生物材料表面性能调控骨髓间充质干细胞分化

结合细胞和生物可降解支架的组织工程和再生医学技术为组织、器官的修复和再生提供了一种新途径。骨髓间充质干细胞(BMSCs)具有多向分化潜能,因其取材简单、来源广泛、增殖能力强,无伦理争议,免疫排斥反应小而备受关注。BMSCs在特定区域定向分化成为靶细胞是干细胞治疗的一个重要前提,尤其受到生物材料表面正负电荷、亲疏水和不同的拓扑结构的影响。材料表面涂层蛋白或接枝多肽能够促进BMSCs的分化能力,而生物材料不同的机械性能、几何形状也会影响BMSCs的分化方向。本文综述了近期生物材料调控BMSCs分化的研究结果,为基于BMSCs的组织工程和再生医学材料的设计提供借鉴和指导。     

5-氮胞苷诱导骨髓间充质干细胞向心肌细胞分化的研究

分离大鼠骨髓间充质干细胞,体外培养呈现成纤维细胞表型,用12μmol//L 5-氮胞苷(5-aza)诱导培养,一周后细胞变为细长形成杆状.两周后培养细胞与临近的细胞融合,三周后形成类肌管状结构.通过RT-PCR检测,5-氮胞苷诱导前,间充质干细胞表达α-actin,desmin和MEF-2D,5-aza诱导后表达β-MHC和GATA4.westem Blot分析结果表明α-actin在诱导前后都表达,而myosin则在诱导后才表达.免疫荧光标记α-actin和β-MHC,证实了上述结果,即在5-氮胞苷诱导前后细胞表达α-actin,诱导后myosin才在细胞质中表达,Myosin和β-MHC是心肌细胞特异表达的蛋白.上述结果表明骨髓间充质干细胞具有向新生心室肌分化的潜能,这些诱导分化的细胞为心肌梗塞移植治疗提供一种潜在的供体细胞.     

中国小型猪骨髓间充质干细胞的蛋白质组研究

报道了骨髓间充质干细胞(MSCs)的蛋白质组表达研究。从体外培养的MSCs提取细胞蛋白,经二维电泳分离后用银染方法可检出蛋白点约1600个,选取48个蛋白点进行胶内酶解及质谱分析,经数据库检索成功鉴定了37个蛋白,并对蛋白功能进行初步分析。本实验数据为进一步分析MSCs增殖、分化或凋亡的分子机理提供相关信息。     

大鼠骨髓间充质干细胞分化过程的比较蛋白质组学研究

从蛋白质组学角度分析大鼠骨髓间充质干细胞(MSCs)体外定向分化为心肌细胞过程中蛋白表达情况, 采用二维电泳分离蛋白, 用PDQuest软件分析蛋白表达差异, 并采用质谱(MALDI-TOF-MS)进行鉴定, 得到了54个蛋白点, 对蛋白的生物功能分析表明, 部分蛋白通过不同的信号途径参与了MSCs的分化过程.     

应用原子力显微镜研究淫羊霍苷诱导人脐带间充质干细胞成骨分化

利用碱性磷酸酶(ALP)染色和钙结节(Vonkossa)染色的方法对诱导21 d的淫羊霍苷诱导人脐带间充质干细胞进行鉴定;应用原子力显微镜(AFM)观察淫羊霍苷的形貌和人脐带间充质干细胞诱导0、5、10、15、21 d后的细胞形貌。结果表明,经成骨诱导分化21 d后,ALP染色呈强阳性,Vonkossa染色可见明显钙结节。AFM分析表明,淫羊霍苷在盖玻片上呈分散状分布,在细胞表面上聚集并呈微米域分布。实验发现,由于吸附在细胞表面时,被细胞膜分子包裹,更有利于在细胞表面的吸附,进入细胞内部,细胞表面的淫羊霍苷颗粒较在盖玻片上时增大,由淫羊霍苷颗粒进入细胞后在细胞表面留下一些小孔,可知其通过进入细胞内部诱导成骨分化。分化后,细胞表面有小突触,是由成骨分化后细胞内形成钙结节造成。     

