甲基丙烯酰化透明质酸/脱细胞华通胶水凝胶支架的制备与表征

背景:随着组织工程为关节软骨损伤修复这一世界难题带来了新的希望,构建成分仿生的光固化3D打印水凝胶支架对软骨组织工程具有重要意义。目的:通过数字光处理3D打印技术构建成分仿生的甲基丙烯酰化透明质酸/脱细胞华通胶水凝胶支架,评价其生物相容性。方法:从人脐带中分离提取华通胶组织后进行脱细胞处理,冷冻干燥后磨成粉末,溶于PBS中制备50 g/L的脱细胞华通胶溶液。制备甲基丙烯酰化透明质酸,冻干后溶于PBS中制备50 g/L的甲基丙烯酰化透明质酸溶液。将脱细胞华通胶溶液与甲基丙烯酰化透明质酸溶液以体积比1∶1混合,加入光引发剂后作为生物墨水。通过数字光处理3D打印技术分别制备甲基丙烯酰化透明质酸水凝胶支架(记为HAMA水凝胶支架)与甲基丙烯酰化透明质酸/脱细胞华通胶水凝胶支架(记为HAMA/WJ水凝胶支架),表征支架的微观结构、溶胀性能、生物相容性与促软骨分化性能。结果与结论:①扫描电镜下见两组支架均呈三维立体的网状结构,其中HAMA/WJ水凝胶支架纤维连接更加均匀;两组支架均在10 h内达到溶胀平衡,HAMA/WJ水凝胶支架的平衡溶胀比低于HAMA水凝胶支架(P<0.05)。②CCK-8实验显示相较于HAMA水凝胶支架,HAMA/WJ水凝胶支架可促进骨髓间充质干细胞的增殖;死活染色显示骨髓间充质干细胞在两组支架上生长良好,并且HAMA/WJ水凝胶支架上的细胞立体分布均匀、细胞数量更多;鬼笔环肽染色显示相较于HAMA水凝胶支架,HAMA/WJ水凝胶支架上的骨髓间充质干细胞黏附与铺展更佳。③将骨髓间充质干细胞接种于两组支架后进行成软骨诱导培养,qRT-PCR检测结果显示,HAMA/WJ水凝胶支架组聚集蛋白聚糖、SOX9、Ⅱ型胶原mRNA表达量均高于HAMA水凝胶支架组(P<0.05,P<0.01)。④结果表明,数字光处理3D生物打印HAMA/WJ水凝胶支架可促进骨髓间充质干细胞的增殖、黏附及成软骨分化。     

季铵盐壳聚糖三维支架复合GNDF载间充质干细胞向神经样细胞分化

背景：壳聚糖类水凝胶因其良好的生物相容性、可降解性及对药物的缓释作用,作为支架材料近年来在组织损伤修复领域逐渐成为研究热点。 目的：探索大鼠骨髓间充质干细胞在季铵盐壳聚糖温敏凝胶支架上生长、向神经样细胞定向分化的可行性,为治疗神经系统损伤寻找理想的组织工程材料。 方法：季铵盐壳聚糖与β-甘油磷酸钠复合制成温敏凝胶,扫描电镜观察凝胶的三维结构,MTT法评价凝胶浸提液对骨髓间充质干细胞活力的影响;将牛血清白蛋白加载于凝胶支架,紫外光谱吸收法分析凝胶支架对牛血清白蛋白的缓释效果。接种大鼠骨髓间充质干细胞于凝胶支架,扫描电镜观察在支架缓释胶质细胞源性神经营养因子作用下,骨髓间充质干细胞的生长、分化情况,免疫荧光技术检测神经元烯醇化酶的表达。 结果与结论：季铵盐化壳聚糖与甘油磷酸钠复合所得凝胶支架,其多孔性特点明显,有温敏特性,对蛋白的缓释效果良好,承载大鼠骨髓间充质干细胞后,对其增殖无明显不利影响。在凝胶支架缓释的胶质细胞源性神经营养因子作用下,骨髓间充质干细胞呈现神经样细胞形态,表达神经元特异性标记物神经元烯醇化酶。说明季铵盐壳聚糖温敏凝胶对胶质细胞源性神经营养因子的缓释效果良好,其凝胶支架具有多孔径、良好生物相容性特点,可承载大鼠骨髓间充质干细胞体外生长和向神经元定向分化。     

