纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性与体内降解研究

对制备的纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性及体内降解情况进行研究,为临床提供实验依据。参照GB/T16886医疗器械生物学评价标准和要求,对纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥进行急性细胞毒性试验、溶血试验、热源试验、急性全身毒性试验及体内植入试验等系列体内外生物学试验研究,以进行有效的生物相容性和安全性评价。纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的溶血率小于国家规定的5%,在体外不引起溶血反应;浸提液注入动物体内后无死亡,活动进食正常;无细胞毒性反应;热原试验动物体温升高均在0.7℃以下,3只兔体温升高值的总数〈1.5℃,无致热作用;材料植入体内初期有轻度炎症反应,随植入时间延长逐渐减轻,材料也逐渐降解吸收。纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥具有良好的生物相容性和降解性能,具有临床开发应用前景。     

纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性与体内降解研究

对制备的纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的生物相容性及体内降解情况进行研究,为临床提供实验依据.参照GB/T16886医疗器械生物学评价标准和要求,对纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥进行急性细胞毒性试验、溶血试验、热源试验、急性全身毒性试验及体内植入试验等系列体内外生物学试验研究,以进行有效的生物相容性和安全性评价.纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的溶血率小于国家规定的5％,在体外不引起溶血反应;浸提液注入动物体内后无死亡,活动进食正常;无细胞毒性反应;热原试验动物体温升高均在0.7℃以下,3只兔体温升高值的总数＜1.5℃,无致热作用;材料植入体内初期有轻度炎症反应,随植入时间延长逐渐减轻,材料也逐渐降解吸收.纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥具有良好的生物相容性和降解性能,具有临床开发应用前景.     

纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥的性能

背景：目前普遍使用的黏合剂对粉碎骨折块进行黏合复位或多或少都存在一些缺陷。 目的：研制具有黏接骨骼作用的生物活性骨水泥。 方法：应用共沉淀法制备纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合材料作为骨水泥的固相粉体,将柠檬酸衍生物配制成溶液作为液相。通过优化实验,从骨水泥的固化时间、抗压强度、抗拉强度、抗稀散性等方面确定最佳配比。 结果与结论：纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠质量比为65/35,其中羧甲基壳聚糖和海藻酸钠质量比为4∶1时复合成粉体,并按固液比为1.0∶0.5(g∶mL)调拌后形成的骨水泥呈膏状,塑形性和抗稀散性能良好,固化时间12~18 min,抗压强度为(4.5±2.1) MPa。体外黏接猪股骨头抗拉强度在不同室温下无显著性差异无显著性意义(P > 0.05),固化后2 h的抗拉强度达到24 h的94%。骨水泥为多孔状结构,孔径为100~300 μm,纳米羟基磷灰石分布较均匀。提示制备的纳米羟基磷灰石/羧甲基壳聚糖-海藻酸钠复合骨水泥具有良好的生物活性、适当的力学强度以及较好的黏合强度。     

壳聚糖/纳米羟基磷灰石分层复合支架的生物相容性研究

制备壳聚糖/纳米羟基磷灰石(CS/nHA)分层复合支架,对其进行细胞毒性评价.分离培养大鼠软骨细胞接种于支架,相差显微镜和扫描电镜观察细胞的黏附及生长情况.动物皮下埋植试验观察其组织相容性.实验结果证实壳聚糖/纳米羟基磷灰石分层复合支架具有良好的生物相容性,有望成为较好的骨软骨组织工程支架.     

经羟基磷灰石涂层的外固定针与骨髓基质细胞生物相容性研究

目的探讨经羟基磷灰石(hydroxyapatite,HA)涂层的外固定材料与骨髓基质细胞(bone marrow stromal cell,BMSc)的生物相容性。方法通过经HA涂层的外固定材料与BMSc体外细胞培养实验,进行细胞形态学观察和细胞增殖、碱性磷酸酶活性测定。结果扫描电镜观察:较空白组和对照组,实验组细胞在涂层表面细胞贴附良好,细胞伸出较多伪足;细胞生长良好;细胞增殖,第5,8,12天有显著差异(<0.05);ALP活性,第6天试验组间有显著性差异(<0.05)。该生物材料对BMSc增殖、分化及分泌功能无抑制作用。结论经HA涂层的外固定材料具有良好的生物相容性。     

