骨组织工程材料的表面修饰和细胞粘附

由于人工合成高分子聚合物材料亲水性差，细胞吸附力差，在材料表面包被，铰链某种蛋白或短肽则有利于细胞在材料上的粘附，并影响细胞的增殖和分化。本重点介绍了与成骨细胞有关的蛋白或短肽，并描述了细胞在材料上的粘附过程。     

结合RGD肽的聚酯材料表面粘附内皮细胞的抗剪切力研究

精氨酸-甘氨酸-天门冬氨酸(RGD)是许多粘附蛋白的高度保守氨基酸序列.生物材料表面结合RGD肽有助于内皮细胞在材料上的粘附、迁移和增殖.本研究在体外流动条件下观察结合RGD肽或纤维粘连蛋白的聚酯材料表面粘附内皮细胞的抗剪切能力,并通过观察肌动蛋白和踝蛋白的表达初步探讨影响细胞粘附稳定性的机制.结果显示材料表面结合RGD或纤维粘连蛋白可以增加细胞的粘附强度,提高抗剪切能力;而RGD和纤维粘连蛋白导致的细胞抗剪切能力增加可能与细胞内肌动蛋白和踝蛋白的表达增加有关.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

VAPG是平滑肌细胞粘附的特异性配体

据GobinAS[JBiomedMaterRes ,2 0 0 3,6 7A(1) :2 5 5 2 97]报道弹性蛋白衍生肽———缬氨酸—丙氨酸—脯氨酸—谷氨酸(VAPG)可成为平滑肌细胞的生物特异性粘附配体。通过嫁接肽序列制成水凝胶材料 ,GobinAS及其同事对它在平滑肌细胞粘附和扩展中的作用进行评价。这些材料是聚乙二醇 (PEG)的丙烯酸盐衍生物 ,即具有光敏聚合性水凝胶。由于PEG含量较高 ,所以这些材料排斥蛋白吸附和细胞粘附性高。然而 ,PEG的双丙烯酸盐衍生物能够与粘附性肽修饰的PEG单丙烯酸衍生物混合 ,可促进细胞粘附。利用光敏聚合作用 ,PEG单丙烯酸盐衍生物…     

BSA和胶原的竞争性吸附与细胞粘附

细胞在材料表面的粘附是贴壁依赖型细胞生长的前提,细胞只有在表面以一定的粘附力发生粘附并铺展后,细胞列哨B生长。当细胞与表面的粘附力较强时,有利于细胞生长,而当粘附力较弱时,则有利于细胞分化。现在普遍认为细胞粘附过程由细胞表面的受体对细胞外基质（ECM）蛋白的特异性识别所调节。胶原等细胞外基质影响细胞的形态和功能,并提供细胞分化和增殖的信号。而培养介质是复杂的溶液,它们中许多有血清或添加的蛋白和表面活性物质。因而细胞在材料表面的粘附受蛋白质竞争性吸附所调节,包括基底预处理、培养基以及细胞分泌等许多不…     

抗粘附肽YIGSR的衍生物对Lewis肺癌侵袭、转移能力的抑制作用

采用高转移肿瘤细胞对基底膜成分的体外粘附、侵袭模型和Lewis肺癌细胞自发肺转移模型 ,研究了YIGSR均叉、杂叉聚合肽的抗肿瘤侵袭、转移活性。实验结果 ,RGD和YIGSR的杂叉肽和YIGSR的均叉肽对PG细胞较对照的YIGSR肽表现了更明显的粘附抑制作用 ,并降低了PG细胞的体外侵袭能力 ,降低了移值Lewis肺癌细胞肺转移小鼠的肺重量或肺转移瘤结节数 ,但对小鼠原发部位移植瘤重量无明显影响 ;YIGSR聚合肽的以上作用有一定的剂量依赖性。     

CCR9亲和短肽的筛选及其结合活性的鉴定

目的获取与CC趋化因子受体9(CCR9)具有亲和力的特定短肽。方法以CCR9的第2个胞外环为靶蛋白,反复筛选Ph.D.-12噬菌体展示肽库获得阳性克隆,通过ELISA测定噬菌体阳性克隆短肽与CCR9的亲和力,共聚焦显微镜检测短肽与CCR9高表达细胞MOLT4结合能力。结果经过3轮淘选、富集,确定了8个噬菌体克隆有插入序列,其中克隆C-4与靶蛋白具有较高亲和力,表达的短肽P1(VHWDFRQWWQPS)可抑制相应C-4克隆与靶蛋白的结合,并可与CCR9高表达的MOLT4细胞结合。结论通过噬菌体肽库筛选出了能与细胞表面分子CCR9结合的短肽序列VHWDFRQWWQPS。     