接枝Nogo-A抗体和多聚赖氨酸的透明质酸水凝胶支架的制备及其与培养海马神经元相容性的研究

为探讨同时接枝Nogo-A受体(NgR)的抗体和多聚赖氨酸(PLL)的透明质酸(HA)水凝胶用于中枢神经系统损伤修复的可行性,本研究在碳二亚胺盐酸盐的介导下用己二酸二酰肼交联的方法制备HA水凝胶,并接枝PLL和NgR抗体。取新生大鼠海马神经元接种于水凝胶支架材料上,分为HA-NgR抗体-PLL水凝胶组和纯HA水凝胶组。培养7d后,用吖啶橙(AO)染色、抗神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)免疫荧光染色和扫描电镜观察两组细胞的生长情况。结果显示:纯HA水凝胶不利于神经细胞在材料上粘附和突起生长;HA-NgR抗体-PLL水凝胶可以显著增加海马神经元的粘附数量、促进神经元突起形成,同时也能使星形胶质细胞在材料表面生长。上述结果提示:接枝NgR抗体和PLL的HA水凝胶与海马神经元相容性良好,为中枢神经系统损伤修复提供了一种较理想的支架材料。     

透明质酸改性的聚乳酸支架

背景：聚乳酸材料不具备细胞外基质材料的良好细胞亲和性能,采用化学方法将透明质酸交联制得的水凝胶具有良好的生物相容性。 目的：以透明质酸对新型多孔隙率聚乳酸支架的进行改性,观察改性后支架的细胞相容性的改变。 方法：采用盐析法制备出高孔隙率聚乳酸支架,采用低浓度NaOH进行表面轻度水解后,利用EDC和透明质酸进行支架的改性。 结果与结论：透明质酸改性聚乳酸支架在扫描电镜下显示为多微孔的三维立体结构,孔壁及界面平滑,孔隙之间可见更细小微孔相连。改性聚乳酸支架水滴渗入较快,改性后多孔支架的保水能力与吸水能力得到明显的改善;透明质酸改性聚乳酸支架上细胞黏附及增殖优于未改性聚乳酸支架。透明质酸改性聚乳酸组软骨细胞生长密度及基质分泌更加旺盛。表明透明质酸改性聚乳酸多孔支架仍保持多孔的三维结构,其水亲和力、吸水能力、保水能力和细胞相容性均得到明显改善。 关键词：透明质酸;聚乳酸;多孔支架;表面改性;水亲和力;吸水能力 doi:10.3969/j.issn.1673-8225.2012.03.023     

携带神经干细胞肌基膜管组织工程支架中神经干细胞的存活分化

背景:多项研究已证实神经干细胞能促进脊髓损伤大鼠神经功能的恢复,肌基膜管具有良好的细胞、组织相容性和降解性,那么能否将二者结合起来构建一个新的神经组织工程支架?目的:以神经干细胞为种子细胞,以肌基膜管为支架,观察携带神经干细胞的肌基膜管组织工程支架中神经干细胞的存活与分化情况。方法:体外分离培养大鼠神经干细胞,并进行鉴定。用化学萃取方法制作去细胞骨骼肌基膜管支架,将神经干细胞移植入肌基膜管支架培养7d后,用免疫组织化学方法检测神经干细胞的存活及分化情况,扫描电镜观察其超微结构。结果与结论:神经干细胞分离培养第5天,Nestin免疫荧光染色可见大量神经球。加血清诱导神经干细胞分化至7d,进行抗NF、抗GFAP免疫荧光染色,镜下可见NF、GFAP阳性细胞,证明培养的神经干细胞具有多项分化潜能。苏木精-伊红染色法显示肌基膜管中肌细胞成分已消失,肌基膜管支架内主要是大致平行的管道。携带神经干细胞的肌基膜管组织工程支架免疫荧光染色证明,神经干细胞在支架内仍具有干细胞特性,并可分化为神经元和神经胶质细胞。扫描电镜显示神经干细胞可以稳固地贴附在肌基膜管内,提示制备的神经组织工程支架具有良好的生物相容性,可以进行体内移植治疗脊髓损伤等神经系统疾病。     