中药龟板提取物化学成分及其调控鼠骨髓间充质干细胞(rMSCs)增殖活性的实验研究

中药龟板提取物浸膏经硅胶柱层析, 用石油醚-乙酸乙酯作洗脱剂, 梯度洗脱, 得到16个组分; 采用MTT法和流式细胞技术研究了它们对鼠骨髓间充质干细胞(rMSCs)的增殖作用, 实验结果显示组分Ts-12具有促进rMSCs增殖的作用(p＜0.05), 而组分Ts-4则表现出抑制rMSCs增殖的作用(p＜0.05), 其它样品对rMSCs的增殖作用不显著(p＞0.05), 不具统计学意义. 采用了GC-MS分析各组分化学成分, 并用HPLC和9个标准物质鉴定确证了组分Ts-12中含有十六酸甲酯(S-1)、十六酸乙酯(S-3)、十八酸甲酯(S-4)和甾醇(S-7)以及十四酸甾醇酯(S-8), 组分Ts-4主要含有十八酸(S-5). 对标准品也采用了MTT法和流式细胞仪研究了它们对rMSCs的增殖作用, 结果表明十四酸甾醇酯和十六酸甲酯具有促进rMSCs增殖的作用, 而十八酸具有抑制作用.     

球形羟基磷灰石纳米颗粒的可控合成及其对间充质干细胞生长分化的影响

本文采用CTAB为添加剂进行球形纳米相羟基磷灰石(nHAP)的可控合成，并采用透射电子显微镜(TEM)、X-射线衍射仪(XRD)、傅立叶变换红外光谱仪(FTIR)和精密接触角测量仪对所制得的纳米颗粒的物性进行了表征。结果表明所制得的纳米颗粒为部分结晶的羟基磷灰石，颗粒为均匀球形，粒径约为20 nm，具有很好的亲水性。由该纳米颗粒构成的生长基质有利于骨髓间充质干细胞的贴壁、增殖以及成骨分化，是一种良好的骨组织工程支架材料。     

Ⅰ型胶原膜的类液晶结构对人脐带间充质干细胞黏附、增殖和分化性能的影响

模拟天然组织中Ⅰ型胶原的有序排列,利用高浓度胶原的液晶态获得表面具有类液晶有序结构的胶原膜,并通过体外培养考察其对人脐带间充质干细胞(hUCMSCs)生物性能的影响.结果表明,当Ⅰ型胶原膜浓度为120 mg/mL时,膜表面的胶原纤维呈定向有序排列,这种有序结构不仅有利于hUCMSCs的黏附和增殖,而且能促进ALP,CollagenⅠ,RUNX2,Ostreix,OCN和OPN等成骨相关基因的表达,表明hUCMSCs在此表面有更强的成骨分化潜能.     

A topographically optimized substrate with well-ordered lattice micropatterns for enhancing the osteogenic differentiation of murine mesenchymal stem cells

Although recently a growing number of reports demonstrate that topography or geometry of the substrate also plays an important role in the fate of the stem cells, most of these studies are usually completed by a few distinct patterns such as simple lines, posts, etc. As a result, there is a lack of quantitative analysis of the relationship between topographical variation and the differentiation of stem cells. Here, the effectiveness of topography variation is studied systematically in several microengineered substrates on osteogenic differentiation. It is found that the effectiveness of the osteogenic differentiation has a peak around 3 μm in the interval length of micropatterns.     

Microtopography based on inverse opal structures regulates the behavior of bone marrow‐derived mesenchymal stem cells

Physical cues from the extracellular microenvironment play an important role in regulating cell behavior, such as adhesion, migration, and differentiation. Many studies have shown that different physical parameters (eg, stiffness and topography) could modulate the in vitro differentiation of mesenchymal stem cells (MSCs), which had multilineage differentiation potential and could be easily isolated from various tissues such as bone marrow, adipose tissue, and the umbilical cord. However, the underlying mechanism of the topographical influence on MSCs and the detailed cell‐substrate interaction remain unclear. Here, we present oriented elliptical inverse opal structures for regulating the morphology and alignment of bone marrow‐derived MSCs. The inverse opal structures were made through a convenient bottom‐up approach of self‐assembly, which is facile and cost effective. MSCs cultured on the oriented structures were highly aligned and extended highly oriented thick lamellipodia. Moreover, the oriented substrates cracked along the lateral boundary of the cells, suggesting that a strong cell‐substrate interaction was induced by the response of MSCs to the oriented topography. These features of the oriented elliptical topography indicated their promising value in stem cell research and tissue engineering.     

