自组装多肽骨修复水凝胶的研究进展

开发具有成骨诱导的骨填充材料是促进骨再生的重要研究方向。自组装多肽水凝胶凭借其高度的仿生人工细胞外基质结构、低免疫原性、易于合成及修饰、载药灵活等优势为骨组织修复提供了一个高效治疗手段。本文讨论了自组装多肽水凝胶的设计原则,报道了自组装多肽结构特征及组装机制,重点介绍了自组装多肽骨修复水凝胶在递送干细胞、血管内皮细胞、骨形成蛋白、成骨因子以及小分子化合物等方面的最新研究进展,总结了限制自组装多肽水凝胶发展的瓶颈和未来发展方向,为构建高成骨性能凝胶递送系统提供理论参考。     

BMSCs在功能化自组装多肽水凝胶中定向分化神经细胞研究

目的:探讨骨髓基质干细胞(BMSCs)在功能化自组装多肽水凝胶中定向分化为神经细胞及分化后细胞的功能特征。方法:制备功能化自组装多肽水凝胶,分别将BMSCs接种于RADA16自组装多肽水凝胶(对照组)与功能化自组装多肽水凝胶(实验组)表面,观察细胞迁移情况。用预诱导剂b FGF和EGF及定向诱导剂(SHH+RA)时序诱导,应用Nestin、MAP2、GFAP、Ch AT和VAT染色,比较分化后神经样细胞的形态和功能标志的差异。结果:BMSCs在功能化自组装多肽水凝胶中共培养及诱导剂的特定组合和时序的诱导下增殖分化呈现神经元细胞样改变,MAP2阳性细胞百分率较对照组显著提高(P<0.05),GFAP阳性细胞百分率较对照组显著降低(P<0.05),且功能标志Ch AT和VAT只在实验组表达。结论:BMSCs在功能化自组装多肽水凝胶中能定向诱导分化为具有功能的神经细胞。     

RGD多肽纳米纤维凝胶的自组装及其与脂肪干细胞生物相容性

目的 研究精氨酸-丙氨酸-天门冬氨酸(RGD)多肽纳米纤维凝胶在体外的自组装及其与脂肪干细胞的生物相容性.方法 固相法合成含RGD序列的多肽,加入含0.5 mol/L Ca2+的DMEM诱导其自组装,透射电镜观察自组装效果.将其与体外分离培养的大鼠脂肪干细胞复合培养,倒置显微镜下观察细胞的生长情况,Calcein-AM...     

功能化自组装多肽水凝胶与嗅鞘细胞生物相容性研究

Olfactory ensheathing cell (OEC) transplantation is a promising or potential therapy for spinal cord injury (SCI). However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell migration. In this study, we made a new peptide hydrogel scaffold which named GRGDSPmx by mixing the pure RADA16 and designer-peptide RADA16-GRGDSP solution, and we examined the molecular integration of the mixed nanofiber scaffolds using Atomic force microscopy (AFM). In addition, we have studied the behavior of OECs in GRGDSPmx condition as well as on RADA16 scaffold by analyzing their phenotypes such as cell proliferation, apoptosis, survival and morphology. The experimental results showed that GRGDSPmx could be self-assembled to form a hydrogel. Inverted optical microscope and Scanning electron microscope (SEM) analysis showed that OECs are viable and they proliferate within the nanostructured environment of the scaffold. MTT assay demonstrated that OECs proliferation rate was increased on GRGDSPmx scaffold compared to the pure RADA16 scaffold. In addition, OECs on GRGDSPmx scaffolds also showed less apoptosis and maintained the original spindle-shape morphology. Calcein-AM/PI fluorescence staining revealed that OECs cultured on GRGDSPmx grew well and the viable cell count was 95%. These results suggested that this new hydrogel scaffold provided an ideal substrate for OEC 3D culture and suggested its further application for SCI repair.     

多肽水凝胶复合支架对大鼠脂肪干细胞体外增殖和分化的影响

目的:研究多肽水凝胶复合骼金(nano-hydroxyapatite/collagen, nHAC)对大鼠脂肪干细胞(adipose-derived stem cells,ADSCs)体外增殖和早期骨向分化的作用?方法:用1%的多肽水凝胶与nHAC制备复合支架,未复合水凝胶的nHAC为对照组,使用扫描电子显微镜(field emission scanning electron microscope,FESEM)和共聚焦显微镜(Confocal laser scanning microscope,CLSM)进行表征,将ADSCs接种到两种支架材料上,以CCK-8检测1?3?5?7 d的增殖情况,以碱性磷酸酶(ALP)活性为早期骨向分化指标,检测其3?5?7 d 的活性?结果:多肽水凝胶在nHAC表面形成涂层,ADSCs在有涂层的nHAC上黏附?铺展得更好,两种支架上的ADSCs在1?3?5?7 d都持续增殖,其中两组间在1?3 d无差异,5?7 d有差异,实验组增殖更快(P < 0.05);ALP值在3?5?7 d都持续增高,实验组更高,两组间有差异(P < 0.05),但在3?5?7 d时两两比较差异不显著(P > 0.05)?结论:多肽水凝胶复合支架材料能显著促进ADSCs的黏附增殖,对细胞早期骨向分化有一定的促进作用,但不如对细胞增殖的促进作用显著?     