低聚乙二醇富马酸酯水凝胶中骨髓来源间充质干细胞的生物学行为

文章快速阅读：  文题释义：水凝胶：是以水为分散介质的凝胶,具有网状交联结构的水溶性高分子中引入一部分疏水基团和亲水残基,亲水残基与水分子结合,将水分子连接在网状内部,而疏水残基遇水膨胀的交联聚合物。是一种高分子网络体系,性质柔软,能保持一定的形状,能吸收大量的水。凡是水溶性或亲水性的高分子,通过一定的化学交联或物理交联,都可以形成水凝胶。这些高分子按其来源可分为天然和合成两大类。天然的亲水性高分子包括多糖类(淀粉、纤维素、海藻酸、透明质酸,壳聚糖等)和多肽类(胶原、聚L-赖氨酸、聚L-谷胺酸等)。合成的亲水高分子包括醇、 丙烯酸及其衍生物类(聚丙烯酸,聚甲基丙烯酸,聚丙烯酰胺,聚N-聚代丙烯酰胺等)。低聚乙二醇富马酸酯水凝胶：是由聚乙二醇和延胡索酸酯与聚乙二醇二丙烯酸酯化学交联而成,具有良好的组织相容性和生物可降解性。实验证明低聚乙二醇富马酸酯水凝胶随着相对分子质量的升高其溶胀度降增加,溶胀比增加引起水凝胶降解速度加快及力学强度减弱。故研制与筛选具有合适溶胀比的低聚乙二醇富马酸酯水凝胶材料是进行骨组织工程支架材料研究的前提。   背景：低聚乙二醇富马酸酯水凝胶是一种具有良好生物相容性及可注射性和可降解性的生物材料。不同相对分子质量水凝胶之间的特性有所差异,将骨髓间充质干细胞包裹其中并诱导细胞成骨分化,相对分子质量相当的水凝胶更有利于细胞增殖和分化,所以采用该材料为骨组织工程支架提供了新的选择。目的：探讨不同相对分子质量的低聚乙二醇富马酸酯水凝胶材料体外包裹大鼠骨髓间充质干细胞的增殖和分化的影响。方法：低聚乙二醇富马酸酯通过氧化还原基团引发系统产生交联,制备出相对分子质量为1000,3000,10000,35 000的低聚乙二醇富马酸酯水凝胶,对水凝胶的溶胀和降解性能进行检测。将骨髓间充质干细胞包裹到4种相对分子质量的水凝胶中,在成骨培养液中诱导1-3周,通过组织学染色(苏木精-伊红染色和茜素红染色)和免疫荧光染色检测水凝胶材料对骨髓间充质干细胞形态的影响以及成骨分化的效果。 结果与结论：①随着水凝胶相对分子质量的增加,成胶时间变短,凝胶的溶胀度明显增加,且随着时间的推移,水凝胶的降解速率与相对分子质量成正比;②细胞复合水凝胶支架材料的组织学与免疫荧光染色结果表明,细胞经过诱导后,在具有适当溶胀与降解特性的相对分子质量为3 000与10 000的水凝胶中所形成的矿化结节数量显著多于在其它两种相对分子质量中的,说明有利于细胞的增殖与分化;③结果表明,低聚乙二醇富马酸酯水凝胶具有良好的生物相容性,且相对分子质量为3 000与     10 000的水凝胶对间充质干细胞的成骨分化有一定的良性调节作用。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程 ORCID:0000-0002-0616-3754(魏丽君)     

