Experimental Study Of β-tricalcium Phosphate Modified By Bone Marrow Mesenchymal Stem Cell Specific Affinity Cyclic Peptide In Treatment Of Early Osteonecrosis Of The Femoral Head

Part 1 Biopanning of bone marrow mesenchymal stem cell specific affinity cyclic peptideBackgroundOsteonecrosis of the femoral head(ONFH)is a refractory disease.In the treatment of this disease,more and more attention has been paid to tissue engineering strategy.The three basic elements of tissue engineering are scaffolds,signal factors and seed cells.In orthopaedic tissue engineering,mesenchymal stem cells are a reliable source of candidate cells.The current gold standard is bone marrow-derived mesenchymal stem cells(BMSCs).The activity and quantity of BMSCs decreases in pathological tissues,which is an important obstacle to choose BMSCs as seed cells for tissue engineering treatment.Therefore,whether to recruit BMSCs to necrotic lesions of the femoral head effectively to promote repair has been a major concern.In tissue engineering therapy,BMSCs can be fully recruited by scaffolds modified by BMSC specific adhesion ligands.This provides a novel method to improve the therapeutic effect of ONFH.Phage display is a powerful tool for finding cell-specific adhesion ligands.It is an in vitro screening technique.We intend to use phage display technique to find specific affinity peptide towards BMSCs and to lay a foundation for the construction of scaffolds with the function of recruiting BMSCs.PurposesTo screen specific affinity cyclic peptide of BMSCs by phage display technique and provide the basis for the construction of functional scaffold.Methods1.Specific affinity cyclic peptide of BMSCs was screened using phage display technology selecting C57BL/6 mouse BMSCs as target cells.2.Fluorescence cytochemistry and flow cytometry were used to verify the affinity of the screened cyclic peptide for C57BL/6 mouse BMSCs.3.Fluorescence cytochemistry was used to futher study the species specificity of the affinity of screened cyclic peptide for BMSCs selecting human and rabbit BMSCs as the research object.Results1.A specific affinity cyclic peptide of BMSCs,CDNVAQSVC,was successfully identified using phage display technology selecting C57BL/6 mouse BMSCs as target cells.2.The results of fluorescence cytochemistry and flow cytometry showed that the cyclic peptide CDNVAQSVC has high affinity towards C57BL/6 mouse BMSCs.3.Selecting human and rabbit BMSCs as the research object,the result of fluorescence cytochemistry showed that the cyclic peptide CDNVAQSVC could bind to human and rabbit BMSCs without species specificity.Conclusions1.The cyclic peptide CDNVAQSVC with specific affinity for BMSCs was successfully identified by phage display technology.2.The identified cyclic peptide CDNVAQSVC can bind to human,rabbit and mouse BMSCs without species specificity.Part 2Construction of functional β-tricalcium phosphate scaffold and its effect on cytological behavior of bone marrow mesenchymal stem cells in vitroBackgroundTissue engineering applies the principles and technologies of biology and engineering to develop functional substitutes for damaged tissues in order to repair injury and reconstruct function.Scaffolds,signal factors and seed cells are three basic elements of tissue engineering.The development of tissue engineering has changed the traditional medical model and provided new ideas for the treatment of refractory diseases such as osteonecrosis of the femoral head(ONFH).In tissue engineering,scaffolds are used as carriers of seed cells and both combine to promote the repair of pathological tissues.The biological characteristics such as biocompatibility,biodegradability and bioactivity of scaffolds are particularly important.β-tricalcium phosphate(β-TCP)is a totally absorbable bioceramic with excellent biocompatibility and osteo-conductivity.Cells can grow,differentiate and reproduce on it.However,as an inorganic material,the application of P-TCP has been challenged due to its faster degradation rate and lack of intrinsic biological stimulatory activity.Surface modification is a widely used strategy to improve the properties of biomaterials.Therefore,in this part,we intend to select β-TCP as the scaffold.The functional P-TCP scaffolds were constructed by binding the affinity cyclic peptide of BMSCs identified in part 1 to β-TCP.The functional P-TCP scaffolds were endowed with the ability to recruit BMSCs and their ability to repair was enhanced.Purposes1.To study the construction method of functional P-TCP scaffolds modified byBMSC affinity cyclic peptide CDNVAQSVC.2.To study the effect of functional β-TCP scaffolds on adhesion,spreading and proliferation of BMSCs in vitroMethods1,The functional β-TCP scaffolds modified by BMSC affinity cyclic peptide CDNVAQSVC were constructed using an adsorption/freeze-drying strategy.The adsorption of BMSC affinity cyclic peptide molecules on the surface of functionalβ-TCP scaffolds was observed by scanning electron microscope(SEM)ImageXpress Micro Confocal was used to detect whether the functional β-TCP scaffolds were successfully constructed2.The effect of functional β-TCP scaffolds on the adhesion of C57BL/6 mouse BMSCs was detected by CCK-8 assay and compared with that of unmodifiedβ-TCP scaffolds3.The spreading state of C57BL/6 mouse BMSCs on functional β-TCP scaffolds was observed by SEM and compared with that of unmodified β-TCP scaffolds.4.The proliferation of C57BL/6 mouse BMSCs on functional β-TCP scaffolds was detected by CCK-8 assay and compared with that of unmodified β-TCP scaffolds.Results1.The result of SEM showed that the BMSC affinity cyclic peptide molecules were successfully adsorbed onto the surface of β-TCP using an adsorption/freeze-drying strategy.The result of ImageXpress Micro Confocal showed that β-TCP adsorbing FITC-labeled cyclic peptide molecules exhibited