Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration

Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by approximately 41% and migration by approximately 15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.     

Calcification by MC3T3-E1 cells on RGD peptide immobilized on titanium through electrodeposited PEG

The effect of a cell-adhesive peptide containing Arg-Gly-Asp (RGD) immobilized through poly(ethylene glycol) (PEG) on titanium (Ti) on calcification by MC3T3-E1 cells was investigated to develop a new surface modification technique using biofunctional molecules. RGD was immobilized on Ti through PEG, both terminals of which were terminated with –NH₂ and –COOH to combine with the Ti surface and RGD. PEG was immobilized on Ti with electrodeposition, and RGD, with immersion. For comparison, glycine was employed because it is the simplest molecule containing both –NH₂ and –COOH at its terminals. MC3T3-E1 cells were cultured and differentiation-induced on each specimen, and the cell calcification properties were investigated. As a result, there was no significant difference in the morphology and extension of MC3T3-E1 cells cultured on each specimen, while the number of cells cultured on RGD/PEG/Ti was the largest. After differentiation-induction, there was no significant difference in the ALP activity among all specimens. On the other hand, the level of cell calcification on RGD/PEG/Ti was the highest. Therefore, the hard tissue compatibility of Ti is improved by immobilizing RGD through functional molecules which have a long molecular chain.     

Retinal ganglion cell polarization using immobilized guidance cues on a tissue-engineered scaffold

Cell transplantation therapies to treat diseases related to dysfunction of retinal ganglion cells (RGCs) are limited in part by an inability to navigate to the optic nerve head within the retina. During development, RGCs are guided by a series of neurotrophic factors and guidance cues; however, these factors and their receptors on the RGCs are developmentally regulated and often not expressed during adulthood. Netrin-1 is a guidance factor capable of guiding RGCs in culture and relevant to guiding RGC axons toward the optic nerve head in vivo. Here we immobilized Netrin-1 using UV-initiated crosslinking to form a gradient capable of guiding the axonal growth of RGCs on a radial electrospun scaffold. Netrin-gradient scaffolds promoted both the percentage of RGCs polarized with a single axon, and also the percentage of cells polarized toward the scaffold center, from 31% to 52%. Thus, an immobilized protein gradient on a radial electrospun scaffold increases RGC axon growth in a direction consistent with developmental optic nerve head guidance, and may prove beneficial for use in cell transplant therapies for the treatment of glaucoma and other optic neuropathies.     

Endothelial cell migration on surfaces modified with immobilized adhesive peptides

Endothelial cell (EC) migration has been studied on aminophase surfaces with covalently bound RGDS and YIGSRG cell adhesion peptides. The fluorescent marker dansyl chloride was used to quantify the spatial distribution of the peptides on the modified surfaces. Peptides appeared to be distributed in uniformly dispersed large clusters separated by areas of lower peptide concentrations. We employed digital time-lapse video microscopy and image analysis to monitor EC migration on the modified surfaces and to reconstruct the cell trajectories. The persistent random walk model was then applied to analyze the cell displacement data and compute the mean root square speed, the persistence time, and the random motility coefficient of EC. We also calculated the time-averaged speed of cell locomotion. No differences in the speed of cell locomotion on the various substrates were noted. Immobilization of the cell adhesion peptides (RGDS and YIGSRG), however, significantly increased the persistence of cell movement and, thus, the random motility coefficient. These results suggest that immobilization of cell adhesion peptides on the surface of implantable biomaterials may lead to enhanced endothelization rates.     

3D自组装肽纳米纤维支架对胰岛细胞分泌功能的影响

背景：3D自组装肽纳米纤维支架能很好模拟体内微环境,给予细胞必要的结构模式,促进细胞外基质的正确组成及细胞的生长,改善细胞功能。 目的：体外观察3D自组装肽纳米纤维水凝胶支架对胰岛细胞分泌功能的影响。 方法：将3D自组装肽纳米纤维水凝胶支架与成年大鼠胰岛细胞共培养,AO-PI荧光染色法检测胰岛细胞的活性及生存率;放射免疫法测定胰岛细胞的分泌功能;扫描电镜观察胰岛细胞包裹在3D纳米支架中成三维立体的生长状态。 结果与结论：在3D纳米支架培养环境中胰岛细胞纯度≥80%;3D纳米支架组胰岛生存率及胰岛细胞分泌功能明显高于无支架组(2D培养组)(P < 0.05);扫描电镜显示自组装肽纳米纤维支架形成了具有几何形状的纳米级薄层,将胰岛细胞包裹在3D纳米支架中,胰岛细胞成三维立体生长。表明3D自组装肽纳米纤维支架可为胰岛细胞体外生存提供3D培养环境,改善胰岛细胞的活性、分泌功能及形态,延长胰岛细胞体外生存期。     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

The effect on osteoblast function of colocalized RGD and PHSRN epitopes on PEG surfaces

Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg-Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro-His-Ser-Arg-Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD-PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor-ligand interactions initiating signal transduction cascades that down-regulate ECM production.     