高分子水凝胶修复关节软骨损伤:安全与有效性评价

背景:高分子水凝胶与关节软骨的细胞外基质组成相似,可促进软骨细胞增殖、分化,形成软骨板,促进关节软骨的再生和修复。目的:阐述几种可降解天然高分子水凝胶及其在关节软骨修复组织工程中的最新研究进程及成果。方法:以"natural polymers,biodegradable polymers,hydrogel scaffold,articular cartilage,regeneration;关节软骨,水凝胶,天然聚合物,组织工程"为检索词,应用计算机检索从1994年1月至2013年7月PubMed数据库、Springer数据库、Sciencedirect数据库、Ovid数据库及CNKI数据库发表的天然高分子水凝胶材料相关文献。结果与结论:天然高分子水凝胶材料包括蛋白质类(胶原蛋白、明胶)及多糖类(壳聚糖、透明质酸)等。改性后天然高分子水凝胶不但具备关节软骨再生的理化特性,而且具有良好的生物特性,即组织相容性、低免疫原性、低细胞毒性、自身可降解性,同时可促进细胞黏附、增殖与分化,具备推动新组织再生的能力,甚至能够作为药物、生长因子等的缓释载体,在关节软骨再生及修复领域有着可观的应用前景。     

水凝胶材料和间充质干细胞在组织工程中的应用进展

随着组织工程学的发展, 人们越来越关注将水凝胶作为支架材料并与细胞3D培养相结合用于组织器官再生与修复。水凝胶由亲水性聚合物、共聚物或可以形成大分子链的单体大分子交联而成, 可吸收大量水分并保持3D结构, 具有良好的生物相容性、可包埋细胞和有效的递送生物活性分子等特点, 因而被广泛用于生物医药领域的药物输送和组织工程等领域。间充质干细胞可以从骨髓、脂肪、脐带等多种组织中获取, 具有低免疫原性及多向分化潜能, 是细胞3D培养以及细胞治疗的首选。目前间充质干细胞主要是2D培养模式, 该培养模式下的间充质干细胞繁殖率低, 且无法模拟体内的生长环境。水凝胶材料作为3D细胞培养支架具有良好的相容性, 可以模拟体内的生长环境, 在修复受损软骨、骨、皮肤和心脏等组织中有巨大潜力。概述水凝胶、间充质干细胞以及间充质干细胞和水凝胶材料在组织工程中的应用, 展示水凝胶材料与间充质干细胞的3D培养在不同组织再生和修复中的发展趋势和可能性, 以期为后续水凝胶和干细胞的深入应用研究提供参考。     

透明质酸水凝胶包裹骨髓间充质干细胞改善心肌梗死大鼠的心功能(Ⅱ)

文题释义：透明质酸：是一种线性的糖胺聚糖,在多种天然组织细胞外基质中含量丰富,参与细胞黏附、迁移、增殖及分化,在机体组织水分保持、关节润滑和损伤修复过程中发挥重要作用。透明质酸通过化学交联方法能形成具备一定特性和机械性能的水凝胶。可注射水凝胶修复梗死心肌的机制：①作为细胞移植的载体,通过其本身的黏滞性阻止因心脏跳动和静脉回流导致的细胞逃逸,提高移植细胞的滞留,并能为移植的细胞提供合适的三维立体生长环境,防止细胞的失巢凋亡,提高细胞的存活率;②作为蛋白或者生长因子的控制释放载体,防止这些生物活性分子被体内的酶降解,阻止其一过性释放,延长生物活性分子在体内的作用时间,并实现局部用药;③水凝胶通过材料堆积增加梗死区室壁厚度,提供力学支撑;④性能优良的水凝胶能为内源性修复创造条件,为干细胞植入提供附着支架;⑤水凝胶降解的活性生物片段招募内源性干细胞,促进心肌再生。背景：作者前期的研究结果显示透明质酸水凝胶包裹骨髓间充质干细胞可以改善大鼠心肌梗死后心功能。目的：探索透明质酸水凝胶包裹骨髓间充质干细胞对心肌梗死大鼠心肌组织学的影响。方法：分离培养雄性SD大鼠骨髓间充质干细胞,然后用透明质酸水凝胶包裹骨髓间充质干细胞在培养皿中进行体外三维培养。结扎雌性SD大鼠左冠状动脉前降支制作心肌梗死模型,1周后行超声检测,将符合条件的大鼠随机分为4组：①PBS组(n=8);②水凝胶组(n=8);③细胞组(n=29);④细胞+水凝胶组(n=29)。造模1周后将模型鼠行二次开胸,按照分组将PBS、透明质酸水凝胶、骨髓间充质干细胞、透明质酸水凝胶包裹骨髓间充质干细胞注射到梗死边缘区及梗死区。移植后4周,苏木精-伊红染色、Masson染色及免疫组化染色评价心脏的血管再生、心肌保护以及心室重塑。结果与结论：①移植后4周,与PBS组相比,水凝胶组的梗死区室壁较厚,细胞组的心肌细胞肥大减轻及血管密度增高,水凝胶+细胞组的心室重塑减轻、存活心肌增多以及血管密度增高;②结果表明,在组织学水平,透明质酸水凝胶包裹骨髓间充质干细胞取得修复梗死心肌的最大效应。ORCID:0000-0002-3332-2642(商青青) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