纳米羟基磷灰石复合骨形态发生蛋白2与骨髓基质干细胞的体外培养*☆

背景：纳米级羟基磷灰石支架材料复合骨形态发生蛋白2因子组织构建模式组合后对骨髓基质干细胞的作用是叠加促进或抑制作用以及其生物相容性如何？ 目的：验证纳米羟基磷灰石复合骨形态发生蛋白2与骨髓基质干细胞在体外共同培养下的生物相容性和成骨性。 方法：将兔骨髓基质干细胞体外培养、传代和扩增,经鉴定后将第3代细胞分别接种在对照组、骨形态发生蛋白2组、纳米羟基磷灰石组、纳米羟基磷灰石/骨形态发生蛋白2组4种培养液中。 结果与结论：对照组和纳米羟基磷灰石组的细胞倍增时间较骨形态发生蛋白2组、纳米羟基磷灰石/骨形态发生蛋白2组两组长;骨形态发生蛋白2组和纳米羟基磷灰石/骨形态发生蛋白2组细胞增殖能力及碱性磷酸酶测量值明显大于对照组和纳米羟基磷灰石组(P < 0.01)。提示纳米羟基磷灰石复合骨形态发生蛋白2的组织构架不仅与骨髓基质干细胞之间具有良好的生物相容性,还可发挥骨形态发生蛋白2促进骨髓基质干细胞增殖和定向分化成骨的作用。 关键词：纳米羟基磷灰石;骨形态发生蛋白2;骨髓基质干细胞;生物相容性;支架;碱性磷酸酶 doi:10.3969/j.issn.1673-8225.2012.12.009 中图分类号: R318  文献标识码: B       

羧乙基壳聚糖-纳米羟基磷灰石复合材料细胞相容性和生物力学性能的实验研究

制备羧乙基壳聚糖-纳米羟基磷灰石(NCECS/nHA)复合材料,研究其生物力学性能以及与气管软骨细胞的生物相容性。方法 气管软骨片段取自8周龄大耳白兔,Ⅱ型胶原酶消化,将所获得软骨细胞传代培养。将体外制备的NCECS/nHA复合材料分别进行干态标本和湿态标本的生物力学检测。将第3代软骨细胞种植到NCECS/nHA复合材料,分别计算材料表面软骨细胞在2h、6h、12h细胞贴壁率,并用噻唑蓝(MTT)法测定细胞增殖活性。结果 NCECS/nHA复合材料具有良好的生物力学性能。兔气管软骨细胞在NCECS/nHA复合材料表面上12h的贴壁率达(88.4±2.1)％,与其他组差异无统计学意义(P＞0.05)。同时MTT显示气管软骨细胞在NCECS/nHA复合材料表面生长状态良好。扫描电镜结果显示软骨细胞在NCECS/nHA薄膜上增殖和分化良好。结论 NCECS/nHA复合材料具备良好的细胞相容性和适宜的生物力学强度,作为一种具有开发潜力的生物材料,可用于组织工程气管的体外构建。     

生物玻璃-纳米羟基磷灰石梯度涂层的生物相容性研究

目的对生物玻璃-纳米羟基磷灰石(BG-nHA)梯度涂层的生物相容性作初步的评价。方法用低温烧结法在钛合金表面制备生物玻璃纳米羟基磷灰石梯度涂层,利用L929细胞检测梯度涂层材料的细胞毒性。取体外分离培养扩增的人骨髓基质干细胞(hBMSC),接种于涂层材料上,通过绿色荧光蛋白染色、扫描电镜和MTT法观察细胞的黏附和生长情况。结果生物玻璃-纳米羟基磷灰石梯度涂层的细胞毒性分级为1级,人骨髓基质干细胞可以在涂层材料表面黏附和生长。结论生物玻璃-纳米羟基磷灰石梯度涂层有良好的生物相容性,具有潜在的临床应用前景。     