成纤维细胞在PHB可降解材料上的粘附与生长研究

聚羟基丁酸酯（PHB）多孔材料可作为组织工程用的支架,然而细胞在此材料表面不易粘附生长,这与粘附蛋白在聚合物材料表面的吸附有关。基于材料表面的粘附与细胞生长、增殖、分化和组织发育密切相关,粘附强度可影响工程组织的最终结构与功能,本文通过对PHB材料进行多聚赖氨酸衣被,有效地实现了在PHB可降解支架上种植细胞来构建工程组织。用离心法制备厚度为20um的透明PHB薄膜,用多聚赖氨酸衣被,对照组为不衣被的PHB薄膜和玻璃片。接种成纤维细胞系NIH3T3,相差显微镜观察细胞的粘附生长过程。细胞在多聚赖氨酸衣被表面迅速粘…     

利用噬菌体肽库筛选HeLa细胞表面与人CD59结合的活性短肽

目的寻找HeLa细胞表面与人CD59特异性结合的短肽序列。方法分别用非转基因HeLa细胞和转染CD59基因的HeLa细胞的裂解蛋白进行5轮亲和筛选,并通过竞争结合实验,然后用ELISA方法筛选噬菌体阳性克隆。提取单克隆DNA测序,并对其进行DNA序列分析推导出短肽序列。结果随机挑选的16个单克隆中有10个克隆对HeLa细胞有特异性结合力,经测序得到一条高度同源的多肽序列。结论通过噬菌体随机肽库对肿瘤细胞进行全细胞蛋白筛选,得到了噬菌体多肽能高特异性与肿瘤细胞结合的短肽序列,为下一步设计肿瘤逃逸相关的CD59活性位点的短肽药物提供了参考依据。     

壳聚糖水凝胶的研制及其与C2C12细胞粘附、支持作用的实验研究

研制可注射的壳聚糖水凝胶材料,并对该材料的理化特性进行检测,同时检验其是否能粘附并支持C2C12细胞生长。对不同温度下的壳聚糖溶液进行pH值的测定,并以壳聚糖溶液和β-甘油磷酸钠溶液为原料,制备温敏性壳聚糖水凝胶,对该材料的粘度及37℃成胶时间进行测定,显微镜下观察水凝胶材料的结构,通过C2C12细胞在其表面上的粘附与生长情况,判定该材料是否具有良好的细胞相容性。结果表明：该材料具有温度敏感性,即室温下为液态,37℃变为固态。当壳聚糖溶液浓度为2%时,加入45%β-甘油磷酸钠溶液后的成胶作用时间最短,约为15min。C2C12细胞在壳聚糖水凝胶材料上的粘附与生长情况良好。壳聚糖水凝胶可作为可注射性组织工程化心肌的支架材料,用于携带有效细胞进行缺血性心脏病的治疗。     

RGD modified polymers: biomaterials for stimulated cell adhesion and beyond

Since RGD peptides (R: arginine; G: glycine; D: aspartic acid) have been found to promote cell adhesion in 1984 (Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule, Nature 309 (1984) 30), numerous materials have been RGD functionalized for academic studies or medical applications. This review gives an overview of RGD modified polymers, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on polymers. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed.     

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H(2)N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG(2)PLA(20)) and the thiol-reactive derivative (MP-NH-PEG(2)PLA(40)) were modified with cyclic alphavbeta3/alphavbeta5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell-polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD-integrin-interactions.     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

Cell adhesion peptide modification of gold-coated polyurethanes for vascular endothelial cell adhesion

