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DMOG And PTHrP Controlled Release Microspheres Combined With 3D Porous Scaffold Loading BMSCs To Construct Tissue Engineering Cartilage

Posted on:2022-08-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:L ChenFull Text:PDF
GTID:1524306551473084Subject:Surgery (orthopedics)
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Background and objective:Due to the structural characteristics of cartilage,once damaged,it is difficult to heal itself.Although there are some surgical treatments for cartilage injury,they all have certain limitations,which lead to the cartilage injury repair has become a clinical problem in orthopedics and remain to be solved.In recent years,cartilage tissue engineering has gradually become a promising strategy for treating cartilage injury.Bone marrow mesenchymal stem cells(BMSCs)are currently commonly used seed cells for cartilage tissue engineering.Dimethyloxalylglycine(DMOG)is a prolyl hydroxylase inhibitor,which has been shown to improve the protein level of HIF-1α,simulate the hypoxic environment of cells,and promote the chondrogenic differentiation of BMSCs.Furthermore,it has been found that parathyroid hormonerelated protein(PTHr P)has the ability to promote chondrocytes to synthesize extracellular matrix and maintain the cartilage phenotype.However,there has been no study on the combined application of DMOG and PTHr P in cartilage tissue engineering research,and it is not clear whether it can more effectively promote chondrogenesis.Therefore,the aim of this study is to first explore the effect of the combined application of DMOG and PTHr P on the chondrogenic differentiation of BMSCs.Then the poly(l-lactic acid)(PLLA)nanofiber 3D porous scaffold incorporating poly(lactic-co-glycolic acid)(PLGA)microspheres for controlledrelease of DMOG and PTHr P will be constructed.Based on the 3D porous structure of scaffold and appropriate controlled-release mechanism of microspheres for DMOG and PTHr P,can realize the combination of multiple factors to promote the chondrogenic differentiation of BMSCs,and the possibility of constructing tissue engineering cartilage can be evaluated by implantation in vivo.Methods:1.BMSCs were isolated and obtained by centrifuge and adhesive culture methods,then the morphological characteristics of BMSCs were observed.The proliferative activity of BMSCs was detected,and the multidirectional differentiation potential of BMSCs was verified through osteogenic,adipogenic and chondrogenic induction.The effects of different concentration gradients of DMOG and PTHr P on the proliferation activity and chondrogenic differentiation of BMSCs were tested respectively,then the optimal concentrations of DMOG and PTHr P were screened.The optimal combination of DMOG and PTHr P was determined through the different time sequence combination,and further evaluated whether the combined application of DMOG and PTHr P had the advantage of promoting the chondrogenic differentiation of BMSCs.2.Fructose was used to prepare sugar sphere,then the PLLA nanofiber 3D porous scaffold was fabricated by phase-separation and sugar sphere leaching.Subsequently,we tested the characteristics of the scaffold including structure,density,porosity,compressive modulus,degradation,and compatibility.Three kinds of PLGA with different ratios of lactide(LA)and glycolide(GA)(PLGA5050: LA/GA=50/50;PLGA6535: LA/GA=65/35;PLGA8515:LA/GA=85/15)were selected to prepare microspheres loading DMOG and PTHr P.The encapsulation efficiency and degradation of the microspheres were tested,and were seeded on the scaffold to test the effect on the proliferation activity of BMSCs,then the PLGA microsphere carriers suitable for DMOG and PTHr P were screened by in vitro release test,respectively.3.Four groups were designed for the third part experiment,including PLLA scaffold as the control group(PLLA),PLLA scaffold/DMOG microsphere group(PLLA/PLGA-D),PLLA scaffold/PTHr P microsphere group(PLLA/PLGA-P)and PLLA scaffold/DMOG microsphere/PTHr P microsphere group(PLLA/PLGA-DP).According to the experimental groups,PLGA microspheres loaded with DMOG and PTHr P were respectively or simultaneously composited onto PLLA scaffolds,and then each group was compounded with BMSCs for chondrogenic differentiation in vitro.RT-PCR,glycosaminoglycan(GAG)quantification,hydroxyproline(HYP)quantification,histological assay,immunofluorescence,and western blot were performed to evaluate the effect of PLLA nanofiber 3D porous scaffold/PLGA microsphere controlled-release DMOG and PTHr P on the chondrogenic differentiation of BMSCs.The protein expression of HIF-1α,yes-associated protein(YAP)and