生物玻璃-纳米羟基磷灰石梯度涂层的生物相容性研究

目的 对生物玻璃-纳米羟基磷灰石(BG-nHA)梯度涂层的生物相容性作初步的评价.方法 用低温烧结法在钛合金表面制备生物玻璃纳米羟基磷灰石梯度涂层,利用L929细胞检测梯度涂层材料的细胞毒性.取体外分离培养扩增的人骨髓基质干细胞(hBMSC),接种于涂层材料上,通过绿色荧光蛋白染色、扫描电镜和MTT法观察细胞的黏附和生长情况.结果 生物玻璃-纳米羟基磷灰石梯度涂层的细胞毒性分级为1级,人骨髓基质干细胞可以在涂层材料表面黏附和生长.结论 生物玻璃-纳米羟基磷灰石梯度涂层有良好的生物相容性,具有潜在的临床应用前景.     

壳聚糖-羧甲基壳聚糖复合膜的研制及其生物相容性评价

壳聚糖膜降解较慢^[1]，为了加快其降解速度，研制了壳聚糖-羧甲基壳聚糖复合膜(以下简称复合膜)并进行了生物学评价。结果表明，该复合膜具有很好的生物相容性，可以满足体内植入膜的基本要求。     

Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels

Alginate is a biodegradable, immunocompatible biopolymer that is capable of immobilizing viable cells and bioactive factors. Few investigations have analyzed the efficacy of alginate gels as substrata for cell attachment and proliferation. Here we have compared the adhesion and subsequent growth of human and rat bone marrow stromal fibroblastic cells on unmodified alginate hydrogel surfaces. It was found that, in contrast to rat cells, human cells did not readily attach or proliferate on unmodified alginates. In attempts to enhance these features, or collagen type I was incorporated into the gels, with no significant improvements in prolonged human cell adherence. However, alginate gels containing both collagen type I and beta-tricalcium phosphate were found to enhance human cell adherence and proliferation. Furthermore, interactions between the collagen and beta-tricalcium phosphate prevented loss of the protein from the hydrogels. These results indicate that alginate gels containing collagen have potential uses as vehicles for delivery of adherent cells to a tissue site. In addition, gels containing beta-tricalcium phosphate, with or without collagen type I incorporation, have potential to support cell growth and differentiation in vitro before implantation. This study emphasizes the limitations of the uses of cells derived from experimental animals in certain model studies relating to human tissue engineering.     

Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.     

可注射藻酸盐支架与人骨髓基质干细胞的体外复合培养*☆

背景：目前可注射组织工程骨的研究主要限于动物实验,若人骨髓基质干细胞与藻酸盐生物相容性良好,可注射组织工程骨将是极具前途的临床治疗手段。 目的：体外观察人骨髓基质干细胞与可注射支架藻酸钙凝胶的生物相容性。 方法：实验组将第2代人骨髓基质干细胞与藻酸钙凝胶复合培养,对照组单纯接种骨髓基质干细胞。倒置相差显微镜、扫描电镜观察各组细胞形态及增殖情况,MTT法半定量检测细胞增殖情况。 结果与结论：倒置显微镜下见实验组细胞生长良好,与对照组无明显差异。扫描电镜见骨髓基质干细胞在藻酸钙表面贴附、增殖良好,第6天时细胞已跨越微孔表面或向孔内生长。MTT法显示与对照组相比,实验组细胞增殖能力不受影响。结果初步表明藻酸钙与人骨髓基质干细胞体外生物相容性较好。      

骨组织工程的种子细胞--骨髓基质细胞的研究进展

骨髓基质细胞作为骨组织工程的种子细胞具有广阔前景.许多实验证实骨髓基质细胞具有间充质干细胞特性,表现为较强的增殖能力和向多种间充质细胞分化的潜能.目前已建立了体外培养骨髓基质干细胞的方法,而且正在摸索进一步纯化的方法和诱导分化的条件.已有利用其成骨特性体内移植实验,表明在适当的条件下,接种在组织工程材料上的骨髓基质细胞可以形成新骨.     