Gold-coated polyurethanes were chemisorbed with three cell-adhesion peptides having an N-terminal cysteine: cys-arg-gly-asp (CRGD), cys-arg-glu-asp-val (CREDV), and the cyclic peptide cys-cys-arg-arg-gly-asp-try-leu-cys (CCRRGDWLC). The peptides were selected based on their presumed preferential interactions with the cell-surface integrins on vascular endothelial cells. The ability of the surfaces to support the preferential adhesion of human vascular endothelial cells was studied by comparing in vitro adhesion results for these cells with those from mouse 3T3 fibroblasts. Surface modification with the peptides was confirmed by water-contact angles and XPS. Surface morphology was determined by AFM and SEM. In vitro cell-culture studies in conjunction with plasma-protein adsorption and immunoblotting were performed on the various modified surfaces. The data suggest that peptide-modified surfaces have significant potential for supporting cell adhesion. Little or no cell adhesion was noted on gold- or cysteine-modified control surfaces. Human vascular endothelial cells showed the greatest adhesion to the CCRRGDWLC-modified surfaces, and the 3T3 fibroblasts adhered best to the CREDV-modified surfaces. Protein adsorption studies suggest that the preferential adsorption of the cell-adhesive proteins fibronectin and vitronectin is not likely mediating the differences noted. It is concluded that the cell-adhesive peptide-modified gold-coated polymers have significant potential for further development both as model substrates for fundamental studies and for use in biomaterials applications.     

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.     

Protein adsorption and cellular adhesion and activation on biomedical polymers

The design and development of new biomedical polymers for clinical application in devices, prostheses, and artificial organs requires a basic and fundamental understanding of biological interactions with biomedical polymers. Efforts in our laboratory have been directed towards appreciating the humoral and cellular interactions which govern protein adsorption and cellular adhesion and activation on biomedical polymers. Information and data are presented on protein adsorption from whole human blood, complement activation and receptors, and monocyte/macrophage adhesion and activation with growth factor release. Supported by experimental evidence, concepts regarding protein/polymer, cell/polymer, cell/protein/polymer, and cell/cell interactions as they are related to in vivo events are presented.     

Human microvascular endothelial cell growth and migration on biomimetic surfactant polymers

Successful engineering of a tissue-incorporated vascular prosthesis requires cells to proliferate and migrate on the scaffold. Here, we report on a series of "ECM-like" biomimetic surfactant polymers that exhibit quantitative control over the proliferation and migrational properties of human microvascular endothelial cells (HMVEC). The biomimetic polymers consist of a poly(vinyl amine) (PVAm) backbone with hexanal branches and varying ratios of cell binding peptide (RGD) to carbohydrate (maltose). Proliferation and migration behavior of HMVEC was investigated using polymers containing RGD: maltose ratios of 100:0, 75:25 and 50:50, and compared with fibronectin (FN) coated glass (1 microg/cm2). A radial Teflon fence migration assay was used to examine HMVEC migration at 12 h intervals over a 48 h period. Migration was quantified using an inverted optical microscope, and HMVEC were examined by confocal microscopy for actin and focal adhesion organization/ arrangement. Over the range of RGD ligand density studied (approximately 0.19-0.6 peptides/nm2), our results show HMVEC migration decreases with increasing RGD density in the polymer. HMVEC were least motile on the 100% RGD polymer (approximately 0.38-0.6 peptides/nm2) with an average migration of 0.20 mm2/h in area covered, whereas HMVEC showed the fastest migration of 0.48+/-0.06 mm2/h on the 50% RGD surface ( approximately 0.19-0.30 peptides/nm2). In contrast, cell proliferation increased with increasing surface peptide density; proliferation on the 50% RGD surface was 1.5%+/-0.06/h compared with 2.2%+/-0.07/h on the 100% RGD surface. Our results show that surface peptide density affects cellular functions such as growth and migration, with the highest peptide density supporting the most proliferation but the slowest migration.     

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.     

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.     

New antitumor agent: In vitro activity on breast carcinoma cells

Previous work showed that established interactions between water-soluble polymers and cell membrane receptors can lead to modulate cell proliferation and differentiation in vitro. These polymers can be considered as bioactive. The aim of this work was to establish the consequences of the interactions between human breast cancer cells MCF7 and polymers of various chemical compositions regarding cell adhesion and proliferation onto tissue culture plate. Water soluble copolymers were synthesized by radical polymerization and are composed of methacrylic acid and sodium styrene sulphonate units. The modulation of the MCF7, biological-induced by these polymers of various compositions, was evaluated. The influence of the polymers chemical composition on the kinetics of cell proliferation, as well as cell morphology and spreading, were studied. A polymer concentration-dependent inhibition effect was observed. One hundred microgram per liter polymers solutions induced strong inhibition of cell proliferation, as well as a change of the MCF7 cells morphology, which can be related to an inhibition of cell spreading. The polymers/MCF7 cells interactions are modulated by the chemical composition of the copolymers and then the respective rate in sulphonate and carboxylate groups distributed along the macromolecular chain.