The Treatment Of Mandibular Bone Defect Using Adipose Stem Cells Combined With Titanium Alloy Scaffold

Objective:Regeneration of mandibular defects remains a major challenge for maxillofacial surgeons due to the irregularity of the shape of the maxillofacial bones.The 3D printed titanium alloy scaffold is a good material for mandibular defect repair because of its good histocompatibility and corrosion resistance.However,it is very difficult for bone to grow inside the 3D printed titanium alloy scaffold which has not been optimized.Furthermore,lack of sufficient number of stem cells in the local bone defect area makes the bone tissue regeneration more difficult.In this study,we use the adipose-derived mesenchymal stem cells(ASC)to repair the mandibular defect.The cells were fully packed in the hydrogel and combined with 3D-printed titanium alloy scaffolds.Its osteogenic potential was evaluated both in vitro and in vivo.Material and Methods:We first loaded ASCs into hydrogels to provide a 3D culture environment for stem cells.Then,we evaluated the osteogenic potential of stem cells in the 3D culture environment in vitro.To confirm the osteogenic potential of ASCs under the 3D culture environment in vitro,we used cell morphology,cytotoxicity,alkaline phosphatase,alizarin red staining,Western blot,RT-PCR and other methods to analyze cell differentiation,cell proliferation,cell activity,and the bone related protein and gene.Then,we use rat mandibular defect model to exam the osteogenic effects in vivo.We wrapped the ASCs into hydrogel and then perfused into 3D-printed titanium alloy scaffold.All the rats were randomly divided into three groups:control group(BS),only the 3D-printed titanium alloy scaffold was loaded into the bone defect;Injection group(SC),250μL phosphate buffer saline containing 1×106 ASCs was injected after implantation.Scaffold+gel group(SCH).Before implantation,1×106 ASCs CellMatrix hydrogel were filled into the 3D-printed titanium alloy scaffold.At 12 weeks after implantation,micro-CT and VG staining of hard tissue sections were performed on the mandibular bone defect to observe the effect of 3D-printed titanium alloy scaffold loaded with ASCs on mandibular bone defect repair.Results:We found that ASCs could form bone in 3D culture environment,and had better osteogenic potential than in 2D culture environment in vitro.There was no significant difference in the proliferation effect of the cells at the 1st,3rd,5th and 7th day in the 3D culture environment compared with that in the 2D culture environment,indicating that the 3D culture had good biocompatibility and could make the cells obtain good biological activity.Using alkaline phosphatase and alizarin red assay,we found that after 14 days of osteogenic induction culture,ASCs in 3D culture environment could well mineralize into bone,and the expression of alkaline phosphatase was higher than 2D.The detection of osteogenic related proteins and genes such as Runx2,OPN and OCN of the cells were highly expressed in ASCs cultured in 3D environment,which again confirmed that stem cells could better differentiate into osteoblasts in 3D culture environment.Then,in vivo results confirmed this hypothesis,we used the micro-CT and found that ASCs loaded 3D-printed titanium alloy scaffolds in vivo had the better osteogenesis effects,no obvious osteogenesis was found in the control group,while only a small amount of bone formation in the SC group,and the SCH group have the maximum bone in the 3D-printed titanium alloy scaffold.Then we confirmed the above phenomenon by VG staining.Conclusion:We demonstrated that 3D-printed titanium alloy scaffold loaded 3D cultured adipose mesenchymal stem cells can promote bone regeneration both in vitro and in vivo.In vitro experiments have confirmed that 3D-cultured ASCs can achieve better bone differentiation effect.In vivo experiments have shown that 3D cultured adipose mesenchymal stem cells can enhance bone regeneration and bone integration effect of 3D-printed titanium alloy scaffolds.The results provide a theoretical foundation for clinical application.This study indicated that the 3D-cultured ASCs combined with 3D-printed scaffold is safe and effective for mandibular bone defects.