脱细胞骨基质对骨髓间充质干细胞成骨及示踪的观察

目的观察脱细胞骨基质材料（ACBM）与骨髓基质干细胞（MSCs）复合,并种植到动物体内后细胞的成骨及定位情况。方法分离培养绿色荧光蛋白（GFP）小鼠MSCs,体外扩增后种植到脱细胞骨基质材料并观察材料对细胞粘附的影响;复合物植入裸鼠骶部肌袋作为实验侧,并以单纯ACBM材料植入作为对照侧,4周后进行大体观察、组织学检查x线及荧光检测。结果细胞材料共培养后6小时观察见材料表面粘附细胞伸展、之后细胞间出现连接、活性表达;体内植入后4周,X线及组织学观察实验侧植人物为中心成骨明显,荧光显微镜下见新生骨组织中BCD阳性细胞较多。对照侧材料逐渐吸收。结论 ACBM作为支架材料有助于MSCs的粘附、生长和成骨活性的表达。     

骨髓间充质干细胞复合仿生骨支架成骨效应的研究

目的 构建骨髓间充质干细胞(BMSCs)复合生物支架材料组织工程骨,探讨低温3D打印联合冷冻干燥法制备的三维仿生复合支架材料内BMSCs增殖、分化、成骨特异性基因表达情况.方法 利用低温3D打印结合真空冷冻干燥法制备三维支架,提取新西兰大白兔的BMSCs,用倒置显微镜及免疫组化鉴定BMSCs的形态及特征.将第3代BMS...     

自组装多肽在医学领域应用研究进展

在现代医学中,随着材料学和组织工程不断发展,出现了许多新型的生物材料.自组装多肽是一种新型纳米层次的生物材料,可以存在于不同环境中,具有独特结构的医学生物再生材料.根据其理化性质,自组装多肽可随之变化成多种结构,每种结构都有相对应的功能.它不仅可作为细胞及组织再生的支架,还可作为药物靶向治疗的载体减少药物不良反应等,给...     

Lysine-Cyclic RGD肽对骨髓间充质干细胞黏附、增殖和分化的影响

目的 研究物理电荷与生物配基两种因素相结合的新型表面修饰剂Lysine-Cyclic RGD(LcRGD)肽对骨髓间充质干细胞(mesenchymal stem cells,MSCs)的黏附、增殖及分化的影响.方法 以光学接触角仪衡量LcRGD肽修饰表面的亲水性;以离心黏附实验、震荡黏附实验评价LcRGD肽对MSCs的黏附性能;以CCK-8实验评价LcRGD表面改性DBM上MSCs的增殖活力;以Western blot法检测LcRGD表面改性脱钙骨基质(decalcified bone matrix,DBM)上MSCs的Runx2和OCN蛋白的表达.结果 LcRGD肽修饰表面接触角为(29.33±4.06)°,远低于普通cRGD肽修饰表面的接触角(63.72±8.65)°,提示LcRGD肽具有更好的亲水性(P＜0.01);离心黏附实验和震荡黏附实验显示相对于普通cRGD肽,LcRGD肽从细胞黏附早期开始便具有更好的细胞黏附性能;CCK-8实验提示MSCs在LcRGD修饰的DBM支架材料上的增殖活力高于普通cRGD肽修饰的DBM支架材料(P＜0.05);Western blot结果提示相对于普通cRGD肽修饰的DBM,MSCs在LcRGD修饰的DBM上表达更高的成骨标志蛋白水平,提示LcRGD肽具有更好的骨诱导性.结论 相对于普通的cRGD肽,物理电荷与生物配基相结合的LcRGD肽具有更好的细胞黏附、促增殖和骨诱导性能.     