green fluorescence2.The result of cell adhesion assay showed that the number of C57BL/6 mouse BMSCs adhering to functional β-TCP scaffolds was significantly higher than that of unmodified β-TCP scaffolds and the difference was statistically significant(P<0.01).3.The result of cell spreading assay showed that the spreading morphology of C57BL/6 mouse BMSCs growing on functional β-TCP scaffolds was significantly better than that of unmodified β-TCP scaffolds.4.The result of cell proliferation assay showed that the number of C57BL/6 mouse BMSCs growing on functional β-TCP scaffolds increased with increased culture time and the difference was statistically significant(P<0.05).The number of cells on functional β-TCP scaffolds was significantly higher than that of unmodifiedβ-TCP scaffolds 7 days after seeding and the difference was statistically significant(P<0.05)Conclusions1.The functional β-TCP scaffolds modified by BMSC affinity cyclic peptide CDNVAQSVC can be successfully constructed by an adsorption/freeze-drying strategy2.Compared with unmodified β-TCP scaffolds,functional β-TCP scaffolds can promote the adhesion,spreading and proliferation of BMSCsPart 3Experimental study of core decompression combined with implantation of functional β-tricalcium phosphate scaffolds in treatment of early osteonecrosis of the femoral headBackgroundOsteonecrosis of the femoral head(ONFH)is infarction of the bone caused by ischemia,which can lead to collapse of the femoral head and secondary osteoarthritis of the hip joint.It can cause pain and joint dysfunction.ONFH is one of the common refractory diseases in orthopedics.It is estimated that 10,000 to 20,000 new patients of ONFH are diagnosed annually in the United States,150,000 to 200,000 in China and 2500 to 3000 in Japan.This disease is an increasingly serious worldwide health problem.ONFH is a progressive disease,which leads to the collapse of the femoral head gradually and secondary osteoarthritis of the hip joint eventually.Eventually,the patients were treated with total hip arthroplasty(THA).These patients are relatively young and the treatment with THA indicates that they may need to undergo arthroplasty more than once.The early diagnosis and treatment of ONFH can significantly improve its natural progress and bring hope to patients with hip preservation.Magnetic resonance imaging(MRI)can diagnose ONFH before collapse without subchondral fracture,which makes it possible to choose appropriate methods to treat early ONFH and delay the progress of the disease.Core decompression(CD)is a common treatment method for ONFH before collapse of the femoral head.It can reduce elevated intraosseous pressure and significantly relieve pain.But some scholars believe that although CD can relieve pain,it doesn’t halt the progress of the disease.Therefore,CD combined with implantation of bone or bone-graft substitutes has appeared in order to enhance the therapeutic effect of pure CD.Hence,in this part,we intend to treat early ONFH using CD combined with implantation of functional β-TCP scaffolds constructed in part 2 and study the therapeutic effect of the method in order to provide a new idea and choice for the treatment of early ONFH.PurposesTo study the effect of core decompression combined with implantation of functionalβ-TCP scaffolds on the treatment of early ONFH.Methods1.The rabbit model of early ONFH was constructed by liquid nitrogen freezing method.The sections of the femoral head were stained with hematoxylin and eosin to observe whether the model of early ONFH was successfully constructed.2.Pure CD,CD combined with implantation of unmodified β-TCP scaffolds or CD combined with implantation of functional β-TCP scaffolds were used to treat early ONFH in rabbits.The therapeutic effect of three methods on early ONFH in rabbits was evaluated by radiographic and histological examinationResults1.4 weeks after modeling with liquid nitrogen freezing method,the sections of the femoral head were stained with hematoxylin and eosin.It was found that the number of vacant bone lacunae of the femoral heads after modeling was significantly higher than that of untreated normal femoral heads.The vacant bone lacunae percentage of the femoral heads after modeling was more than 50%.The result indicated that the rabbit model of early ONFH was successfully constructed2.12 weeks after surgery,the result of Micro-CT showed that the femoral heads in CD group and CD combined with implantation of unmodified β-TCP scaffolds group became flatten and the load area collapsed.The shape of the femoral heads in CD combined with implantation of functional β-TCP scaffolds group was complete and smooth.The bone mineral density(BMD)of the femoral heads in CD combined with implantation of functional β-TCP scaffolds group was significantly higher than that of CD group(P<0.01)and CD combined with implantation of unmodified β-TCP scaffolds group(P<0.05),and the difference was statistically significant.3.12 weeks after surgery,the vacant bone lacunae percentage among the trabecular matrix of femoral heads in CD combined with implantation of functional β-TCP scaffolds group was significantly lower than that of CD group and CD combined with implantation of unmodified β-TCP scaffolds group.The expression content of type Ⅰ collagen(COL1)and osteocalcin(OCN)in CD combined with implantation of functional β-TCP scaffolds group was significantly higher than that of CD group and CD combined with implantation of unmodified β-TCP scaffolds group.The difference was statistically significant(P<0.01).Conclusions1.The rabbit model of early ONFH can be successfully constructed by liquid nitrogen freezing method2.The therapeutic effect of CD combined with implantation of functional β-TCP scaffolds on the treatment of early ONFH is superior to CD and CD combined with implantation of unmodified β-TCP scaffolds.