The Research Of Platelet-rich Plasma Combined With ADSCs Mediates Decellularized Bone Matrix Osteointegration In Critical Bone Defects

Background:At present,the main treatment of large bone defects caused by trauma,infection,tumor or congenital diseases is still based on autogenous or allogeneic bone graft.However,severe donor site complications,high risk of infection,and lack of donors have prevented them from being a sustainable option for bone repair.Although considerable progress has been made in bone graft-related surgical techniques,it has not been possible to provide a consistent and effective solution for routine clinical use.Clearly,new treatment strategies need to be established to meet patient needs.In most cases,bone tissue has an inherent ability to regenerate itself.This extraordinary inherent ability is based on a complex healing process that ultimately results in the regeneration of skeletal tissue and the complete restoration of bone function.Activation and enhancement of bone regeneration can develop more effective therapies for complex clinical cases(e.g.,nonunion,avascular necrosis)or challenging cases(e.g.,diabetics,smokers).Obviously,this depends largely on understanding the cytokines involved in bone regeneration and the factors' physiological healing processes and their regulation.Treatment plans that enhance bone tissue self-repair capabilities provide opportunities for acellular products that can induce and drive tissue repair by recruiting appropriate endogenous cell subsets and regulating their metabolic activity.As long as it is biologically valid,ready-made standardized materials are biologically and commercially more attractive than cell-based products.A promising example of implementing this concept is the use of cell-free extracellular matrix.In these methods,cell-free extracellular matrix is often based on products of natural origin(eg,decellularized bone matrix)or by combining sensing molecules with appropriate scaffold materials to synthesize bone-regenerating materials of specific characteristics.However,after thorough decellularization and protein removal,Decellularized bone matrix scaffolds(DBMs)cannot retain natural osteoinductive factors,so they cannot achieve the same clinical effects as autologous bone grafts.The main methods to solve this problem focus on the use of drugs such as growth factors and mesenchymal stem cells(MSCs).MSCs can differentiate along the osteoblast lineage,so as to promote bone regeneration.In this study,we selected adipose derived stem cells(ADSCs)from MSCs as our seed cells.Compared with other MSCs,ADSCs have many advantages,such as abundant sources,less invasive collection methods,less ethical issues,and satisfactory proliferation potential.Platelet rich plasma(PRP)is defined as a plasma product containing relatively high concentrations of various growth factors.It has been proved that the abundant growth factors in PRP can promote the regeneration of bone,cartilage and blood vessels,and it is a good growth factor donor with simple technology and extraction process.The hypothesis of this study is that PRP and DBMs with ADSCs can construct a composite system to promote bone regeneration,which is highly osteoinductive and can promote bone defect regeneration.To test this hypothesis,DBMs were combined with ADSCs and PRP into rabbit critical radius critical size bone defect(CSD)to evaluate its potential to promote angiogenesis and bone regeneration.In this study,ADSCs and PRP were used as seed cells and osteogenic drugs to explore the osteointegration effect of CSD using the DBMs complex system equipped with ADSCs and PRP as the biologically active interface.Methods:1.Preparation of acellular bone matrix scaffold with good mechanical properties and low immunogenicity;making platelet-rich plasma with good biocompatibility and rich growth factors as growth factor donors and cell carriers.2.Scanning electron microscope was used to observe the appearance of the stent;the mechanical properties of the stent were explored using a universal testing machine.3.Cell experiments in vitro The proliferation and viability of ADSCs in the composite system were observed using Cell Counting Kit-8(CCK-8)and live-dead cell staining.4.The in vitro test was performed in a rabbit model with a critical large radius bone defect,divided into five groups: blank group(B),DBMs group,DBMs filled with ADSCs group(DBMs/A),and DBMs filled with PRP group(DBMs/P)And DBMs equipped with PRP and ADSCs group(DBMs/A/P).The scaffold systems of each group were implanted into the middle radius CSD,and the effects of this scaffold composite system on CSD bone regeneration and osseointegration were further studied by imaging and histological examination.Results:1.In this study,the pore diameter of DBMs is 300-500 ?m,the maximum compressive force is 295.11 N,the compressive elastic modulus is 0.003 N mm-2,and the compressive strength is 14.440 N mm-2,which are all close to the parameters of natural bone.2.Compared with the plate group and DBMs/A group,the ADSCs implanted in the DBMs/A/P composite system showed good proliferative activity,showing that the system has good biocompatibility,which is beneficial to the proliferation of ADSCs.3.The composite system was implanted into rabbit CSD lesions,and bone tissue was obtained after 6 and 12 weeks.The X-ray display is compared to the blank group,DBMs,DBMs/A,and DBMs/P groups.The DBMs/A/P group showed higher bone density and bone regeneration volume,and the histological score showed that the degree of bone regeneration in the DBMs/A/P group(11.35 ± 0.52)was significantly higher than the DBMs/A group(9.01 ± 0.70)and DBMs/P group(8.15 ± 0.72)and DBMs group(4 ± 0.81)and blank group(1.25 ± 0.95).Conclusion:Compared with autologous and allogeneic bone grafts,the DBMs prepared by physiochemical methods of heterogeneous cancellous bone have more raw materials sources,but their osteoinductive capacity is reduced.But the addition of PRP can improve cell adhesion and osteointegration of DBMs.To our knowledge,this is the first time that PRP and DBMs have been used in combination to handle CSD.In addition,PRP improved ADSCs regeneration and differentiation ability,and promoted bone regeneration and osseointegration of DBMs in rabbit radial CSD model.This study may inspire other researchers to further develop xenogeneic cancellous bone,thus providing new options for clinical treatment of large bone defects.