Simultaneous injection of bone marrow cells and stromal cells into bone marrow accelerates hematopoiesis in vivo

We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra-bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.     

骨髓间充质干细胞与纳米羟基磷灰石支架材料的体外相容性研究

目的　探讨兔骨髓间充质干细胞(rBMSCs)与纳米羟基磷灰石(nano-HA)支架材料的相容性,进一步验证nano-HA材料作为骨组织工程支架材料的可行性。 方法　将rBMSCs与nano-HA支架材料在体外复合培养,通过倒置显微镜、扫描电镜观察细胞与材料的复合情况,用MTT法、碱性磷酸酶活性检测法检测材料对细胞增殖、分化的影响。 结果　rBMSCs可以在nano-HA支架材料表面及孔隙中良好的黏附、迁移、增殖和分化,复合培养5 d后nano-HA支架材料对rBMSCs的增殖分化表现出一定的促进作用。 结论　rBMSCs与nano-HA支架材料具有良好的生物相容性, nano-HA支架材料可以作为rBMSCs良好的载体。     

Osteogenic differentiation and angiogenesis with cocultured adipose-derived stromal cells and bone marrow stromal cells

The purpose of this study was to determine the influence of cocultured adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation and angiogenesis of bone marrow stromal cells (BMSCs) as well as the underlying mechanism and the optimal ratio. Two in vitro coculture models, segregated cocultures using transwell and mixed cocultures, were employed to assess the indirect and direct effects of coculture respectively. Coculture was carried out for 14 days using 1 × 10⁵ BMSCs and ASCs of variable number. BMSCs, ASCs, or both were seeded in PLGA scaffold and implanted in the subcutaneous tissue of 25 nude mice for in vivo analysis of angiogenesis. To evaluate the orthotopic bone formation, critical size calvarial defects were created on 20 mice, and implanted with hydroxyapatite/β-tricalcium phosphate granules plus BMSCs, ASCs, or both. From both transwell and mixed coculture model, 1 × 10⁵ BMSCs cocultured with 0.5 × 10⁵ ASCs showed significantly greater osteogenic differentiation and mineralization than BMSCs alone. The mixed ASC/BMSC coculture at or above a ratio of 0.5/1 showed increased secretion of vascular endothelial growth factor (VEGF), and induced effective tube formation from human umbilical vein endothelial cells, which were comparable to ASCs. Cytokine profiling assay and gene expression study showed elevated levels of angiogenic factors VEGF and CXCL1, osteogenic factor Wnt5a as well as transforming growth factor (TGF)-βR1 and SMAD3 from BMSCs when cocultured with ASCs. After 5 weeks of implantation, polylactic-co-glycolic acid (PLGA)-ASCs-BMSCs had a number of vascular structures comparable to PLGA-ASCs and significantly greater than PLGA-BMSCs. Calvarial defects treated with ceramic/BMSCs/ASCs had greater area of repair and better reconstitution of osseous structure than the defects treated with ceramic/ASCs or ceramic/BMSCs after 10 weeks. In conclusion, ASCs added to BMSCs promoted osteogenesis and angiogenesis at the optimal ASC/BMSC ratio of 0.5/1.     

Graft materials and bone marrow stromal cells in bone tissue engineering

Bone augmentation procedures rely on osteogenic/osteoconductive properties of bone graft material (BGM). A further improvement is represented by use of autologous bone marrow stromal cells (BMSC), expanded in vitro and seeded on BGM before implantation in the bone defect. The effect of different BGMs on BMSC osteogenic differentiation was evaluated. BMSC were cultured in vitro in the presence of different BGM (natural, synthetic, and mixed origins). Cellular morphology was analyzed with scanning electron microscopy. The capability of BMSC to differentiate was determined in vitro by alkaline phosphatase gene expression and enzyme activity at different time points (7, 14, and 28 days) and in vivo by ectopic bone formation of implanted tissue constructs in an immunodeficient murine model. BGM supports the cell adhesion and osteogenic differentiation of BMSC developing a useful tool in the bone tissue engineering.