Ceria Nanoparticles Promote Endochondral Ossification-based Critical-sized Bone Defect Regeneration

Background:Bone defect refers to the destruction of the structural integrity of the bone,mainly due to trauma,infection,tumor,debridement of osteomyelitis,bone tumor resection and various congenital diseases,which is one of the common clinical diseases.In 1986,SchmitzJP proposed the concept of critical bone defect,and the length of the bone defect reached 1.5times of the long bone diameter as the critical value of the bone defect.When the bone defect reached or exceeded the critical value,the bone defect could not naturally heal.According to this,many scholars refer to bone defects greater than 1.5 times the diameter of long bones as large segmental bone defects.Large segmental bone defects can cause loss of motor function and seriously affect the quality of life of patients,and its repair has always been a clinical problem.Traditional clinical treatment of large segmental bone defects mainly relies on bone transplantation,but the limited source of bone graft and the donor-recipient immune rejection have greatly limited the wide application of this method in clinical.At present,the rapid development of bone tissue engineering has brought new methods for the treatment of large segmental bone defects,and has become a research hotspot.Bone tissue engineering technology can not only repair large segmental bone defects,but also can be prepared in large quantities according to the shape of the injury zone.It overcomes the shortcomings of donor site damage caused by autologous and allogeneic bone transplantation,immune rejection and pathogenicity in the transplanted area.It is recognized as an ideal method for repairing large segmental bone defects.There are two main ways to repair bone defects in vivo by tissue engineering:intramembranous ossification and endochondral ossification.Recent studies have confirmed that compared with intramembranous ossification,endochondral ossification is better for the repair of large segmental bone defects because of its rich blood supply.During endochondral ossification development,hypertrophic chondrocytes produce avascular cartilaginous matrix enriched in type X collagen and secrete matrix metalloproteinase 13（MMP13）and vascular-endothelial growth factor（VEGF）to direct the formation of mineralized matrix and attract blood vessels.Osteoblasts,osteoclasts,and hematopoietic precursors that accompany vascular invasion mediate resorption of the cartilaginous template and formation of vascularized bone.Therefore,the regulation of hypertrophic chondrocytes is a key link in endochondral ossification.Inadequate hypertrophy of chondrocytes can hinder vascular invasion and matrix mineralization during endochondral ossification and delay bone formation.Ensuring adequate hypertrophy of mesenchymal stem cells（MSCs）is the key to bone regeneration based on endochondral ossification.In-depth study of its specific molecular mechanism will provide a new theoretical basis and treatment scheme for clinical treatment of large segmental bone defects.In recent decades,nanomaterials have become a research hotspot in tissue regeneration due to their unique biomimetic characteristics and biological characteristics.More and more studies have shown that some nanomaterials can play a key role in bone tissue engineering by regulating proliferation and differentiation of stem cells.Among these nanomaterials,ceria nanoparticles（CNPs）because of the coexistence of Ce³⁺and Ce⁴⁺on the lattice surface,the low reduction potential make them can easily switch back and forth.This interchangeable property between Ce³⁺and Ce⁴⁺makes them have unique regenerative properties and radical scavenging capacity,which has attracted great attention and been widely used in nanobiology and regenerative medicine.Currently,it has been reported that CNPs are enriched in bone tissue,enhance the adhesion and proliferation of MSCs through their antioxidant properties,and further stimulate the paracrine of MSCs to promote the vascularization of endothelial progenitor cells during bone formation.However,the effect of CNPs on the multi-directional differentiation potential of MSCs and its role in bone repair is still unclear.Revealing the specific mechanism of CNPs regulating the multi-directional differentiation of MSCs will provide a theoretical basis for bone repair based on CNPs as biomaterials,and is of great significance for the clinical treatment of large segmental bone defects.Objective:This study was to explore the role and mechanism of bone tissue engineering based on CNPs as the biological interface material in the repair of large segmental bone defects on the basis of the study of the effect of CNPs on the multi-directional differentiation potential of bone mesenchymal stem cells（BMSCs）.Materials and Methods:1.The CNPs were synthesized by microemulsion method,and the Alendronate（AL）was used as an anchor molecule to stably bind to the surface of CNPs by multidentate coordination.The amino terminus of AL can effectively bind to carboxylated PEG chains,thereby modifying the surface of the CNPS with PEG.2.To investigate the specific effects of PEG-modified CNPs on osteogenic,chondrogenic and adipogenic differentiation of BMSCs.