【作者】 陈力；

【导师】 项舟；

【作者基本信息】 四川大学 ， 外科学（骨科）， 2021， 博士

【摘要】 研究背景与目的:由于软骨的自身结构特点,一旦出现损伤,难以实现自身愈合。尽管有一些手术方法可对软骨损伤进行治疗,但都存在一定的局限性,导致其成为了骨科临床的一个难题且仍未得到很好的解决。近年来,软骨组织工程技术逐渐成为治疗软骨损伤中一种颇具前途的方法。骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)是目前常用于软骨组织工程的种子细胞。二甲基乙二酰基甘氨酸(dimethyloxalylglycine,DMOG)是一种脯氨酰羟化酶抑制剂,可导致缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)的蛋白水平升高,模拟细胞低氧环境,从而促进BMSCs的成软骨分化。此外,甲状旁腺激素相关蛋白(parathyroid hormone-related protein,PTHr P)具有促进软骨细胞合成细胞外基质并维持其软骨表型的作用。然而截止目前,尚未见DMOG和PTHr P联合应用于软骨组织工程的研究,其是否能更有效地促进成软骨尚不明确。因此,本研究的目的是首先探寻DMOG和PTHr P联合应用对于BMSCs成软骨分化的作用。然后制备左旋聚乳酸(poly(l-lactic acid),PLLA)纳米纤维三维多孔支架及聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)微球控释DMOG和PTHr P的复合系统。可在利用支架三维多孔结构的基础上,通过微球适宜的控制释放机制联合应用DMOG和PTHr P,并负载BMSCs,实现多因素的结合来诱导体外成软骨,并植入体内进一步验证,从而综合评估其构建组织工程软骨的可能性。方法:1.采用离心贴壁法分离获取BMSCs,观察其形态学特征;检测BMSCs的增殖活性,并通过成骨、成脂、成软骨诱导分化验证其多向分化潜能;分别检测不同浓度梯度的DMOG和PTHr P对BMSCs增殖活性及成软骨分化的影响,筛选出DMOG和PTHr P的最佳浓度;通过不同作用时间顺序的组合,确定DMOG和PTHr P联合应用的最优组合方案,并进一步评估DMOG和PTHr P联合应用是否具有促进BMSCs成软骨分化的优势。2.选择果糖制备糖球致孔剂,然后利用热致相分离法结合糖球致孔剂浸出技术制备PLLA纳米纤维三维多孔支架,并检测其宏观及微观结构特征、密度、孔隙率、压缩模量、体外降解性能以及与细胞的相容性;选择3种不同丙交酯(lactide,LA)与乙交酯(glycolide,GA)比例的PLGA(PLGA5050:LA/GA=50/50;PLGA6535:LA/GA=65/35;PLGA8515:LA/GA=85/15)制备成微球,均分别负载DMOG和PTHr P,检测其包封率、体外降解性能以及复合PLLA支架后对BMSCs增殖活性的影响,再通过体外释放试验分别筛选出适合DMOG和PTHr P的PLGA微球载体。3.将实验分成4组:单纯PLLA支架组(PLLA)作为对照组、PLLA支架/DMOG微球组(PLLA/PLGA-D)、PLLA支架/PTHr P微球组(PLLA/PLGAP)和PLLA支架/DMOG微球/PTHr P微球组(PLLA/PLGA-DP);按照分组设计,将载有DMOG和PTHr P的PLGA微球分别或同时复合到PLLA支架上,然后接种BMSCs进行体外成软骨分化;通过RT-PCR、糖胺聚糖(glycosaminoglycan,GAG)定量、羟脯氨酸(hydroxyproline,HYP)定量、组织学检测、免疫荧光以及Western blot,来综合比较评估DMOG和PTHr P的PLGA控释微球结合PLLA纳米纤维三维多孔支架负载BMSCs的体外成软骨能力;通过Western blot检测HIF-1α、Yes相关蛋白(yes-associated protein,YAP)及其磷酸化蛋白(P-YAP)的表达情况,并观察HIF-1α的蛋白翻译抑制剂(KC7F2)作用后,HIF-1α、YAP和P-YAP蛋白表达的变化。