天然水凝胶的改性研究及在组织工程中的应用

天然高分子因其生物相容性好、生物降解能力强等优点,被广泛应用于组织工程、生物医药等领域。但天然高分子材料的降解性等缺点限制了其在组织工程,特别是人类临床医学上的应用,天然高分子水凝胶的改性研究成为组织工程学的热点研究领域。文章主要概述了以透明质酸、海藻酸盐、羧甲基壳聚糖、胶原蛋白为基础的天然水凝胶的改性研究及改性后的新型水凝胶的初步生物学评价,以及对未来可能应用领域的探讨,并展望了此类水凝胶的发展方向。     

Evaluation of biomimetic scaffold of gelatin-hydroxyapatite crosslink as a novel scaffold for tissue engineering: biocompatibility evaluation with human PDL fibroblasts, human mesenchymal stromal cells, and primary bone cells

Biomimetic gelatin (gel)-hydroxyapatite (HA) composites have been prepared for studying hard tissue engineering scaffolds. However, the biocompatibility test of this form of material using these three cell types, which are periodontal ligament (PDL) fibroblast cells, human mesenchymal stromal cells (HMSc) and primary cells from human hip bone (HBc) has never been evaluated. The objective of this article is to prepare and evaluate the biocompatibility of gel-HA crosslinked scaffold for tissue engineering. Two different scaffolds were prepared: preparation (1), 2.5% gel/2.5% HA; preparation (2), 2.5% gel/5% HA. Three cell types including PDL, HMSc, and HBc were used. Assessment of biocompatibility and osteoblastic cellular responses was evaluated using a three-dimensional cell culture method and scanning electron microscopy (SEM). From SEM, it was observed that scaffold (1) exhibits stable porous formation with well-blended and dispersed HA powder. All three cell types were able to proliferate in both scaffolds. The HMSc and HBc got attached to the scaffolds to a significantly higher degree and subsequently proliferated more than PDL. The alkaline phosphatase (ALP) activities of HMSc and HBc were stronger when cultured in scaffold (S1) than (S2). It was seen that the two scaffold preparations show good biocompatibility with all three cell types tested. The better cellular responses with scaffold (S1) than (S2) might be due to the different structural and morphological characteristics, that is, scaffold (S1) retained more small-sized apatite crystals and a better developed pore configuration than scaffold (S2). Based on these findings, the biomimetically synthesized composite scaffolds have the potential to be used in hard tissue regeneration and tissue engineering fields.     

