Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells

The regulation of mouse embryonic stem cell (mESC) fate is controlled by the interplay of signaling networks that either promote self-renewal or induce differentiation. Leukemia inhibitory factor (LIF) is a cytokine that is required for stem cell renewal in mouse but not in human embryonic stem cells. However, feeder layers of embryonic fibroblasts are capable of inducing stem cell renewal in both cell types, suggesting that the self-renewal signaling pathways may also be promoted by other triggers, such as alternative cytokines and/or chemical or physical properties of the extracellular matrix (ECM) secreted by feeder fibroblasts. We have recently used a synthetic polyamide matrix (Ultra-Web) whose three-dimensional (3D) nanofibrillar organization resembles the ECM/basement membrane. Growth of mESCs on this nanofibrillar surface greatly enhanced proliferation and self-renewal in comparison with growth on tissue culture surfaces without nanofibers, despite the presence of LIF in both systems. Enhanced proliferation and self-renewal of the stem cells on nanofibrillar surfaces were correlated with the activation of the small GTPase Rac, the activation of phosphoinositide 3-kinase (PI3K) pathway, and the enhanced expression of Nanog, a homeoprotein required for maintenance of pluripotency. Inhibitors of PI3K reduced the expression level of Nanog in mESCs cultured on 3D nanofibrillar surfaces. These results provide support for the view that the three-dimensionality of the culture surface may function as a cue for the activation of Rac and PI3K signaling pathways, resulting in stem cell proliferation and self-renewal.     

Electrospun nanofibrillar surfaces promote neuronal differentiation and function from human embryonic stem cells

In this study, the impact of randomly oriented electrospun polyamide nanofibrous architecture on neurogenic differentiation of human embryonic stem cells (hESCs) compared with the lack of nanofibrous features in vitro in a neural-inducing condition was examined. Flow cytometry analysis of hESC-derived neural ectoderm (NE) showed nanofibrous surfaces capable of supporting NE by expression of higher percentages of related markers NESTIN, SOX1, and PAX6 in addition to significantly greater total cell proliferation as shown by Ki67 in the neurogenic condition. After replating hESC-derived NE, the differentiated cells expressed higher neuronal markers (TUJ1 and MAP2) and motor neuron markers (HB9, ISL1, and ChAT) at both the protein and mRNA levels on nanofibers. The presence of developed spread neurites and plausible neurite connections were shown by scanning electron microscopy. Additionally, Na(+) and Ca(2+) currents in differentiated neurons on nanofibers were significantly greater than both control and generated action potentials. Moreover, less duration of inward currents, greater negative resting membrane potential, and enhanced expression and functionality of ionic channel genes were observed in neuronal cells on nanofibers. These results indicated that a nanofibrillar surface along with neurogenic growth factors provided a better environment for hESC neurogenic differentiation and function, which holds great promise in prospective tissue engineering applications.     

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.     

Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts

Microcontact printing techniques were used to pattern circles (diameters 10. 50, 100, and 200 microm) of N1[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS) borders on borosilicate glass, a model substrate. The DETA regions were further modified by immobilization of either the cell-adhesive peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS) and Lysine-Arginine-Serine-Arginine (KRSR) or the non-adhesive peptides Arginine-Aspartic Acid-Glycine-Serine (RDGS) and Lysine-Serine-Serine-Arginine (KSSR). After four hours under standard cell culture conditions but in the absence of serum, adhesion of either osteoblasts or fibroblasts on surfaces patterned with the non-adhesive peptides RDGS and KSSR was random and low. In contrast, both osteoblasts and fibroblasts adhered and formed clusters onto circles modified with the adhesive peptide RGDS, whereas only osteoblasts adhered and formed clusters onto the circles modified with KRSR, a peptide that selectively promotes adhesion of osteoblasts. These results provide evidence that patterning of select peptides can direct adhesion of specific cell lines exclusively to predetermined regions on material surfaces.     

Selective adhesion of astrocytes to surfaces modified with immobilized peptides.

Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.     

