Effect Of L-WNT3A On Osteogenesis Of Autologous Bone Grafts

ObjectiveBone defects are partial bone loss caused by trauma,infection or tumor,disease and bone resection,and the clinical incidence is higher.The treatment is still a relatively difficult problem in plastic surgery.Currently available methods include traditional bone transplantation,tissue engineering technology combining autogenous materials and artificial materials,membrane-guided tissue regeneration technology originally produced in periodontal treatment,genetic engineering technology that can improve the efficiency of growth factors,etc.Bone transplantation is the main method.Similar with other tissue transplantation,clinically,bone transplantation is mainly divided into autogenous bone transplantation,allogeneic bone transplantation,xenogeneic bone transplantation,artificial bone transplantation and tissue engineering bone transplantation according to the source of the graft.Autogenous bone grafts are heterogeneous complexes of stem cells,osteogenic scaffolds and growth factors,and maintaining the activity of autogenous bone grafts is the key to maintaining their osteogenic potential.Clinicians believe that it is important to shorten the time for autologous bone grafts to be preserved in vitro.In addition,due to the rich content of bone progenitor cells in cancellous bone,it has stronger osteogenic ability than cortical bone.However,there are many factors that affect the bone formation ability of autogenous bone grafts.Although scholars at home and abroad have conducted a lot of research on it,there are still many controversies.After obtaining the bone graft,it is more able to maintain its activity in a solution compared to a dry environment,but the effect of different solutions on the activity of the bone graft is unclear.At the same time,the temperature at which the graft is stored is also controversial.No matter what kind of factors,maintaining the activity of autologous bone graft is the key to its osteogenesis.The Wnt signaling pathway is a classic osteogenesis-related signaling pathway that can be activated by endogenous Wnt signaling molecules to regulate osteogenic differentiation of mesenchymal stem cells.Cells that can recognize endogenous Wnt signaling molecules are called Wnt-responsive cells.Wnt protein is an extracellular protein with multiple biological functions and participates in the regulation of many physiological functions in the body.As a protein that regulates human bone mass and fracture healing,its expression can activate the Wnt signaling pathway to regulate the osteogenic differentiation of mesenchymal stem cells,reduce the apoptosis and necrosis of osteoblasts,promote the proliferation and migration of osteoblasts,and improve autologous Bone graft activity.There are 19 kinds of Wnt proteins.Existing data suggest that Wnt3a,Wnt4,Wnt5 a,Wnt7b,Wnt10 b,Wnt16 can affect the activity of osteoblasts.Among them,Wnt3a is secreted at the fastest rate,so it is the easiest to prepare and purify.Studies have pointed out that Wnt3a can activate β-catenin,which in turn activates the classic Wnt / β-catenin signaling pathway.However,contrary to previous understanding,Wnt3a activates the classical Wnt / β-catenin signaling pathway,promotes the proliferation of mesenchymal stem cells,inhibits their osteogenic differentiation,and can maintain the activity of mesenchymal stem cells.Therefore,this study intends to use the Wnt3a protein to activate the Wnt signaling pathway in autogenous bone grafts.Wnt3a protein is a lipid-modified protein with high hydrophobicity,so a specific medium is required for transport.Wnt protein in mammals is transferred between cells through lipid vesicles,maintaining the activity of Wnt protein.To simulate this transport mode,we used a liposomal preparation of human Wnt3a protein(L-WNT3A)to maintain its stability at physiological temperature.This experiment intends to clarify the source of the autogenous bone graft’s osteogenesis ability and the effect of time,temperature and solution stored on the autogenous bone graft after detachment,and then explore the adjustment of various parameters during autogenous bone grafting and the feasibility of improving the bone-forming ability of autogenous bone grafts.This experiment helps to develop a clinical treatment method to improve the efficacy of autogenous bone transplantation and accelerate bone repair.Method1.Experimental animals: Lewis rats were purchased from Charles River and used for spinal fusion experiments.All other experiments used mice.In order to simulate autologous bone grafting,syngeneic mice were used for bone transplantation.Wild type,β-actin-GFP and Axin2LacZ/+ mice were obtained from Jackson Labs,Axin2 CreERT2/+;R26RmTmG/+ mice were generated from Axin2CreERT2/+ and R26RmTmG/+ mice from Jackson Labs.Mouse bone maturity is 8-16 weeks old and aged mice are >12 months old.2.Surgical methods: SRC bone graft,humeral single cortical defect bone graft,cranial bone graft,maxillary edentulous defect bone graft,OVX surgery.3.Detection methods: histology: aniline blue,ALP,TRAP activity and five-color staining;immunohistochemical staining;cell activity assay: TUNEL staining,trypan blue staining,Caspase3 activity;qRT-PCR;endocytosis assay;Wnt Determination of activity.4.Statistical methods: Results are expressed as mean ± standard deviation.Two-tailed Student’s t-test and one-way ANOVA,two methods were used to determine significant differences between data sets.A P value <0.05 was considered statistically significant.Result1.After autologous bone graft transplantation,surviving cells are enriched around the mineralized matrix.Compared with the bone marrow cell part,the mineralized matrix part of the bone graft showed obvious proliferative capacity and osteogenic activity.2.Although the bone fragments in the mineralized matrix part and DBM are both inactivated and surrounded by dense cell populations,there are few Runx2 positive cells in DBM and the lowest ALP activity,compared with autogenous bone grafts,its bone formation ability is poor.3.Wnt-responsive cells mainly exist on the surface of the mineralized matrix of bone grafts,mainly bone progenitor cells.During repair,these cells participate in the process of osteogenic differentiation and directly promote bone regeneration,the number of which is affected by the donor age of the autogenous bone graft.4.As the time in vitro increases,the number of apoptotic cells continues to increase,cell viability decreases,and the amount of new bone produced by the same volume of bone graft obtained from the same animal also decreases.As the time in vitro increases,the Wnt responding cell population gradually undergoes apoptosis.5.When the bone graft is away from the body immediately at 4 ℃,cell necrosis is at the lowest level.When the bone graft was isolated at 37 ℃ for 1 hour,cell necrosis was highest.Despite differences in the degree of cell necrosis,bone grafts stored at 37 ℃ for 1 hour produced the same amount of new bone as bone grafts stored at 23 ℃.However,if the bone graft is stored at 4 ℃,the amount of new bone formed compared to the bone graft stored at 23 ℃ increases.Compared with bone grafts stored at 4 ℃,bone grafts stored at 23 ℃ and 37 ℃ have fewer Wnt-responsive cells,and the bone-forming ability of the bone graft is also reduced.6.Preserving autologous bone graft in 4 ℃ normal saline can maintain cell viability.Conversely,storing autogenous bone graft in 37℃ normal saline will reduce cell viability.Regardless of storage temperature,the bone-forming ability of bone grafts incubated in DMEM solution containing 10% FBS is stronger than that of bone grafts stored in physiological saline.7.The autologous bone graft treated with L-WNT3 A shows stronger osteogenic ability and higher expression of osteogenic protein,and can restore the osteogenic ability of osteoporotic animals.Conclusion1.Wnt responsive cells attached to mineralized stroma in autologous bone grafts are osteoprogenitor cells and are sources of osteogenic capacity of autologous bone grafts.2.At physiological temperature,L-WNT3 A can greatly enhance the cell activity,proliferative capacity and osteogenic potential of autogenous bone grafts,and improve the osteogenic capacity of autogenous bone grafts.