Hyaluronic acid-poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury

Brain tissue engineering in the postinjury brain represents a promising option for cellular replacement and rescue, providing a cell scaffold for either transplanted or resident cells. In this article, a hyaluronic acid (HA)-poly-D-lysine (PDL) copolymer hydrogel with an open porous structure and viscoelastic properties similar to neural tissue has been developed for brain tissue engineering. The chemicophysical properties of the hydrogel with HA:PDL ratios of 10:1, 5:1, and 4:1 were investigated by scanning electron microscopy (SEM) and X-ray photoelectron spectrometry. Neural cells cultured in the hydrogel were studied by phase-contrast microscope and SEM. The incorporation of PDL peptides into the HA-PDL hydrogel allowed for the modulation of neuronal cell adhesion and neural network formation. Macrophages and multinucleated foreign body giant cells found at the site of implantation of the hydrogel in the rat brain within the first weeks postimplantation decreased in numbers after 6 weeks, consistent with the host response to inert implants in numerous tissues. Of importance was the infiltration of the hydrogel by glial fibrillary acidic protein-positive cells-reactive astrocytes-by immunohistochemistry and the contiguity between the hydrogel and the surrounding tissue demonstrated by SEM. These findings indicated the compatibility of this hydrogel with brain tissue. Collectively, the results demonstrate the promise of an HA-PDL hydrogel as a scaffold material for the repair of defects in the brain.     

Characterization of neural stem cells on electrospun poly(epsilon-caprolactone) submicron scaffolds: evaluating their potential in neural tissue engineering

Development of biomaterials with specific characteristics to influence cell behaviour has played an important role in exploiting strategies to promote nerve regeneration. The effect of three-dimensional (3D) non-woven electrospun poly(epsilon-caprolactone) (PCL) scaffolds on the behaviour of rat brain-derived neural stem cells (NSCs) is reported. The interaction of NSCs on the randomly orientated submicron (PCL) fibrous scaffolds, with an average fibre diameter of 750 +/- 100 nm, was investigated. The PCL scaffolds were modified with ethylenediamine (ED) to determine if amino functionalisation and changes in surface tension of the fibrous scaffolds affected the proliferation and differentiation characteristics of NSCs. Surface tension of the fibrous scaffold increased upon treatment with ED which was attributed to amine moieties present on the surface of the fibres. Although surface treatment did not change the differentiation of the NSCs, the modified scaffolds were more hydrophilic, resulting in a significant increase in the number of adhered cells, and increased spreading throughout the entirety of the scaffold. When the NSCs were seeded on the PCL scaffolds in the presence of 10% FBS, the stem cells differentiated primarily into oligodendrocytes, indicating that electrospun PCL has the capacity to direct the differentiation of NSCs towards a specific lineage. The data presented here is useful for the development of electrospun biomaterial scaffolds for neural tissue engineering, to regulate the proliferation and differentiation of NSCs.     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