its phosphorylated protein(P-YAP)was detected by western blot,and the changes of HIF-1α,YAP and P-YAP protein expression were observed after the application of KC7F2.4.Based on the study in the third part,each group was compounded with BMSCs and implanted subcutaneously into nude mice after chondrogenic differentiation for 28 days in vitro.At 4 weeks and 8 weeks after surgery,the implants were grossly observed and histologically examined to comprehensively compare and evaluate the ability of PLLA nanofiber 3D porous scaffold/PLGA microsphere controlled-release DMOG and PTHr P compounding with BMSCs to form cartilage in vivo.Results:1.BMSCs had clear cell contours under the microscope,showing fibroblast-like morphology,strong cell proliferation activity and positive staining for osteogenic,adipogenic and chondrogenic differentiation.DMOG and PTHr P can significantly up-regulate the expression of BMSCs chondrogenic marker genes at200μM and 10ng/ml,respectively.Safranin O-fast green staining showed that the combination of DMOG and PTHr P in the form of DMOG(0-day14)/ PTHr P(0-day28)had more positive staining and deeper staining.In addition,the combined application of DMOG and PTHr P can more effectively promote the expression of chondrogenic marker genes,inhibit the expression of cartilage hypertrophic marker genes,and increase the production of GAG.2.The PLLA nanofiber 3D porous scaffold had an interpore opening size of115.8±19.1μm,an interpore opening ratio of 19.2%±2.4%,a porosity of97.83%±0.07% and a compressive modulus of 183.2±2.3KPa.Scanning electron microscope,live/dead staining and cytoskeleton staining observed that BMSCs presented a good growth state after seeding to the PLLA scaffold.PLGA microspheres had a high encapsulation efficiency,and did not affect the internal structure of scaffold and the proliferation activity of stem cells after composited with PLLA scaffold.PLLA/PLGA5050 had the fastest release rate and shortest release time of DMOG or PTHr P in vitro,while PLLA/PLGA8515 had the slowest release rate and longest release time of DMOG or PTHr P.3.Compared with control group(PLLA),PLLA/PLGA-D group and PLLA/PLGAP group,the PLLA/PLGA-DP group was more effective in upregulating the expression of chondrogenic marker genes,inhibiting the expression of cartilage hypertrophic marker genes,increasing the production of GAG and HYP,and resulting in more positive staining in histological assay.Immunofluorescence showed that it can more effectively promote the protein expression of collagen II and inhibit the protein expression of collagen I.Western blot showed that it can effectively up-regulate the protein expression of collagen II,aggrecan,HIF-1αand YAP,and down-regulate the protein expression of P-YAP.After adding KC7F2,the protein expression of HIF-1α and YAP decreased,and the expression of P-YAP increased.4.Compared with control group(PLLA),PLLA/PLGA-D group and PLLA/PLGAP group,the PLLA/PLGA-DP group was found to be more similar to natural cartilage by gross observation,with milky white,smooth surface,rich luster and stronger elasticity.Moreover,the results of histological examination showed that there were more mature cartilage lacuna,more uniform distribution,more positive staining areas and deeper staining in PLLA/PLGA-DP group.Conclusion:1.BMSCs have good proliferative activity and multidirectional differentiation potential.The optimal combination of DMOG and PTHr P is to use DMOG in the first half of the chondrogenic differentiation cycle and PTHr P in the whole process.The combined application of DMOG and PTHr P can more effectively promote the chondrogenic differentiation of BMSCs.2.PLLA nanofiber 3D porous scaffold has a good performance in porosity,mechanical property,degradability and promoting the adhesion and proliferation of BMSCs.PLGA microspheres have high encapsulation efficiency and do not affect the internal structure of scaffold after composited with the scaffold.PLGA5050 and PLGA8515 microspheres are suitable for releasing DMOG and PTHr P,respectively.3.DMOG and PTHr P controlled release PLGA microspheres combined with PLLA nanofiber 3D porous scaffold loading BMSCs has a good chondrogenesis ability in vitro,and can be made into tissue engineering cartilage complex.4.DMOG and PTHr P controlled release PLGA microspheres combined with PLLA nanofiber 3D porous scaffold loading BMSCs has a good ability to construct tissue engineering cartilage through ectopic in vivo.
Keywords/Search Tags:cartilage injury, bone marrow mesenchymal stem cells, dimethyloxalylglycine, parathyroid hormone-related protein, chondrogenesis, tissue engineering
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