Intramarrow injection of β-catenin-activated,but not na?ve mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with β-catenin-activated MSCs. These results reveal that naïve MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/β-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.     

Biodegradable thermogel as culture matrix of bone marrow mesenchymal stem cells for potential cartilage tissue engineering

Poly（lactide-co-glycolide）-poly（ethylene glycol）-poly（lactide-co-glycolide）（PLGA-PEG-PLGA） triblock copolymer was synthesized through the ring-opening polymerization of LA and GA with PEG as macroinitiator and stannous octoate as catalyst. The amphiphilic copolymer self-assembled into micelles in aqueous solutions, and formed hydrogels as the increase of temperature at relatively high concentrations（〉 15 wt%）. The favorable degradability of the hydrogel was confirmed by in vitro and in vivo degradation experiments. The good cellular and tissular compatibilities of the thermogel were demonstrated. The excellent adhesion and proliferation of bone marrow mesenchymal stem cells endowed PLGA-PEGPLGA thermogelling hydrogel with fascinating prospect for cartilage tissue engineering.     

Polypyrrole thin films formed by admicellar polymerization support the osteogenic differentiation of mesenchymal stem cells

The objective of this study was to evaluate the attachment, proliferation, and differentiation of rat mesenchymal stem cells (MSC) toward the osteoblastic phenotype seeded on polypyrrole (PPy) thin films made by admicellar polymerization. Three different concentrations of pyrrole (Py) monomer (20, 35, and 50 x 10(-3) M) were used with the PPy films deposited on tissue culture polystyrene dishes (TCP). Regular TCP dishes and PPy polymerized on TCP by chemical polymerization without surfactant using 5 x 10(-3) M Py, were used as controls. Rat MSC were seeded on these surfaces and cultured for up to 20 d in osteogenic media. Surface topography was characterized by atomic force microscopy, X-ray photoelectron spectroscopy, and static contact angle. Cell attachment, proliferation, alkaline phosphatase (ALP) activity, and calcium content were measured to evaluate the ability of MSC to adhere and differentiate on PPy-coated TCP. Increased monomer concentrations resulted in PPy films of increased thickness and surface roughness. PPy films generated by different monomer concentrations induced drastically different cellular events. A wide spectrum of cell attachment characteristics (from excellent cell attachment to the complete inability to adhere) were obtained by varying the monomer concentration from 20 m to 50 x 10(-3) M. In particular the 20 x 10(-3) M PPy thin films demonstrated superior induction of MSC osteogenicity, which was comparable to standard TCP dishes, unlike PPy films of similar thickness prepared by chemical polymerization without surfactant. Adhesion of mesenchymal stem cells on tissue culture plates (TCP) coated with polypyrrole thin films made by admicellar polymerization.     

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.     

Mechanical stretch promotes proliferation of rat bone marrow mesenchymal stem cells

Tissues and cells in the body are continuously exposed to a complex mechanical environment. Mechanical stimulations are critical to morphological, developmental and functional states of living cells, and the fashion of the mechanical stimulation applied to the cells is supposed to be extremely important for the induced cell response and function. In this study, we investigated whether mechanical stretch regulates and promotes proliferation of rat bone marrow mesenchymal stem cells (rMSCs) in vitro. rMSCs from rat bone marrow were isolated, purified and subjected to a cyclic equiaxial stretch treatment, and then MTT assay was adopted and expression of c-fos gene was measured by RT-PCR to access cell proliferation. The results demonstrated that OD values of rMSCs increased in a time-dependent and magnitude-dependent manner after exposure to 1 Hz stretch within 15–60 min and 2–8% strain. Expression of c-fos gene in rMSCs subjected to stretch treatment (1 Hz, 8% strain and 60 min) is significantly higher than that of unstimulated control cells. These results suggest that mechanical stretch plays an important role in regulating the cell growth and proliferation, and an appropriate mechanical stretch treatment could promote proliferating capacity of rMSCs.