骨髓间充质干细胞在淫羊藿苷/羟基磷灰石/聚乳酸-羟基乙酸共聚物支架上的成骨

文题释义：纳米结构：是尺寸介于分子和微米尺度间的物体结构。当纳米羟基磷灰石与高分子材料物理混合后,羟基磷灰石会发生自聚,从而在材料表面产生纳米结构。这种纳米结构有利于细胞(如骨髓充间质干细胞)的黏附,是骨修复材料表面细胞增殖和后期成骨分化的基础。成骨分化：当干细胞接受诱导时可以向成骨细胞转变。淫羊藿苷高分子复合支架与间充质干细胞共培养一段时间后,其骨分化标志物碱性磷酸酶和骨钙素的活性增高,同时成骨相关基因和蛋白(Runx-2、COLⅠ)表达水平上升,即细胞在淫羊藿苷诱导下发生了成骨分化。  摘要背景：近年来,骨组织工程技术为临床治疗骨缺损提供了全新的思路和模式。该研究首次将传统中药与组织工程支架的纳米结构结合,以期探索并构建一种可用于骨缺损治疗的新型骨组织替代材料。目的：研究淫羊藿苷(icariin,ICA)/羟基磷灰石(hydroxyapatite,HA)/聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)复合支架的成骨活性。方法：将HA与PLGA通过物理共混的方式制成HA/PLGA复合支架,然后将其浸泡于不同浓度的ICA溶液中,从而得到ICA/HA/PLGA支架。利用兔骨髓间充质干细胞分别对复合支架的细胞黏附、增殖、成骨作用和细胞毒性进行评价。细胞黏附、细胞增殖和细胞毒性采用MTT法进行检测,碱性磷酸酶活性和骨钙素活性采用ELISA法进行检测,成骨相关基因和蛋白表达水平分别用荧光定量PCR和Western blot法进行检测。结果与结论：①PLGA中加入适量HA可以提高支架的力学强度,且在HA含量为10%时效果最佳,拉伸强度为(1.67±0.37) MPa;压缩模量为(4.17±1.62) MPa,且会在支架表面形成纳米结构;该微结构可以促进骨髓间充质干细胞在支架表面的黏附;②ICA不会影响骨髓间充质干细胞在复合支架上的增殖,且1.00 µmol/L ICA水溶液浸泡后的ICA/HA/PLGA复合支架具有最优的成骨分化功能,其碱性磷酸酶活性、骨钙素活性、成骨相关基因和蛋白(Runx-2和COLⅠ)的表达水平均最高;③ICA/HA/PLGA复合支架无细胞毒性;④结果表明,HA(10%)/ICA(1.00 µmol/L)/PLGA支架具有良好的机械性能、成骨作用和生物相容性,是一种具有良好应用潜力的骨组织工程支架。ORCID: 0000-0002-9770-9109(王德欣) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Silicon nanowires enhanced proliferation and neuronal differentiation of neural stem cell with vertically surface microenvironment

Owing to its biocompatibility, noncytotoxicity, biodegradability and three-dimensional structure, vertically silicon nanowires (SiNWs) arrays are a promising scaffold material for tissue engineering, regenerative medicine and relevant medical applications. Recently, its osteogenic differentiation effects, reorganization of cytoskeleton and regulation of the fate on stem cells have been demonstrated. However, it still remains unknown whether SiNWs arrays could affect the proliferation and neuronal differentiation of neural stem cells (NSCs) or not. In the present study, we have employed vertically aligned SiNWs arrays as culture systems for NSCs and proved that the scaffold material could promote the proliferation and neuronal differentiation of NSCs while maintaining excellent cell viability and stemness. Immunofluorescence imaging analysis, Western blot and RT-PCR results reveal that NSCs proliferation and neuronal differentiation efficiency on SiNWs arrays are significant greater than that on silicon wafers. These results implicate SiNWs arrays could offer a powerful platform for NSCs research and NSCs-based therapy in the field of neural tissue engineering.     

Growth and metabolism of human hepatocytes on biomodified collagen poly(lactic-co-glycolic acid) three-dimensional scaffold

Hepatic tissue engineering offers a promising approach toward alleviating the need for donor liver, yet many challenges must be overcome including choice of scaffold, cell source, and immunologic barriers. Poly(lactic-co-glycolic acid) (PLGA) polymers are innovative biodegradable materials that have been shown to be useful as scaffolds for seeding and culturing various types of cells. In this study, a porous sponge scaffold of modified PLGA polymer with collagen was investigated for its ability to improve the growth and metabolism of human hepatocytes. We evaluated the biocompatibility of collagen-modified PLGA (C-PLGA) scaffolds with hepatocytes isolated from human liver. Cell adhesion and function (cell density, culture lifespan, albumin synthesis, urea synthesis, and ammonia elimination and diazepam clearance) were assessed during different culture periods. The number of hepatocytes cultured in C-PLGA scaffolds was higher compared with those cultured in PLGA scaffolds without collagen modification, and the lifespan of hepatocytes cultured in C-PLGA scaffolds was longer than that of cells cultured in PLGA scaffolds. Albumin and urea synthesis and ammonia elimination from attached hepatocytes were greater in C-PLGA than in PLGA scaffolds, with the exception of diazepam clearance. Collagen-modified PLGA scaffold is a promising biomaterial for hepatic tissue engineering.     