（1）The localization of CNPs in BMSCs was observed by transmission electron microscope.（2）CCK-8 was used to detect the effect of CNPs on cell proliferation.（3）The effects of CNPs on chondrogenic differentiation and hypertrophic differentiation of BMSCs were detected by different methods such as toluidine blue,Safranin O,immunohistochemical staining.The expression of chondrogenic related genes Sox9,COMP and Col2a1 were detected by qRT-PCR and Western blot at 7 and14 days after induction,and the expression of hypertrophic related genes Runx2,MMP13 and Col10a1 were detected by qRT-PCR and Western blot at 21 and 28 days after induction.（4）Alkaline phosphatase（ALP）and alizarin red staining were used to detect the formation of alkaline phosphatase and calcium nodules in osteogenic differentiation,respectively,and cells were collected at 7,14,and 21 days after osteogenic induction.The expression of osteogenic related genes Runx2,Osterix2,Col1a1,and ALP was detected by qRT-PCR and Western blot.（5）Oil red O staining was used to detect the influence of CNPs on the formation of lipid droplets in the adipogenic differentiation of BMSCs.The expression of adipogenic related genes PPARγ,C/EBPαand C/EBPβwas detected by qRT-PCR and Western blot at 10 and 21days after induction.3.The effect of PEG-modified CNPs as an interface modification material for tissue engineered bone on the repair of large segmental bone defects was studied in animal models.（1）The CNPs were used as interface modification materials to construct a tissue-engineered bone scaffold on the cancellous bone.The cells were planted on the scaffold and the biocompatibility of the constructed tissue engineered bone scaffold was assessed by scanning electron microscope（SEM）,laser scanning confocal microscope（LSCM）and neutral red staining.（2）A subcutaneous ectopic osteogenesis model of nude mice was established.The constructed tissue-engineered bone scaffolds were implantedin the back of the mouseand were harvested 6 and 12 weeks after implantation.Samples were sectioned for histological staining to observe the osteogenesis and detect the expression of osteogenic related genes.（3）A femur defect model of approximately 3 mm was developed in FVB/N mice.The constructed scaffolds were implanted into the defects for 12 weeks.After the mice were sacrificed,femurs were collected for micro-CT analysis and histological analysis to observe the osteogenesis and detect the expression of osteogenic related genes.4.To explore the mechanism of PEG-modified CNPs to promote the repair of large segmental bone defects based on endochondral ossification.We previously found that CNPs promote the expression of DHX15 in human gastric cancer cells and it is reported that DHX15 can activate p38 MAPK signaling pathway.It is known that p38 MAPK play a positive role in the chondrogenic and hypertrophic differentiation of BMSCs.In this part of the study,qRT-PCR and Western blot were used to detect the changes of DHX15 expression in chondrogenic and hypertrophic differentiation of BMSCs induced by CNPs;DHX15shRNA was used during the chondrogenic and hypertrophic differentiation of BMSCs,qRT-PCR,immunofluorescence and Western blot were used to confirm whether DHX15 plays a regulatory role;in order to detect whether CNPs can regulate the chondrogenic and hypertrophic differentiation of BMSCs by affecting the expression of DHX15 and thereby regulate the process of endochondral ossification,qRT-PCR,immunofluorescence,Western blot were used.Results:1.This study stably synthesized CNPs by microemulsion method,and the particle size is about 3 nm.After PEG modification,CNPs was better dispersed in solvent.2.The chondrogenic differentiation of BMSCs showed that CNPs promoted the chondrogenic related genes Sox9,COMP,Col2a1 and hypertrophic related genes Runx2,MMP13 and Col10a1 expression.In osteogenic differentiation of BMSCs,CNPs inhibited the formation of calcium nodules and alkaline phosphatase and the expression of osteogenic related genes Runx2,Osterix2,and Col1a1.The adipogenic differentiation of BMSCs demonstrated that CNPs suppressed the formation of lipid droplets and the expression of adipogenic related genes PPARγ,C/EBPαand C/EBPβ.3.Subcutaneous ectopic osteogenesis in nude mice indicated that CNPs promotedangiogenesis and new bone formation.4.Repairment of large segmental bone defects in the middle femur of mice showed that CNPs enhanced bone defect regeneration based on endochondral ossification.5.The phosphorylation of p38 MAPK was activated by DHX15.Interfere with DHX15inhibited chondrogenic and hypertrophic differentiation of BMSCs.CNPspromoted the expression of DHX15 and partially restored down-regulated gene expression.Conclusion:1.CNPspromoted differentiation of BMSCs into chondrocytes,and inhibited osteogenic differentiation and adipogenic differentiation.2.CNPs promoted subcutaneous ectopic osteogenesis and in situ large segmental bone defect repair by endochondral ossification.3.DHX15 regulated chondrogenic and hypertrophic differentiation of BMSCs by activating p38 MAPK phosphorylation.4.CNPs may regulate chondrogenic and hypertrophic differentiation of BMSCs,and further endochondral ossification by regulating DHX15/p38 MAPK signaling pathway.