Experimental Study On Repairing Skull Defect With Decalcified And Decellul Arized Cortical Bone

BackgroundBone defects caused by fractures,diseases or congenital defects are still medically important problems to be solved.As the population ages,the number of patients with bone defects caused by frequent orthopaedic diseases is increasing.Bone tissue has a certain self-healing ability,but it is difficult to heal when the bone defect size exceeds the threshold of self-healing.At this time,most of the clinical treatments using bone defect repair materials are used to promote the repair of bone defects.Therefore,the demand for bone defect repair materials in clinical work is also increasing.At present,there are three main types of bone defect repair materials commonly used in clinical practice:allogeneic bone,autologous bone and artificial bone.However,some of the repair materials are used to treat bone defect diseases and are limited.It is urgent to develop and select suitable and effective solutions for bone defect implant materials.It has been reported in the literature that many products have been developed to meet the requirements of different bone implant materials,but only a small number of products have been successfully used clinically to treat bone defect diseases.Some of the biological materials studied now have more or less defects.Therefore,it is necessary to further explore the development of new bone biomaterials with potential applications without the need to add exogenous growth factors or cells.Expand basic research on biomaterials and provide a theoretical basis for the development of new osteoinductive biomaterials for clinical applications.ObjectiveThe osteogenic effects of the decalcified acellular cortical bone matrix prepared in this experiment were studied by in vitro cell experiments and in vivo animal experiments,and the structural characteristics and cytotoxicity of the prepared materials were observed.To explore the osteogenesis of cytotoxicity and experimental animals by the materials prepared in this experiment,and provide theoretical basis for the clinical application of xenogeneic decalcified and decellularized cortical bone matrix to repair non-bearing bone.Decellularized decalcified bone is rich in source,simple in preparation,and has good bone repair effect.Method(1)Material preparation:fresh rabbit bone was taken to remove bone marrow and its attached soft tissue,and decalcified by 10%EDTA.It was made into decalcified bone(DBM),then frozen in liquid nitrogen for 10 min,left at room temperature for 10 min,and repeated three times.Triton X-100 was treated with 0.5%concentration for 24 h,then treated with 0.5%SDS for 24 h.After removing the washing solution,wash the rabbit bone with PBS and wash it thoroughly for more than 10 times.This process was carried out on a 37?constant temperature shaker(300 rpm/min).Rabbit cortical bone was prepared by the above treatment to prepare decalcified and decellularized bone matrix(DCDBM).(2)Material properties:Scanning electron microscopy,DNA content determination,HE section staining of the treated materials were used to evaluate the microstructure and decellularization effect of the material.(3)In vitro cell experiments:BMSCs were extracted from 2-3 days old SD rats and cultured to the third passage with 1×10~4 cells/well to 96-well plates.The prepared material was immersed in 75%alcohol.After disinfection,3-4 times during immersion in sterile PBS for 24 h.The sterilized materials were divided into two groups:the decalcification group and the decalcification and decellularization group were immersed in complete medium for 24 hours.The BMSCs medium was aspirated and replaced with two infusions for testing for cytotoxicity.Cell viability was tested with CCK8 at 1,2,3,and 5d,respectively.(4)In vivo animal experiment:A total of 24 skull defects models were constructed with SD rats,8 rats in each group,divided into 3 groups,and 4 rats died in each group at 8 and 12 weeks.They were:blank group,decalcification group,decalcification and decellularization group.Take samples from 8 weeks and 12 weeks.The bone defect repair effect was evaluated by Micro CT scanning.Result(1)Material properties:The color of the decalcified and decellularized bone matrix material prepared is lighter than that of decalcified bone,and the color of decalcified bone is yellowish.Scanning electron microscopy showed that the cell mass on the surface of the cell was completely removed after decellularization treatment.After the mineral was removed,the structure of the collagen fiber reached intact retention after decellularization,revealing that the larger pores were favorable.The migration of cells is attached.The DNA content was significantly reduced and the HE sections showed that the cells were completely cleared.(2)Cell experiments showed that the acellular bone matrix material soaking solution had no toxic effect on BMSCs.(3)The SD rat skull defect model showed that both decalcified bone and acellular bone had osteogenic effects,and the former was better than the latter.ConclusionAfter decalcification and decellularization of rabbit cortical bone,the original cells were cleared,the bone matrix structure was preserved intact and the immunogenicity was reduced.Decalcified bone matrix has certain ability to induce bone formation in rats,and can better induce bone formation in vivo after decellularization treatment.It provides an experimental theoretical basis for the clinical application of xenogeneic decalcified acellular cortical bone matrix to repair non-weight-bearing bone.