SHH修饰聚多巴胺涂层纤维蛋白支架对大鼠神经干细胞的影响

目的:构建音猬因子(SHH)修饰的聚多巴胺(PD)涂层纤维蛋白胶(FG),并探究该支架对大鼠神经干细胞(NSCs)的生长及分化的影响。方法:按FG材料表面涂层情况,将实验分为3组:FG表面未处理组(control)、FG表面进行多巴胺涂层组(PD)和FG经多巴胺处理后表面涂有SHH组(PD-SHH)。采用扫描电子显微镜分别观察支架微观结构,用ELISA测量PD-SHH组SHH。分离并培养大鼠神经干细胞,并将神经干细胞分别与该3种支架复合培养,免疫荧光及Western Blot检测各组细胞中胶质纤维酸性蛋白(GFAP)、生长再生相关蛋白(GAP43)和髓鞘碱性蛋白(MBP)的表达。结果:ELISA结果显示PD-SHH缓释支架可以持续释放SHH达到19 d;扫描电子显微镜可见纤维蛋白胶经冷冻干燥后呈海绵型网状结构,多巴胺及SHH微粒粘附在网状结构上;免疫荧光及Western Blot结果显示:PD-SHH组细胞高表达GAP43和MBP等神经细胞相关蛋白而低表达GFAP(P<0.05)。结论:制备的聚多巴胺涂层纤维蛋白支架具有良好的SHH缓释效果,该复合支架对大鼠NSCs分化有明显的促进作用。     

Chitosan/gelatin porous scaffolds containing hyaluronic acid and heparan sulfate for neural tissue engineering

The novel chitosan (Cs)/gelatin (Gel) porous scaffolds containing hyaluronic acid (HA) and heparan sulfate (HS) were fabricated via freeze-drying technique, and their physicochemical characteristics including pore size, porosity, water absorption, and in vitro degradation and biocompatibility were investigated. It was demonstrated that the Cs/Gel/HA/HS composite scaffolds had highly homogeneous and interconnected pores with porosity above 96% and average pore size ranging from 90 to 140?μm and a controllable degradation rate. The scanning electron microscopic images, cell viability assay, and fluorescence microscopy observation revealed that the presence of HA and HS in the scaffolds significantly promoted initial neural stem and progenitor cells (NS/PCs) adhesion and supported long-time growth in three-dimensional environment. Moreover, NS/PCs also maintained mutilineage differentiation potentials with enhanced neuronal differentiation upon induction in the Cs/Gel/HA/HS composite scaffolds in relation to Cs/Gel scaffolds. These results indicated that the Cs/Gel/HA/HS composite scaffolds were suitable for neural cells’ adhesion, survival, and growth and could offer new and important options for neural tissue engineering applications.     

新型脊髓纳米组织工程支架的组织相容性

背景：纳米技术可改善脊髓组织工程生物材料的性能。 目的：分析新型脊髓纳米组织工程支架的组织相容性。 方法：以胶原为原料制备纤维定向排列及非定向排列的纳米纤维膜,培养及鉴定SD大鼠脊髓源性神经干细胞。将两种纳米纤维膜与SD乳鼠脊髓源性神经干细胞共培养,以正常培养的神经干细胞为对照,通过MTT实验检测纳米纤维膜的细胞相容性;以扫描电镜检测细胞在纳米纤维膜表面的黏附及增殖情况;将纳米纤维膜植入SD大鼠体内,通过组织学检查确定其降解情况及组织相容性;通过免疫组织化学实验确定神经干细胞在体内的存活及移动情况。 结果与结论：两种纳米纤维膜表面的神经干细胞黏附及增殖情况良好,MTT实验结果表明纳米纤维膜的细胞相容性佳,电镜结果表明细胞在纳米纤维膜表面黏附良好,增殖情况佳;在体内纳米纤维膜降解情况良好,组织相容性佳;BrdU标定的神经干细胞在SD大鼠体内存活并移动情况良好。结果表明新型纳米组织工程支架具有良好的细胞及组织相容性。