propose a novel strategy of “two-step”BTE and investigate the optimal time point for MSCs transplantation;on the other hand,we focus on the fabrication of a bioactive customized bone graft scaffold.The two-step BTE strategy and the new graft scaffold will help to boost up the medical efficacy of BTE technology and facilitate its clinical transformation.Materials and methods Collagen(Col)is the main component of extracellular matrix.We established a biodegradable collagen scaffold and developed a rat model of bone defects.Then,we investigated the local and systemic inflammatory conditions at different time point after the scaffold implantation.Then we explored the effects of ectopic osteogenesis and in situ osteogenesis with two-step BTE strategy,that is,the early seeding cells on the scaffolds and the local injection of seeding cells 7 days after operation.Besides,we transfected BMSCs by luciferase and observed the dynamic survival of BMSCs after the implantation into the body by Bioluminescence imaging.The long-term(12weeks)bone repair effect was also observed.PLGA was used to prepared simvastatin(SIM)loaded microspheres(MP).The particle size and distribution,encapsulation efficiency and drug release rule of the SIM-loaded MP was observed,and SIM-PLGA-MP were co-cultured with BMSCs to explore the in vitro bioactivity effects.Then,PCL/Col/SIM-PLGA-MP scaffolds were fabricated by rapid prototyping technique and combining freeze-drying technique for rat femur critical size defect treatment.Results There was an obvious inflammatory response after the operation,reached the peak after 3 days,and preliminary returned to normal levels after 7 days.“Two-step”BTE,which was developed by the inoculation of seeding cells on the scaffolds and the injection of seeding cells 7d postoperative,can realize the repair of bone defects.The survival time of the BMSCs with the “two-step” strategy was the longest.After12 weeks of observation,the “two-step” BTE could achieve better bone repair effects.SIM-PLGA-MP were fabricated with spherical in shape and a smooth outer surface.Drug release kinetic study showed that SIM encapsulated in the scaffold was released in a sustained manner for up to 30 days.In vitro bioactivity study showed that the SIM-loaded scaffolds had a strong ability to accelerate cell proliferation and promote their osteogenic differentiation.Moreover,in vivo experiment using a rat femur defect model also documented that PCL/Col/SIM-PLGA-MP scaffolds had a remarkable effect in promoting bone regeneration.Conclusion1.The “two-step” transplantation strategy of BMSCs can improve the survival of seeding cells.2.“Two-step” BTE strategy is feasible.Bone formation of “two-step” BTE is better than that of traditional BTE strategy.3.PLGA-MP prepared can release lipophilic drugs in a sustained manner for up to 4 weeks.Simvastatin can effectively promote the proliferation and differentiation of BMSCs.4.The prepared PCL/Col/SIM-PLGA-MP has good mechanical properties and good performance by combining 3D-printing porous PCL scaffolds and compounding SIM-PLGA-MP-containing collagen,can repair the rat critical bone defect.These findings suggested that the novel SIM-loaded PCL/Col scaffold may provide a promising route for bone repair.