摘要
目的 观察载二甲双胍(Met)聚乳酸—羟基乙酸聚合物(PLGA)/羟基磷灰石(HA)支架复合骨髓间充质干细胞(BMSCs)植入对兔股骨缺损的修复作用。方法 自新西兰兔双侧股骨中段获取BMSCs培养传代,分别以0、0.1、1、10 mmol/L的Met处理BMSCs,检测BMSCs活力及成骨分化能力,筛选Met适宜作用浓度。采用溶剂浇铸—粒子沥滤法制备PLGA/HA支架和PLGA/HA/Met支架,将BMSCs分别与2种支架复合,Hochest染色观察BMSCs的黏附增殖情况。将16只新西兰兔采用钻孔法构建兔股骨骨缺损模型,并随机分为Blank组、PLGA/HA组、PLGA/HA/BMSCs组、PLGA/HA/Met/BMSCs组,每组4只;Blank组不做任何处理,PLGA/HA组植入PLGA/HA支架,PLGA/HA/BMSCs组植入复合1×10^(6)个BMSCs的PLGA/HA支架,PLGA/HA/Met/BMSCs组植入复合1×10^(6)个BMSCs载Met的PLGA/HA支架。支架植入术后4周采用ELISA法检测血清骨钙素;支架植入术后8周观察骨缺损修复情况,碱性磷酸酶染色观察组织切片骨化程度,采用RT-PCR法检测缺损处骨组织中的CollagenⅠ、RUNX-2 mRNA。结果 Met可激活BMSCs并促进其成骨分化,浓度为1 mmol/L时,其促进成骨分化作用最佳。BMSCs可与PLGA/HA支架及PLGA/HA/Met支架黏附并在其内增殖。支架植入术后4周,Blank组、PLGA/HA组、PLGA/HA/BMSCs组、PLGA/HA/Met/BMSCs组血清骨钙素水平依次增高(P均<0.05);支架植入术后8周,四组股骨缺损处骨痂渐明显且骨缺损渐缩小,碱性磷酸酶染色蓝色面积依次增加且渐深,股骨缺损处CollagenⅠ、RUNX-2 mRNA表达水平依次增高(P均<0.05)。结论 Met可激活BMSCs并促进其成骨分化,PLGA/HA支架可作为Met的载体和缓释体,将Met和BMSCs装载于PLGA/HA支架构建PLGA/HA/Met/BMSCs支架可更好地促进兔股骨缺损的修复。
Objective To observe the repair effect of implantation of PLGA/HA scaffold with metformin(Met) and bone marrow mesenchymal stem cells(BMSCs) on the femur bone defect in rabbits.Methods BMSCs were obtained from the bilateral middle femurs of New Zealand rabbits.BMSCs were cultured and passaged.BMSCs were treated with Met solution at concentrations of 0,0.1,1 and 10 mmol/L.The viability and osteogenic differentiation of BMSCs were detected.The optimal concentration of Met was screened out.Solvent casting-particulate leaching method was applied to make PLGA/HA scaffold and PLGA/HA/Met scaffold.BMSCs were combined with these two kinds of scaffolds,respectively.Hochest staining was applied to detect the adhesion and proliferation of BMSCs in these scaffolds under microscope.The bone defect models of were established by drilling hole on 16 New Zealand rabbits' femurs.The models were randomly divided into the Blank group,PLGA/HA group,PLGA/HA/BMSCs group,and PLGA/HA/Met/BMSCs group with four rabbits in each group.The rabbits in the Blank group were not processed.The rabbits in the PLGA/HA group were implanted with PLGA/HA scaffold in bone defect.The rabbits in the PLGA/HA/BMSCs group were implanted with PLGA/HA scaffold loaded 1×10^(6) BMSCs in bone defect.The rabbits in the PLGA/HA/Met/BMSCs group were implanted with PLGA/HA scaffold loaded 1×10^(6) BMSCs and 0.001 mmol Met in bone defect.The serum osteocalcin was measured by enzyme-linked immunosorbent assay(ELISA) at four weeks after the scaffold implantation.The repair of bone defect was observed at eight weeks after scaffold implantation.The degree of ossification of histological section was observed under microscope after alkaline phosphatase staining.RT-PCR was applied to detect the gene expression levels of Collagen Ⅰ and RUNX-2 in the bone tissues.Results Met could activate BMSCs and promote their osteogenic differentiation.At the concentration of 1 mmol/L,Met had the best effect in promoting osteoblast differentiation.BMSCs could adhere to PLGA/HA scaffold and PLGA/HA/Met scaffold and proliferate in these scaffolds.After four weeks,the serum osteocalcin in the Blank group,PLGA/HA group,PLGA/HA/BMSCs group,and PLGA/HA/Met/BMSCs group increased gradually(all P<0.05).After eight weeks,the callus of four groups increased gradually,the bone defects decreased gradually,and the ALP staining area and depth of tissue sections increased gradually,and the gene expression levels of Collagen Ⅰ and RUNX-2 increased gradually(all P<0.05).Conclusions Met can activate BMSCs and promote their osteogenic differentiation.PLGA/HA scaffold can be used as carrier and sustained-release agent of metformin.The PLGA/HA/Met/BMSCs scaffold constructed by loading metformin and BMSCs onto the PLGA/HA scaffold could better promote the repair of bone defects.
作者
韩俊柱
王旭东
安笑言
夏启鑫
HAN Junzhu;WANG Xudong;AN Xiaoyan;XIA Qixin(Department of Orthopaedics,The Second Affiliated Hospital of Bengbu Medical College,Bengbu 233000,China)
出处
《山东医药》
CAS
2023年第20期6-10,共5页
Shandong Medical Journal
基金
安徽省卫生健康委科研项目(AHWJ2021b126)。
