壳聚糖和明胶材料对血管平滑肌细胞的作用

壳聚糖是一种具有发展前景的生物材料。研究了不同脱乙酰度壳聚糖、明胶及壳聚糖与明胶共混材料对血管平滑肌细胞生长的促进作用。在不同材料上培养细胞,采集培养后第1天和第5天的图像．在培养24、72、120h时做MTT实验。此外,还用酶联免疫检测(ELISA)方法测量了材料吸附细胞外基质蛋白的数量,探讨细胞外基质蛋白在血管平滑肌细胞与材料相互作用中所起的作用。结果表明脱乙酰度高的壳聚糖材料能较好地促进血管平滑肌细胞的粘附、铺展和生长,可能是一种具有一定应用前景的血管组织工程研究用材料。     

壳聚糖及其改性材料对骨髓基质细胞的作用

壳聚糖是一种广泛应用的生物可降解材料,该论文研究了几种与壳聚糖相关的材料对骨髓基质细胞生长和分化的作用,主要实验方法是在材料表面培养骨髓基质细胞并对其进行诱导促使其向成骨细胞方向分化。通过对细胞生长和分化情况的观察和测定,对几种材料与骨髓基质细胞的亲和性作出了评价。另外,通过ELISA法测定了细胞外基质分子在材料上的吸附量,测量了各材料的表面接触角以研究细胞在材料表面的铺展和增殖。结果表明尽管壳聚糖本身与骨髓基质细胞并不具有很好的亲和性,但通过与明胶混合,壳聚糖的生物相容性得到了明显提高,是很有应用前景的骨修复材料。     

血清白蛋白在多聚阳离子－壳聚糖共混材料表面的构象变化及其与细胞生长的关系

用L-多聚赖氨酸、聚乙烯亚胺及L-多聚鸟氨酸三种多聚阳离子对壳聚糖进行共混修饰,制备了三种共混材料.在这些材料表面吸附了血清白蛋白,并利用圆二色(CD)光谱研究了白蛋白吸附到材料表面后的构象变化.结果显示,与天然状态相比,白蛋白吸附到共混材料表面后,其α-螺旋、β-折叠及无规则卷曲的含量均发生了明显改变.通过研究MC3T3-E1细胞在这些材料表面的生长情况,发现细胞的增殖与血清白蛋白的构象变化有一定关系,在吸附的白蛋白构象与天然构象最接近的共混材料表面,MC3T3-E1细胞增殖水平最高.     

不同脱乙酰度和分子量壳聚糖的神经亲和性研究

研究了四种不同脱乙酰度和分子量的壳聚糖与神经细胞的亲和性。利用酶联免疫技术（ELISA）测定了人血清白蛋白和纤粘蛋白在四种壳聚糖膜上的吸附量,并利用圆二色柱（CD）测定了人血清白蛋白吸附在四种壳矣糖膜上的二极结构。通过研究它们对胎鼠大脑皮层细胞（FMCC）生长的影响,发现在这四种壳聚糖中,分子量较大的两种壳聚糖更有利于神经细胞的生长,许多轴突聚集成树干状。     

胶原蛋白-壳聚糖支架的构建及其与骨髓基质干细胞复合的实验研究

目的:构建一种组织工程神经支架,并观察体外培养的骨髓基质干细胞在其内部的生长情况,为后续种子细胞的移植提供阶段性实验数据.方法:以Ⅰ型胶原蛋白和壳聚糖为原料通过冷冻干燥技术制备神经支架,扫描电镜观察其内部结构,测量其孔径大小、孔隙率等指标.将体外培养的骨髓基质干细胞与Ⅰ型胶原蛋白-壳聚糖神经支架复合,共培养2天;扫描电镜观察细胞在支架内部的生长情况.结果:构建的神经支架均为圆柱状,内部为纵向平行排列的孔径均匀的微管样结构,细胞紧密贴附在支架微孔内壁上,细胞生长状况良好.结论:Ⅰ型胶原蛋白-壳聚糖支架具有良好的内部三维结构和生物相容性,可与细胞复合后用于修复周围神经缺损.     