4.在第三部分研究基础上,将其各实验组接种BMSCs,体外成软骨诱导28天后植入裸鼠皮下,术后4周和8周分别对植入物进行大体观察和组织学检测;综合比较评估DMOG和PTHr P的PLGA控释微球结合PLLA纳米纤维三维多孔支架负载BMSCs在体内异位成软骨的能力及构建组织工程软骨的可能性。结果:1.分离提取的BMSCs在镜下细胞轮廓清晰,呈现类似成纤维细胞样形态;BMSCs的细胞增殖活性较强,成骨、成脂和成软骨诱导分化后各自的特异性染色呈阳性;DMOG和PTHr P分别在200μM和10ng/ml时能显著上调BMSCs成软骨标志基因的表达;番红O-固绿染色显示,DMOG和PTHr P联合应用以DMOG(0-day14)/PTHr P(0-day28)的组合形式的阳性染色更多、着色更深;DMOG和PTHr P的联合应用,能更有效促进成软骨标志基因的表达、抑制软骨肥大标志基因的表达、提高GAG的生成。2.PLLA纳米纤维三维多孔支架的内连通孔尺寸为115.8±19.1μm,而内孔连通度为19.2%±2.4%,孔隙率为97.83%±0.07%,压缩模量为183.2±2.3KPa,扫描电子显微镜观察、活/死细胞染色以及细胞骨架染色显示BMSCs在PLLA支架上的生长状态良好;PLGA微球的包封率高,复合PLLA支架后并不影响支架的内部结构,且对干细胞的增殖活性无明显的影响;PLLA/PLGA5050负载DMOG或PTHr P的体外释放速率最快,释放时间最短,而PLLA/PLGA8515负载DMOG或PTHr P的体外释放速率最慢,释放时间最长。3.相比于对照组(PLLA)、PLLA/PLGA-D组和PLLA/PLGA-P组,PLLA/PLGA-DP组经RT-PCR检测结果显示其能更有效上调成软骨标志基因的表达以及抑制软骨肥大标志基因的表达;更有效提高GAG和HYP的生成;组织学观察表现出更多的阳性染色;免疫荧光显示其能更有效促进II型胶原的蛋白表达,抑制I型胶原的蛋白表达;Western blot显示其能有效上调II型胶原、蛋白多糖、HIF-1α和YAP蛋白的表达,下调P-YAP蛋白的表达;添加KC7F2后,HIF-1α和YAP蛋白的表达降低,P-YAP蛋白的表达升高。4.相比于对照组(PLLA)、PLLA/PLGA-D组和PLLA/PLGA-P组,PLLA/PLGA-DP组经大体观察发现其标本外观更类似天然软骨,呈乳白色、表面光滑、富有光泽、弹性感更强;组织学检测结果显示,其有更多的成熟软骨陷窝形成、分布更为均匀、基质阳性染色区域更多、着色更深。结论:1.BMSCs具有良好的增殖活性和多向分化潜能;DMOG和PTHr P联合应用的最优组合方式是在成软骨分化周期中,前半程应用DMOG同时全程应用PTHr P;DMOG和PTHr P的联合应用能更有效促进BMSCs的成软骨分化。2.PLLA纳米纤维三维多孔支架具有高孔隙率、良好的机械性能、体外可降解性,仿生细胞外基质的纳米纤维孔壁结构有利于BMSCs的黏附与增殖;PLGA微球具有高包封率的特点,与支架复合后不影响支架的内部结构,PLGA5050和PLGA8515微球分别适合作为DMOG和PTHr P的释放载体。3.DMOG和PTHr P的PLGA控释微球结合PLLA纳米纤维三维多孔支架负载BMSCs,具有良好的体外成软骨能力,可制作成组织工程软骨复合体。4.DMOG和PTHr P的PLGA控释微球结合PLLA纳米纤维三维多孔支架负载BMSCs,具有良好的体内异位构建组织工程软骨的能力。更多还原

【Abstract】 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.更多还原