姜黄素促进大鼠骨髓间充质干细胞的体外增殖及成骨分化

目的：研究姜黄素对大鼠骨髓间充质干细胞（rat bone marrow mesenchymal stem cell,rBMSC）生物学行为的影响。方法：将含不同浓度梯度姜黄素溶液的完全培养基与rBMSC共培养,通过CCK?8法检测细胞增殖情况,确定姜黄素溶液的最适浓度;将rBMSC与4 μg/mL姜黄素共孵育,0 μg/mL姜黄素处理组作为对照,通过碱性磷酸酶（alkaline phosphatase,ALP）染色和活性检测评估其早期成骨分化水平;使用茜素红染色评估成骨分化晚期细胞外基质矿化情况;利用实时荧光定量逆转录PCR检测细胞成骨诱导7、14 d时成骨指标骨形态发生蛋白（bone morphogenetic protein?2,Bmp2）、核心结合因子α1（core binding factor alphal α1,Runx2）、骨钙素（osteocalcin,Ocn）、骨桥蛋白（osteopontin,Opn）、成骨细胞特异基因（osterix,Osx）的表达。结果：CCK?8结果显示含不同浓度姜黄素溶液的培养基中的rBMSC均能持续增殖,4 μg/mL姜黄素组对rBMSC增殖的促进作用最为显著（与对照组相比,P < 0.001）;ALP活性检测及染色结果显示姜黄素组细胞的ALP活性高于对照组（P < 0.001）;茜素红染色结果显示姜黄素组的细胞外基质矿化水平高于对照组;实时荧光定量逆转录PCR结果显示姜黄素组细胞的相关成骨基因Bmp2、Runx2、Ocn、Opn、Osx的表达均高于对照组（P < 0.05）。结论：姜黄素能够促进rBMSC的体外增殖及成骨分化。     

Compatibility of olfactory ensheathing cells with functionalized self-assembling peptide scaffold in vitro

Background Olfactory ensheathing cell （OEC） transplantation is a promising or potential therapy for spinal cord injury （SCI）. However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell miqration.     

新型自组装多肽水凝胶对成骨细胞前体细胞作用的体外研究

目的 观察新型自组装肽TRSAWmx水凝胶对在体外小鼠成骨细胞前体细胞MC3T3-E1细胞粘附效果、增殖能力及成骨分化能力的影响.方法 通过原子力显微镜观察新型水凝胶的自组装性能;以倒置显微镜对贴壁细胞计数,比较不同材料细胞的粘附;以CCK-8检测和评价不同材料上细胞的增殖;通过激光共聚焦显微镜观察细胞在材料表面的生长;茜素红-S染色观察钙结节形成;检测材料上培养的细胞碱性磷酸酶(alkaline phosphatase,ALP)活性以及细胞ALP、骨钙蛋白(osteocalcin,OCN)、血管内皮生长因子(vascular endothelial growth factor,VEGF)等基因的表达.结果 TRSAWmx能自组装成纳米纤维结构;在接种30、60、90 min时,TRSAWmx组细胞粘附数明显高于空白组(P＜0.05);3、5、7d时TRSAWmx组细胞有着更佳的增殖活力(P＜0.05);在成骨诱导培养3、7、14 d时,TRSAWmx组ALP活性明显高于RADA16-Ⅰ组(P＜0.05);培养14 d后,TRSAWmx组茜素红-S染色可见钙结节形成多,成骨相关基因ALP、OCN、VEGF表达水平更高(P＜0.05).结论 新型自组装多肽纳米水凝胶TRSAWmx在体外能有效提高MC3T3-E1细胞粘附、增殖和成骨分化能力.     

利用软骨细胞外基质来源的多孔支架与骨髓基质干细胞体外长期培养组织工程化软骨的试验研究

研究背景 关节软骨损伤临床常见,但损伤后自我修复能力极差,目前的修复方法均有其局限性。在先前的研究中,我们研制了关节软骨细胞外基质来源的多孔支架（Cartilage ECM-derived porous scaffold, CEDPS）并观察了并在裸鼠体内异位构建软骨。但在进一步可能的临床应用之前,应进一步评估其体外培养组织工程软骨的特点及可行性。 方法 本研究利用关节软骨细胞外基质来源的支架及骨髓基质干细胞体外长时间培养构建软骨组织。粉碎人关节软骨,脱细胞处理后差速离心法收集细胞外基质悬液,采用冷冻干燥技术制备三维多孔支架。扫描电镜及Micro-CT观察其微观结构,并进行细胞毒性试验,组织学观察,生化成分定量检测其胶原、氨基葡聚糖（GAG）、DNA含量,生物力学方法测量其干性及覆水状态下压缩弹性模量;骨髓基质干细胞经含TGF-β1, bFGF的条件培养基成软骨诱导后鉴定,种植到支架上,荧光显微镜及扫描电镜观察细胞黏附情况,Dead/Live免疫荧光染色观察支架内部细胞活性,体外培养1,3周后观察大体形态和组织学形态变化,同时行II型胶原免疫组织化学分析。 结果 制备的CEDPS支架无细胞碎片残留,软骨细胞外基质特异性染色阳性,具有相互贯通的三维孔隙结构;支架生化成分定量检测：总胶原含量为708.2?44.7μg/mg,GAG含量为254.7?25.9μg/mg;支架纵向压缩弹性模量E = 1.226?0.288MPa,覆水后压缩弹性模量E = 0.052?0.007MPa。体外培养的BMSCs-CEDPS复合体形成了类软骨样组织,Dead/Live染色表明支架内部均为活细胞,电镜检查结果表明细胞广泛均匀的分布在支架内部,呈圆形或椭圆形,在支架上增殖显著,细胞基质分泌明显。组织学结果表明蕃红花“O”、Ⅱ型胶原免疫组化染色阳性,表明随着培养时间的增加,细胞在支架中增殖显著,细胞外有大量基质分泌。 结论 CEDPS支架在生化组成和结构上与软骨细胞外基质成分类似,去细胞彻底,具有良好的生物力学特性,是一种较为理想的软骨组织工程支架载体;成软骨诱导的BMSCs与CEDPS支架在体外可初步构建类软骨样组织。     