通过研究改性壳聚糖与细胞的相互作用评价其生物相容性

利用细胞生物学的方法, 研究了四种不同的细胞在经过改性的壳聚糖（ＣＨＩＴＯＳＡＮ） 膜上的生长,测定了细胞相对黏附力、细胞初始黏附率, 并利用ＦＤＡ 实验测定了细胞活力,从而从多个方面评价了这几种不同材料的生物相容性。实验结果表明,与明胶交联的壳聚糖膜明显比其它两种膜有利于细胞的黏附和生长,为进一步对材料进行筛选奠定了基础。     

L型多聚赖氨酸和D型多聚赖氨酸对神经细胞分化的影响

目的:比较L型多聚赖氨酸(L-PL)和D型多聚赖氨酸(D-PL)浸泡的玻片培养小鼠大脑皮层神经元细胞对其分化的影响。方法:取昆明白小鼠的大脑皮层细胞,分别用L-PL和D-PL进行培养,统计和比较细胞的分化比率、突起数量以及突起生长长度。结果:L-PL和D-PL培养的神经细胞第1、2天的分化率比较差异具有统计学意义(P0.05),而突起数和突起长度比较差异无统计学意义(P0.05)。当L-PL、D-PL的浓度分别为20μL/m L、5μL/mL时,其对1、2天细胞的分化产生显著影响,并且D型多聚赖氨酸培养的细胞分化率高于L型。500、100、20μg/m L的L-PL中,100μg/ml L-PL培养细胞的分化率最高,而125、25、5μg/m L的D型多聚赖氨酸培养细胞分化率比较差异虽然无统计学意义,但25μg/ml D-PL培养神经细胞的分化率总体趋势高于其他浓度。D-PL所形成的粒径大小与L-PL不同,且D-PL的总体趋势稍大于L-PL。结论:L型多聚赖氨酸和D型多聚赖氨酸会在神经细胞生长早期对分化率产生影响,但不影响突起数和突起长度。     

乳珍对不同给氧条件下大脑神经细胞培养的影响

已有报道,大鼠服用牛初乳制品“乳珍”后,迷宫学习训练次数减少;也有实验表明,服用后,人鼠耐受缺氧的能力得到增强。本研究以体外培养的方法,观察乳珍是否能促进大脑神经细胞的发育并提高神经细胞耐受缺氧的能力。 1 材料和方法 1.1 神经细胞的制备和给氧条件的控制 本实验采用的细胞分离和培养方法已有描述。取8日龄来亨鸡胚前脑制备成细胞悬液,然后接种于预先涂以多聚赖氨酸(Sigma)的24孔培养板(Costar)孔内,4     

壳聚糖基角膜细胞载体的制备及其细胞相容性

为探讨羟丙基壳聚糖基共混膜作为组织工程技术中角膜细胞培养载体的可行性, 分别制备了羟丙基壳聚糖/硫酸软骨素、羟丙基壳聚糖/明胶/硫酸软骨素以及羟丙基壳聚糖/氧化透明质酸/硫酸软骨素三种共混膜。测定其透光率、含水量和蛋白吸附性能; 在共混膜上培养兔角膜上皮细胞, 通过观察角膜上皮细胞在不同载体膜上的生长状态、贴附情况, 测定细胞活性以及上清液中乳酸脱氢酶的活性, 研究三种壳聚糖基载体膜片与角膜上皮细胞的相容性。膜片理化性质测定结果表明三种共混膜片具有良好的透明度, 适宜的含水量和较强的蛋白吸附性能; 细胞相容性实验结果表明羟丙基壳聚糖/明胶/硫酸软骨素共混膜对细胞的损伤最小, 有利于细胞在膜上的贴附和生长, 表现出良好的细胞相容性, 有望作为角膜细胞载体体外构建组织工程化角膜。     

神经肽对神经母细胞瘤细胞分裂和代谢的影响

神经-内分泌-免疫系统作为机体的整合系统,在衰老变化的发生和发展过程中起着重要的作用。一些资料表明,伴随神经系统的老化,脑和脑脊液中一些神经肽也显著变化。本实验采用小鼠神经母细胞瘤细胞无血清培养建立神经细胞老化实验研究模型,以流式细胞光度术研究神经肽对无血消培养的神经母细胞瘤细胞的细胞周期和细胞总蛋白的影响。以了解神经肽对神经细胞老化过程中的DNA和细胞总蛋白的影响。1 材料与方法     

Survival and differentiation of neuroepithelial stem cells on chitosan bicomponent fibers