The reactivity of alpha-chymotrypsin immobilized on radiation-grafted hydrogel surfaces.

The enzymatic activity of alpha-chymotrypsin (CT), immobilized on hydrogel-coated polymer film supports, has been investigated. The support was prepared by radiation-graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAAc) on silicone rubber films. The enzyme was covalently coupled to the carboxylic group of MAAc via the N-hydroxysuccinimide (NHS) ester active intermediate. Increasing MAAc contents of the hydrogel resulted in increased attachment of CT. The integrity of the CT active site after attachment was assessed by an active site titration with diisopropyl fluorophosphate (DFP). As the MAAc content of the hydrogel was increased, an increasing fraction of the attached CT retained its activity to DFP. A greater fraction of CT was active towards DFP when adsorbed than when coupled. The rates of hydrolysis of some synthetic model substrates by the immobilized CT were also measured. The negative charge on the hydrogel had a large effect on the rates of these hydrolyses. The pH optimum for the hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) by immobilized CT was higher than that of free CT. Increasing MAAc content of the hydrogel resulted in larger shifts in the pH optimum. The maximum rates of ATEE hydroylsis per mg CT declined sharply with increasing MAAc content of the hydrogel. This is probably related to the increasing repulsive force between the ATEE (negatively charged above congruent to pH 9.5) and the hydrogel with increasing MAAc content. The activity of immobilized CT to ATEE is small compared to that of free CT, partly due to this charge effect. Conversely, the rate of hydrolysis of BAEE, a positively charged substrate, by immobilized CT at pH 11, is almost fourfold greater than that by free CT at its pH optimum.     

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.     

Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness

Our current understanding of 3-dimensional (3D) cell migration is primarily based on results from fibrous scaffolds with randomly organized internal architecture. Manipulations that change the stiffness of these 3D scaffolds often alter other matrix parameters that can modulate cell motility independently or synergistically, making observations less predictive of how cells behave when migrating in 3D. In order to decouple microstructural influences and stiffness effects, we have designed and fabricated 3D polyethylene glycol (PEG) scaffolds that permit orthogonal tuning of both elastic moduli and microstructure. Scaffolds with log-pile architectures were used to compare the 3D migration properties of normal breast epithelial cells (HMLE) and Twist-transformed cells (HMLET). Our results indicate that the nature of cell migration is significantly impacted by the ability of cells to migrate in the third dimension. 2D ECM-coated PEG substrates revealed no statistically significant difference in cell migration between HMLE and HMLET cells among substrates of different stiffness. However, when cells were allowed to move along the third dimension, substantial differences were observed for cell displacement, velocity and path straightness parameters. Furthermore, these differences were sensitive to both substrate stiffness and the presence of the Twist oncogene. Importantly, these 3D modes of migration provide insight into the potential for oncogene-transformed cells to migrate within and colonize tissues of varying stiffness.     

Bacterial adhesion and osteoblast function on titanium with surface-grafted chitosan and immobilized RGD peptide

Biomaterials-associated infections remain a source of serious complications in modern medicine. When a biomaterial is implanted in the body, the result of successful tissue integration or implant infection depends on the race for the surface between bacteria and tissue cells. One promising strategy to reduce the incidence of infection is the functionalization of the biomaterial surface to inhibit bacterial adhesion and encourage the growth of cells. In this in vitro study, the surface of titanium alloy substrates was first functionalized by covalently grafted chitosan (CS). The cell-adhesive Arg-Gly-Asp (RGD) peptide was then immobilized on the CS-grafted surface through covalent binding of peptide to the free NH(2) groups of CS. Both these functionalized surfaces showed a decrease in adhesion of Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) compared with the pristine substrate. A significant increase in osteoblast cell attachment, proliferation, and alkaline phosphatase activity was observed on the surface with the immobilized Arg-Gly-Asp peptide. Thus, utilizing surface-grafted chitosan in conjunction with the cell-adhesive peptide to modify the metal surface provides a promising means for enhancing tissue integration of implants by reducing bacterial adhesion and promoting osteoblast functions.     

