摘要
目的探讨氨等离子体锚定甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(Gly-Arg-Gly-Asp-Ser,GRGDS)短肽的消旋聚乳酸(poly-D,L-lactide acid,PDLLA)骨支架修复大鼠股骨干缺损的成骨效能。方法采用氨等离子体锚定短肽技术,在PDLLA表面以酰胺键锚定GRGDS活性短肽,制备锚定短肽PDLLA(peptides anchoredaminated-PDLLA,PA/PDLLA)骨支架。取4只4周龄SD大鼠双侧股骨及胫骨骨髓,采用全骨髓贴壁法分离、培养BMSCs,取第3～6代成骨诱导的BMSCs分别接种至PA/PDLLA支架及PDLLA支架。45只成年雄性SD大鼠,体重350～500 g,制备右侧股骨干8 mm长骨缺损模型,随机分为3组(n=15)。骨缺损分别采用BMSCs-PA/PDLLA支架复合物(PA/PDLLA组)、BMSCs-PDLLA支架复合物(PDLLA组)填充修复,空白对照组不作处理。术后观察大鼠一般情况,4、8、12周分别对骨缺损部位进行大体观察、X线片检查、组织学观察、Micro-CT扫描及实时荧光定量PCR检测。结果术后2只实验动物死亡,予以补充;其余均存活至实验完成。术后各时间点大体及X线片观察示PA/PDLLA组骨缺损部位骨修复重建较PDLLA组快,空白对照组未见骨修复。X线片评分示各时间点PA/PDLLA组评分最高,PDLLA组次之,空白对照组最低;除术后4周空白对照组与PDLLA组比较差异无统计学意义(P>0.05)外,其余各时间点组间比较,差异均有统计学意义(P<0.05);且随术后时间延长,PDLLA组及PA/PDLLA组X线片评分均呈增加趋势(P<0.05)。组织学观察及Micro-CT检查示,PA/PDLLA组成骨质量最好,PDLLA组次之,空白对照组最差;骨密度、骨体积与选取感兴趣区域体积比值、骨小梁数量、结构模型指数组间比较差异均有统计学意义(P<0.05)。实时荧光定量PCR检测示PA/PDLLA组成骨相关基因骨钙素、Ⅰ型胶原、ALP、BMP-2、骨桥蛋白的表达均显著高于其余两组(P<0.05)。结论氨等离子体锚定GRGDS短肽活性修饰的PDLLA骨支架,能够加速SD大鼠股骨干缺损模型的骨修复重建过程。
Objective To study the osteogenic effects of a new type of peptides anchored aminated-polyoD, L-lactide acid （PA/PDLLA） scaffold in repairing femoral defect in rats. Methods The PDLLA scaffolds were treated by ammonia plasma and subsequent anchor of Gly-Arg-Gly-Asp-Ser （GRGDS） peptides via amide linkage formation. Thus PA/PDLLA scaffolds were prepared. The bone marrow was harvested from the femur and tibia of 4 4-week-old Sprague Dawley （SD） rats, and bone marrow mesenchymal stem cells （BMSCs） were isolated and cultured by whole bone marrow adherence method. BMSCs-scaffold composites were prepared by seeding osteogenic-induced BMSCs at passages 3-6 on the PA/PDLLA and PDLLA scaffolds. The right femoral defects of 8 mm in length were prepared in 45 adult male SD rats （weighing, 350-500 g） and the rats were divided into 3 groups （n=lS） randomly. BMSCs-PA/PDLLA （PA/PDLLA group） or BMSCs-PDLLA （PDLLA group） composites were used to repair defects respectively, while defects were not treated as blank control （blank control group）. General state of the rats after operation was observed. At 4, 8, and 12 weeks after operation, general, radiological, histological, micro-CT observations and real-time fluorescent quantitative PCR were performed. Results Two rats died after operation, which was added; the other rats survived to the end of the experiment. At each time point after operation, general and radiological observations showed more quick and obvious restoration in PA/PDLLA group than in PDLLA group; no bone repair was observed in blank control group. The X-ray scores were the highest in PA/PDLLA group, higher in PDLLA group, and thelowest in blank control group; showing significant difference in multiple comparison at the other time （P 〈 0.05） except between blank control group and PDLLA group at 4 weeks （P 〉 0.05）. The X-ray scores showed an increasing trend in PDLLA group and PA/PDLLA group with time （P 〈 0.05）. Histological and micro-CT observations showed the best osteogenesis in PA/PDLLA group, better in PDLLA group, and worst in blank control group. Comparison between groups had significant differences （P 〈 0.05） in bone mineral density, bone volume/total volume of range of interest, trabecular number, and structure model index. Significant differences （P 〈 0.05） were found in the expression levels of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, collagen type I, bone morphogenetic protein 2, and osteopontin when compared PA/PDLLA group with the other groups by real-time fluorescent quantitative PCR analysis. Condusion The PA/PDLLA scaffolds can accelerate the repair of femoral defects in rats.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2013年第5期520-528,共9页
Chinese Journal of Reparative and Reconstructive Surgery
基金
福建省自然科学基金资助项目(2012J05142)
广东省自然科学基金资助项目(S2011010003091)~~
关键词
组织工程骨
消旋聚乳酸
氨等离子体
骨缺损
大鼠
Tissue engineered bone Poly-D, L-lactide acid Ammonia plasma Bone defect Rat