In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.     

髓核去细胞基质复合骨髓间充质干细胞体外构建组织工程髓核的研究?

目的：体外培养兔骨髓间充质干细胞(rBMSCs)-髓核去细胞基质支架(NPAMS)复合体(rBMSCs-NPAMS)构建组织工程髓核。方法制备若干 NPAMS,接种 BMSCs 至 NPAMS 体外培养分为 NPAMS 组、实验组和正常对照组(正常髓核)。肉眼及显微镜观察复合体形态变化,并行扫描电镜(SEM)、苏木素-伊红(HE)染色、免疫组织化学、实时定量 PCR(qRT-PCR)、支架的生物力学等检测。结果肉眼下 rBMSCs-NPAMS 形态接近正常髓核;SEM 显示细胞在支架表面大量黏附、并向深部迁移,表面细胞密度比横截面细胞密度大;HE 染色表明随时间延长,rBMSCs-NPAMS 内细胞量递增,分布更广泛;免疫组织化学显示细胞外基质2型胶原(CollagenⅡ)分泌量随时间递增,且实验组 CollagenⅡ表达量大于 NPAMS 组,小于正常对照组;qRT-PCR结果：NPAMS 组未提取到 mRNA,实验组 CollagenⅡ、聚集蛋白聚糖(Aggrecan)mRNA 相对表达量呈时间依赖性递增,但均小于正常对照组(P <0.01),支架与正常髓核在相同位移下的压缩载荷差异无统计学意义(P >0.05)。结论采用髓核体外 NPAMS复合 rBMSCs 可成功构建组织工程髓核。     

大鼠骨髓间充质干细胞与脱细胞脊髓支架体外共培养

目的 评价脱细胞脊髓支架在大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)体外培养中的作用,探索最佳接种浓度.方法 分离、培养及鉴定SD大鼠BMSCs,诱导BMSCs向神经元样细胞分化.制备脱细胞脊髓支架,将第3代BMSCs同脱细胞脊髓支架共培养,MTT法检测细胞增殖活性,比较与普通培养的差异;取第3代BMSCs以不同浓度[(0.5、1、2、3、4)×106/mL]接种于支架(脊髓支架修整0.5 cm小段),检测细胞在支架上的粘附率及上架细胞数,建立接种浓度与细胞粘附率、上架细胞数的关系回归方程;扫描电镜观察脱细胞脊髓支架形态以及细胞与支架的粘附情况.结果 成功实现BMSCs的分离及培养,BMSCs流式鉴定CD29、CD90阳性表达,向神经元诱导胶质纤维酸性蛋白(GFAP)、巢蛋白(Nestin)呈阳性表达;与普通培养相比,BMSCs在支架上细胞增殖活力显著提高(P＜0.05);细胞与支架的粘附率在接种浓度为2×106/mL最高,达到(79.6±2.7)％,单位体积上架细胞数达到(1.364±0.047)×106/cm3;扫描电镜观察支架空间结构良好,细胞与支架粘附良好,共培养第3天较第1天细胞数量明显增加.结论 脱细胞脊髓支架具有多通道的空间结构,适合BMSCs粘附、存活、增殖,该支架是良好的天然脊髓组织工程材料.当粘附率及细胞上架数基本达到最大时的最佳接种浓度为2×106/mL.     

Neurogenesis of adipose-derived stem cells in hydrogel

Adipose tissue is a readily available source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Peptide hydrogel is a novel biomaterial which provides three-dimensional microenvironments for a variety of cells for tissue grafting. In this study, adipose-derived stem cells (ADSCs) were isolated from rats, seeded into the peptide hydrogel polymer scaffolds and cultured in Neurobasal (NB) media supplemented with B27, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Ten days after the culture, some cells were expanded into clonal populations in which the expression of both Nestin and Brdu was detected but only Brdu expression was detected in the cells that were not expanded into clonal populations. Our results suggested that ADSCs in peptide hydrogel polymer scaffolds can be induced to differentiate into cells capable of expressing the neuron-associated markers, self-renewal and self-propagation.