A calcium-cross-linked hydrogel based on alginate-modified atelocollagen functions as a scaffold material

We have developed a scaffold material consisting of a covalently-bonded structure of alginate and atelocollagen (AtCol). Addition of calcium ions caused the material to form a hydrogel (alginate-modified AtCol gel). The condition of the alginate-modified AtCol gel could be controlled by the feed ratio of alginate and the activating reagent. Measurement of temporal stability in culture medium suggested that covalent bonding between alginate and AtCol might contribute to the structural stability of the alginate-modified AtCol gel. Culture with endothelial cells indicated that cell adhesiveness on the alginate-modified AtCol gel was similar to that on native collagen. Collagenase digestion revealed that the alginate-modified AtCol gel had considerable ability to retain basic fibroblast growth factor. Additionally, active cell migration into alginate-modified AtCol was detected by in vitro assay using endothelial cells. These findings indicate that this gel material can be expected to function as a scaffold for inducing vascular in-growth.     

The use of covalently immobilized stem cell factor to selectively affect hematopoietic stem cell activity within a gelatin hydrogel

Hematopoietic stem cells (HSCs) are a rare stem cell population found primarily in the bone marrow and responsible for the production of the body's full complement of blood and immune cells. Used clinically to treat a range of hematopoietic disorders, there is a significant need to identify approaches to selectively expand their numbers ex vivo. Here we describe a methacrylamide-functionalized gelatin (GelMA) hydrogel for in vitro culture of primary murine HSCs. Stem cell factor (SCF) is a critical biomolecular component of native HSC niches in vivo and is used in large dosages in cell culture media for HSC expansion in vitro. We report a photochemistry based approach to covalently immobilize SCF within GelMA hydrogels via acrylate-functionalized polyethylene glycol (PEG) tethers. PEG-functionalized SCF retains the native bioactivity of SCF but can be stably incorporated and retained within the GelMA hydrogel over 7 days. Freshly-isolated murine HSCs cultured in GelMA hydrogels containing covalently-immobilized SCF showed reduced proliferation and improved selectivity for maintaining primitive HSCs. Comparatively, soluble SCF within the GelMA hydrogel network induced increased proliferation of differentiating hematopoietic cells. We used a microfluidic templating approach to create GelMA hydrogels containing gradients of immobilized SCF that locally direct HSC response. Together, we report a biomaterial platform to examine the effect of the local presentation of soluble vs. matrix-immobilized biomolecular signals on HSC expansion and lineage specification. This approach may be a critical component of a biomaterial-based artificial bone marrow to provide the correct sequence of niche signals to grow HSCs in the laboratory.     

Microfluidic assay of endothelial cell migration in 3D interpenetrating polymer semi-network HA-Collagen hydrogel

Cell migration through the extracellular matrix (ECM) is one of the key features for physiological and pathological processes such as angiogenesis, cancer metastasis, and wound healing. In particular, the quantitative assay of endothelial cell migration under the well-defined three dimensional (3D) microenvironment is important to analyze the angiogenesis mechanism. In this study, we report a microfluidic assay of endothelial cell sprouting and migration into an interpenetrating polymer semi-network HA-Collagen (SIPNs CH) hydrogel as ECM providing an enhanced in vivo mimicking 3D microenvironment to cells. The microfluidic chip could provide a well-controlled gradient of growth factor to cells, whereas the hydrogel could mimic a well-defined 3D microenvironment in vivo. (In addition/Furthermore, the microfluidic chip gives a well-controlled gradient of growth factor to cells) For this reason, three types of hydrogel, composed of semi-interpenetrating networks of collagen and hyaluronic acid were prepared, and firstly we proved the role of the hydrogel in endothelial cell migration. The diffusion property and swelling ratio of the hydrogel were characterized. It modulated the migration of endothelial cells in quantified manner, also being influenced by additional synthesis of Matrix metalloproteinase(MMP)-sensitive remodeling peptides and Arginine–glycine–lycinee (RGD) cell adhesion peptides. We successfully established a novel cell migration platform by changing major determinants such as ECM material under biochemical synthesis and under growth factor gradients in a microfluidic manner.     

A composite hydrogel platform for the dissection of tumor cell migration at tissue interfaces

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and contributes to tumor recurrence. The most rapid mode of tissue infiltration occurs along blood vessels or white matter tracts, which represent topological interfaces thought to serve as “tracks” that speed cell migration. Despite this observation, the field lacks experimental paradigms that capture key features of these tissue interfaces and allow reductionist dissection of mechanisms of this interfacial motility. To address this need, we developed a culture system in which tumor cells are sandwiched between a fibronectin-coated ventral surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that the dorsal surface slows migration through biochemically specific mechanisms rather than simple steric hindrance. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface.     

