机构地区:[1]遵义医科大学口腔医学院,贵州遵义563006 [2]遵义医科大学附属口腔医院牙周病科,贵州遵义563006
出 处:《遵义医科大学学报》2025年第1期9-18,共10页Journal of Zunyi Medical University
基 金:国家自然科学基金资助项目(NO:82060204);贵州省科学技术基金项目[NO:黔科合基础-ZK(2021)一般437];贵州省卫健委科学技术基金资助项目(NO:gzwkj2023-202)。
摘 要:目的初步探究PCL-PEG-HAAM支架的制备方法,以及复合支架对人牙周膜干细胞(PDLSCs)增殖、黏附及成骨分化的影响。方法通过生物酶/化学/机械联合的方法对人羊膜(HAM)进行脱细胞制备人脱细胞羊膜(HAAM),采用0.1%明胶作为黏附介质将聚己内酯-聚乙二醇(PCL-PEG)与HAAM整合,构建PCL-PEG-HAAM复合支架,并通过扫描电镜观察其显微结构。酶消化法培养PDLSCs,流式细胞术及多向诱导分化(成骨、成脂、成软骨)对PDLSCs进行鉴定。实验分组:Control组、PCL-PEG组、HAAM组、PCL-PEG-HAAM组,选取第3代PDLSCs分别进行CCK-8实验、BCIP/NBT染色、茜素红染色以及WB检测成骨相关蛋白(RUNX2、ALP、COL1)的表达;此外,还将第3代PDLSCs负载于PCL-PEG、HAAM、PCL-PEG-HAAM进行细胞粘附实验,通过以上实验检测PDLSCs在各组支架上的生物学性能及成骨分化的能力。结果去除羊膜上皮细胞的HAAM完整保留了基底层和致密层,扫描电镜下见基底层表面完整无剥脱,致密层胶原纤维相互交织呈多孔蜘蛛网状结构;PCL-PEG-HAAM复合支架为双层膜结构,两者间可见明显交界线,HAAM与下方呈“渔网状”的PCL-PEG纳米纤维紧密连接。CCK-8结果显示第7天时PCL-PEG-HAAM组细胞增殖速率高于PCL-PEG组(P<0.05);PCL-PEG-HAAM组的细胞粘附率高于PCL-PEG组(P<0.05);BCIP/NBT染色和茜素红染色结果显示PCL-PEG、HAAM、PCL-PEG-HAAM支架均能促进PDLSCs的成骨分化,其中,PCL-PEG-HAAM组染色面积最深最广;WB结果显示PCL-PEG-HAAM组RUNX2、COL1、ALP成骨蛋白表达量显著高于Control组、PCL-PEG组(P<0.05)。结论PCL-PEG-HAAM复合支架促进了PDLSCs增殖、黏附及成骨分化,是一种具有开发潜力的口腔生物膜。Objective To prepare PCL-PEG-HAAM scaffolds and investigate their effects on proliferation,adhesion and osteogenic differentiation of human periodontal ligament stem cells(PDLSCs).Methods Human acellular amniotic membrane(HAAM)was prepared by enzyme/chemical/mechanical process of Human amniotic membrane(HAM).0.1%gelatin was used as the adhesion medium to integrate poly(caprolactone)-polyethylene glycol(PCL-PEG)into HAAM to construct PCL-PEG-HAAM compound scaffold,and its microstructure was observed by scanning electron microscope(SEM).PDLSCs were cultured by enzymatic digested tissues and identified by flow cytometry and multi-lineage(osteogenic,adipogenic and chondrogenic)differentiation;the experimental groups are as follows:Control group,PCL-PEG group,HAAM group,and PCL-PEG-HAAM group.The third-passaged PDLSCs were applicated to CCK-8 experiments,BCIP/NBT staining,Alizarin red staining,and WB to detect osteogenic proteins(RUNX2,ALP,and COL1);in addition,the third-passaged PDLSCs were loaded on PCL-PEG,HAAM,and PCL-PEG-HAAM for cell adhesion assay.The biological properties and osteogenic differentiation of PDLSCs on each scaffold were assessed by experiments above.Results HAAM preserved the basal and the dense layer completely.The surface of basal layer was intact without stripping,and the collagen fibers of dense layer interwoven with one another,forming a porous spider network structure under SEM.The PCL-PEG-HAAM scaffold was a double-layer membrane with obvious boundary between layers.The HAAM is tightly connected with the PCL-PEG nanofibers in a“fishing network”below.The results of CCK-8 showed that the cell proliferation rate in PCL-PEG-HAAM group was higher than that in PCL-PEG group on the 7th day(P<0.05).The cell adhesion rate in PCL-PEG-HAAM group was higher than that in PCL-PEG group(P<0.05).The results of BCIP/NBT staining and madarin red staining showed that PCL-PEG,HAAM and PCL-PEG-HAAM scaffolds could promote osteogenic differentiation of PDLSCs,and the stained surface of PCL-PEG-HAAM group wa
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