3D Printed Porous Tubular/Hydrogel/Oxygen Generating Microsphere Composite Scaffold For Repairment Of Necrosis Of The Femoral Head In Rabbits

Osteonecrosis of the femoral head(ONFH)is a common disease.Once necrosis of the femoral head occurs,the condition will often get worse if appropriate intervention is not given,and finally joint replacement surgery must be taken.The mechanism of ONFH is not fully understood.ONFH is often accompanied by an imbalance of bone metabolism,which is mainly manifested by reduced bone formation caused by decreased osteoblast activity and increased bone resorption caused by enhanced osteoclast activity.At the same time,fatty tissue in the femoral head proliferates,trabecular decreases and vacuole cells are formed.Further development leads to bone tissue necrosis and collapse of the bearing area of the femoral head.These manifestations are closely related to ischemia and hypoxia caused by necrotic local microcirculation disorder.Hypoxia also affects the survival and function of endothelial cells,aggravates vascular injury and ischemia,is not conducive to vascular regeneration,and thus enters ischemic hypoxic necrosis Vicious circle.The combination of core decompression and tissue engineering technology prolongs the survival rate of the patient's femoral head,but because the reconstruction of blood vessels takes a long time,the implant and bone necrosis area rely on molecular diffusion to obtain oxygen and nutrients.People in the internal necrosis area are in a state of ischemia and hypoxia,which is not conducive to the survival and repair of internal/exogenous seed cells.Based on this,we constructed a long-term slow-release oxygen gelatin/calcium peroxide microsphere(Gelatin/CaO₂),and prepared a multi-layer tubular porous polycaprolactone/nanohydroxyapatite(PCL/nHA)scaffold,Combined with sodium alginate/gelatin hydrogel containing BMSCs,compounded into a decomposable oxygen scaffold with good mechanical properties and biological activity,used to repair steroid induced osteonecrosis necrosis of femoral head(SONFH)in rabbits.The study was completed in three parts:the first part extracted rabbit bone marrow mesenchymal stem cells,then the cells were test by specific molecular markers on the cell surface and the multi-directional differentiation potential.Then BMSCs were transfected by GFP-lentivirus to construct a cell clone labeled with GFP.In the second part,Gelatin/CaO₂ microspheres were prepared by water/oil emulsifying and dispersing method.The oxygen release performance of the microspheres was verified by detecting the dissolved oxygen curve of the microspheres.Then,they were co-cultured with BMSCs in vitro to carry out live death staining and EdU proliferation experiments to understand the microsphere biocompatibility and biological function.Then a 3D printing technique was used to prepare a PCL/nHA porous tubular scaffold,and the biocompatibility of the scaffold was tested by CCK8 after co-culture with BMSCs.Then the calcium ion cross-linking method was used to construct the gelatin/sodium alginate hydrogel,and the confocal was used to detect the cell-carrying performance of the hydrogel.Finally,the microstructures of microspheres,scaffolds,hydrogels and the cell adhesion effect of composite scaffolds were detected by scanning electron microscope.The third part of the experiment was to build rabbit SONFH animal models by methylprednisolone and LPS,then core decompressing and implantation of the composite scaffolds with BMSCs into the femoral head were taken by surgery.12 weeks after operation,micro CT and histological examination were used to evaluate the repair effect of ONFH by these materials.The first part of the results showed that high-purity BMSCs were successfully extracted and a seed cell clone expressing GFP was constructed.The second part of the results showed that oxygen-releasing microspheres can continuously release oxygen for about 4 weeks in vitro,and had good biocompatibility and low biotoxicity,and can improve the proliferation of stem cells under extremely low oxygen conditions.The scaffolds had a porous structure and high penetration with good cell adhesion properties.Hydrogels were highly effective for cell loading and enhanced the cells adhesion.In the third part,composite materials containing oxygen-releasing microspheres showed better performance of repairment effects on SONFH by micro CT and histological examination,and the transplanted seed cells were directly involved in bone repairment by GFP tracking.In summary,the 3D printed porous tubular scaffold/hydrogel/sustainable oxygen generating microsphere composite materials had stable oxygen release performance,which showed better effects on repairing SONFH than non-oxygen releasing materials.This may be related to the beneficial effects on the survival and proliferation of seed cells by oxygen supplement in the early stage after transplantation.Oxygen-releasing materials will provide a new strategy for repair-ment of SONFH.