Spatially directed guidance of stem cell population migration by immobilized patterns of growth factors

We investigated how engineered gradients of exogenous growth factors, immobilized to an extracellular matrix material, influence collective guidance of stem cell populations over extended time (>1 day) and length (>1 mm) scales in vitro. Patterns of low-to-high, high-to-low, and uniform concentrations of heparin-binding epidermal growth factor-like growth factor were inkjet printed at precise locations on fibrin substrates. Proliferation and migration responses of mesenchymal stem cells seeded at pattern origins were observed with time-lapse video microscopy and analyzed using both manual and automated computer vision-based cell tracking techniques. Based on results of established chemotaxis studies, we expected that the low-to-high gradient would most effectively direct cell guidance away from the cell source. All printed patterns, however, were found to direct net collective cell guidance with comparable responses. Our analysis revealed that collective "cell diffusion" down a cell-to-cell confinement gradient originating at the cell starting lines and not the net sum of directed individual cell migration up a growth factor concentration gradient is the principal driving force for directing mesenchymal stem cell population outgrowth from a cell source. These results suggest that simple uniform distributions of growth factors immobilized to an extracellular matrix material may be as effective in directing cell migration into a wound site as more complex patterns with concentration gradients.     

Cell-attachment activities of surface immobilized oligopeptides RGD,RGDS, RGDV,RGDT, and YIGSR toward five cell lines

Tetrapeptides, Arg-Gly-Asp-Ser (RGDS), Arg-Gly-Asp-Val (RGDV), and Arg-Gly-Asp-Thr (RGDT), respectively, appearing in the cell-attachment domains of fibronectin, vitronectin, and collagen, and pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR) appearing in B1 chain of laminin, were synthesized by liquid-phase procedure. Bioactivities of RGD, RGDX (X=S, V and T), YIGSR, and YIGSR-NH₂ as cell recognition determinants were investigated by cell-attachment test using these oligopeptides immobilized to ethylene-acrylic acid copolymer (PEA) film. The cell lines used were A431, NRK, CHO-K1, HeLa.S3, and RLC-16 cells. It was found that the residue X in RGDX plays an important role for cell-attachment activity of RGDX, and, regarding YIGSR, introduction of NH₂ residue at the C-terminal of the pentapeptide enhances the cell-attachment activity.     

RGD肽表面修饰聚苯乙烯及其细胞相容性研究

目的以聚苯乙烯二维平面为模板研究了蛋白表面修饰技术,构建具有生物活性的生物材料表面。方法采用物理包被法依靠疏水作用在PS表面架构明胶、胶原和RGD（精氨酸-甘氨酸-天冬氨酸）多肽的生物活性层。通过光电子能谱（XPS）分析修饰表面的元素含量变化,N元素含量显著提高,说明蛋白分子在表面存在。Bradford方法定量分析明胶、胶原和RGD多肽的表面吸附量。结果XPS证实了表面N原子的引入,存在酰胺键,确定蛋白分子存在于PS表面。结论动态接触角下降显著,证明修饰表面的亲水性得到提高。并在明胶、胶原和RGD多肽修饰表面接种人表皮细胞,对比考察其对细胞行为的影响,提高了细胞的黏附和增殖能力。     

Gene-activated and cell-migration guiding PEG matrices based on three dimensional patterning of RGD peptides and DNA complexes

Essential to the design of genetic bioreactors used in the human body is a consideration of how the properties of biomaterials can combine to envelope, spatially guide, reprogramme by gene transfer, and then release cells. In order to approach this goal, poly(ethylene glycol) (PEG) matrices with modulated structural features and defined spatial patterns of bioactive signals have been designed and produced. In particular, within such PEG matrices, both an adhesive RGD peptide gradient, to directionally attract NIH3T3 cells, and a designed spatial distribution of immobilized poly(ethylenimine) (PEI)/DNA complexes, to obtain a localized transfection, have been realized. These bioactive biomaterials have been designed bearing in mind that cells following an RGD gradient migrate through the matrix, in which they find the bound DNA and become transfected. Both cell migration and transfection have been monitored by fluorescence microscopy. Results show that this system is able to envelope cells, spatially guide them towards the immobilized gene complexes and locally transfect them. Therefore, the system, acting as a genetic bioreactor potentially useful for the regulation of biology at a distance, could be used to directly control cell trafficking and activation in the human body, and has many potential biomedical applications.