Chitosan is a popular biomaterial used in tissue engineering. Fibers of chitosan could provide a favorable anatomical substrate for cell growth which provides a promising application for axonal regeneration during peripheral injury. Neuroepithelial stem cells (NEPs) are the most primitive neural stem cells with multipotential for neuronal and glia differentiation. To assess the biocompatibility between NEPs and chitosan fibers, and to explore whether the NEPs have the ability to differentiation on chitosan fibers, NEPs were harvested from the neural tube and seeded on chitosan fibers in in vitro culture. The biocompatibility of chitosan fibers was tested by MTT assays. The growth and survival were observed by light and scanning electronic microscope at different times in culture. And, the differentiation of NEPs was examined by immunocytochemical staining. The results indicated that NEPs could grow on the chitosan fibers and attach firmly to the surface of fibers. On chitosan fibers, NEPs could differentiate into neurons and glia. Our study demonstrated that chitasan fibers had a good biocompatibility with NEPs which affords a potential alternative for the repair of peripheral nerve injury.     

Effects of chitosan/collagen substrates on the behavior of rat neural stem cells

Spinal cord and brain injuries usually lead to cavity formation. The transplantation by combining stem cells and tissue engineering scaffolds has the potential to fill the cavities and replace the lost neural cells. Both chitosan and collagen have their unique characteristics. In this study, the effects of chitosan and collagen on the behavior of rat neural stem cells (at the neurosphere level) were tested in vitro in terms of cytotoxicity and supporting ability for stem cell survival, proliferation and differentiation. Under the serum-free condition, both chitosan membranes and collagen gels had low cytotoxicity to neurospheres. That is, cells migrated from neurospheres, and processes extended out from these neurospheres and the differentiated cells. Compared with the above two materials, chitosan-collagen membranes were more suitable for the co-culture with rat neural stem cells, because, except for low cytotoxicity and supporting ability for the cell survival, in this group, a large number of cells were observed to migrate out from neurospheres, and the differentiating percentage from neurospheres into neurons was significantly increased. Further modification of chitosan-collagen membranes may shed light on in vivo nerve regeneration by transplanting neural stem cells.     

Proliferation and Differentiation Potential of Human Adipose-Derived Stem Cells Grown on Chitosan Hydrogel

Applied tissue engineering in regenerative medicine warrants our enhanced understanding of the biomaterials and its function. The aim of this study was to evaluate the proliferation and differentiation potential of human adipose-derived stem cells (hADSCs) grown on chitosan hydrogel. The stability of this hydrogel is pH-dependent and its swelling property is pivotal in providing a favorable matrix for cell growth. The study utilized an economical method of cross linking the chitosan with 0.5% glutaraldehyde. Following the isolation of hADSCs from omentum tissue, these cells were cultured and characterized on chitosan hydrogel. Subsequent assays that were performed included JC-1 staining for the mitochondrial integrity as a surrogate marker for viability, cell proliferation and growth kinetics by MTT assay, lineage specific differentiation under two-dimensional culture conditions. Confocal imaging, scanning electron microscopy (SEM), and flow cytometry were used to evaluate these assays. The study revealed that chitosan hydrogel promotes cell proliferation coupled with > 90% cell viability. Cytotoxicity assays demonstrated safety profile. Furthermore, glutaraldehyde cross linked chitosan showed < 5% cytotoxicity, thus serving as a scaffold and facilitating the expansion and differentiation of hADSCs across endoderm, ectoderm and mesoderm lineages. Additional functionalities can be added to this hydrogel, particularly those that regulate stem cell fate.     

Chitins and chitosans for the repair of wounded skin,nerve, cartilage and bone

This review provides a balanced integration of the most recent chemical, biochemical and medical information on the unique characteristics of chitins and chitosans in the area of animal/human tissue regeneration. Hemostasis is immediately obtained after application of most of the commercial chitin-based dressings to traumatic and surgical wounds: platelets are activated by chitin with redundant effects and superior performances compared with known hemostatic materials. To promote angiogenesis, necessary to support physiologically ordered tissue formation, the production of the vascular endothelial growth factor is strongly up-regulated in wound healing when macrophages are activated by chitin/chitosan. The inhibition of activation and expression of matrix metalloproteinases in primary human dermal fibroblasts by low MW chitosans prevents or solves problems caused by metalloproteinase-2 such as the hydrolysis of the basement membrane collagen IV. Experimental biocompatible wound dressings derived from chitin are today available in the form of hydrogels, xerogels, powders, composites, films and scaffolds: the latter are easily colonized by human cells in view of the restoration of tissue defects, with the advantage of avoiding retractive scar formation. The growth of nerve tissue has been guided with chitin tubes covalently coated with oligopeptides derived from laminin. The regeneration of cartilage is also feasible because chitosan maintains the correct morphology of chondrocytes and preserves their capacity to synthesize cell-specific extracellular matrix: chitosan scaffolds incorporating growth factors and morphogenetic proteins have been developed. Impressive advances have been made with osteogenic chitosan composites in treating bone defects, particularly with osteoblasts from mesenchymal stem cells in porous hydroxyapatite-chitin matrices. The introduction of azido functions in chitosan has provided photo-sensitive hydrogels that crosslink in a matter of seconds, thus paving the way to cytocompatible hydrogels for surgical use as coatings, scaffolds, drug carriers and implants capable to deliver cells and growth factors. The peculiar biochemical properties of chitins and chitosans remain unmatched by other polysaccharides.     

Direct transplantation of uncultured hair‐follicle pluripotent stem (hfPS) cells promotes the recovery of peripheral nerve injury

We previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin‐expressing hair‐follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed the hfPS cell area (hfPSCA). Previously, hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential. J. Cell. Biochem. 110: 272–277, 2010. © 2010 Wiley‐Liss, Inc.     

Investigation of MC3T3-E1 cell behavior on the surface of GRGDS-coupled chitosan

The GRGDS (Gly-Arg-Gly-Asp-Ser) peptide has intermediate affinity to alphaVbeta3 and alphaIIbbeta3, which are the integrins most reported to be involved in bone function. In this study, biomimetic chitosan films modified with GRGDS peptide were prepared and were used as a substrate for the in vitro culture of MC3T3-E1 cells in order to investigate the effect of GRGDS modification on MC3T3-E1 cell behavior. The results of electron spectroscopy for chemical analysis (ESCA), attenuated total reflection-Fourier transform infrared spectra (ATR-FTIR), and amino acid analysis (AAA) demonstrated that the chitosan films were successfully modified with GRGDS peptides and that the surface density of the immobilized GRGDS was on the order of 10(-9) mol/cm2. The immobilization of the GRGDS sequence on chitosan as well as the peptide concentration play a significant role in MC3T3-E1 cell behavior. MC3T3-E1 cell attachment, proliferation, migration, differentiation, and mineralization were remarkably greater on GRGDS-coupled chitosan than on unmodified chitosan. Besides, the degree of acceleration of these biological processes was found to be dependent on peptide density. Competitive inhibition of MC3T3-E1 cell attachment using soluble GRGDS peptides indicated that the interaction of MC3T3-E1 cells with the surface of the materials was ligand-specific. Cytoskeleton organization in the fully spread MC3T3-E1 cells was highly obvious on GRGDS-coupled chitosan when compared to the lack of actin fibers noted in the round MC3T3-E1 cells on unmodified chitosan. These results suggest that MC3T3-E1 cell function can be modulated, in a peptide density-dependent manner, by the immobilization of GRGDS peptide on chitosan used for scaffold-based bone tissue engineering.     

Axonal Regeneration and Remyelination Evaluation of Chitosan/Gelatin-Based Nerve Guide Combined with Transforming Growth Factor-β1 and Schwann Cells

Despite efforts in peripheral nerve injury and regeneration, it is difficult to achieve a functional recovery following extended peripheral nerve lesions. Even if artificial nerve conduit, cell components and growth factors can enhance nerve regeneration, integration in peripheral nerve repair and regeneration remains yet to be explored. For this study, we used chitosan/gelatin nerve graft constructed with collagenous matrices as a vehicle for Schwann cells and transforming growth factor-β1 to bridge a 10-mm gap of the sciatic nerve and explored the feasibility of improving regeneration and reinnervation in rats. The nerve regeneration was assessed with functional recovery, electrophysiological test, retrograde labeling, and immunohistochemistry analysis during the post-operative period of 16 weeks. The results showed that the internal sides of the conduits were compact enough to prevent the connective tissues from ingrowth. Nerve conduction velocity, average regenerated myelin area, and myelinated axon count were similar to those treated with autograft (p > 0.05) but significantly higher than those bridged with chitosan/gelatin nerve graft alone (p < 0.05). Evidences from retrograde labeling and immunohistochemistry analysis are further provided in support of improving axonal regeneration and remyelination. A designed graft incorporating all of the tissue-engineering strategies for peripheral nerve regeneration may provide great progress in tissue engineering for nerve repair.