Enhancing the neuronal interaction on fluoropolymer surfaces with mixed peptides or spacer group linkers

Embryonic hippocampal neurons cultured on surface modified fluoropolymers showed enhanced interaction and neurite extension. Poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surfaces were aminated by reaction with a UV-activated mercury ammonia system yielding FEP-[N/O]. Laminin-derived cell-adhesive peptides (YIGSR and IKVAV) were coupled to FEP surface functional groups using tresyl chloride activation. Embryonic (E18) hippocampal neurons were cultured in serum-free medium for up to 1 week on FEP film surfaces that were modified with either one or both of GYIGSR and SIKVAV or GGGGGGYIGSR and compared to control surfaces of FEP-[N/O] and poly(L-lysine)/laminin-coated tissue culture polystyrene. Neuron-surface interactions were analyzed over time in terms of neurite outgrowth (number and length of neurites), cell adhesion and viability. Neurite outgrowth and adhesion were significantly better on peptide-modified surfaces than on either FEP or FEP-[N/O]. Cells on the mixed peptide (GYIGSR/SIKVAV) and the spacer group peptide (GGGGGGYIGSR) surfaces demonstrated similar behavior to those on the positive PLL/laminin control. The specificity of the cell-peptide interaction was demonstrated with a competitive assay where dissociated neurons were incubated in media containing peptides prior to plating. Cell adhesion and neurite outgrowth diminished on all surfaces when hippocampal neurons were pre-incubated with dissolved peptides prior to plating.     

Directing neuronal cell growth on implant material surfaces by microstructuring

For best hearing sensation, electrodes of auditory prosthesis must have an optimal electrical contact to the respective neuronal cells. To improve the electrode-nerve interface, microstructuring of implant surfaces could guide neuronal cells toward the electrode contact. To this end, femtosecond laser ablation was used to generate linear microgrooves on the two currently relevant cochlear implant materials, silicone elastomer and platinum. Silicone surfaces were structured by two different methods, either directly, by laser ablation or indirectly, by imprinting using laser-microstructured molds. The influence of surface structuring on neurite outgrowth was investigated utilizing a neuronal-like cell line and primary auditory neurons. The pheochromocytoma cell line PC-12 and primary spiral ganglion cells were cultured on microstructured auditory implant materials. The orientation of neurite outgrowth relative to the microgrooves was determined. Both cell types showed a preferred orientation in parallel to the microstructures on both, platinum and on molded silicone elastomer. Interestingly, microstructures generated by direct laser ablation of silicone did not influence the orientation of either cell type. This shows that differences in the manufacturing procedures can affect the ability of microstructured implant surfaces to guide the growth of neurites. This is of particular importance for clinical applications, since the molding technique represents a reproducible, economic, and commercially feasible manufacturing procedure for the microstructured silicone surfaces of medical implants.     

Biomimetic materials replicating Schwann cell topography enhance neuronal adhesion and neurite alignment in vitro

It is well established that Schwann cells (SCs) promote and enhance axon guidance and nerve regeneration by providing multiple cues, including extracellular matrix, cell surface molecules, neurotrophic factors and cellular topography. Which of the elements of the complex environment associated with SCs provides the essential information for directed nerve growth is unclear, because, until now, it has been impossible to investigate their contributions individually. Our development of biomimetic materials that replicate the micro- and nanoscale topography of SCs has allowed us to investigate for the first time the role of cellular topography in directing nerve growth. Dorsal root ganglion (DRG) neurons were cultured on flat poly(dimethyl siloxane) (PDMS) and on PDMS replicas with protruding SC topography. Image analysis showed that more neurons adhered to the replicas than to the flat substrates, and that neurite growth on the replicas followed the underlying SC pattern. Neuronal alignment was dependent on cell density. Live SCs derived from the DRG also grew along the replica SC pattern. These results suggest that the combination of micro- and nanoscale topographical cues provided by SCs can influence nerve growth and point toward design parameters for future nerve guidance channels.     

Biomimetic materials replicating Schwann cell topography enhance neuronal adhesion and neurite alignment in vitro

It is well established that Schwann cells (SCs) promote and enhance axon guidance and nerve regeneration by providing multiple cues, including extracellular matrix, cell surface molecules, neurotrophic factors and cellular topography. Which of the elements of the complex environment associated with SCs provides the essential information for directed nerve growth is unclear, because, until now, it has been impossible to investigate their contributions individually. Our development of biomimetic materials that replicate the micro- and nanoscale topography of SCs has allowed us to investigate for the first time the role of cellular topography in directing nerve growth. Dorsal root ganglion (DRG) neurons were cultured on flat poly(dimethyl siloxane) (PDMS) and on PDMS replicas with protruding SC topography. Image analysis showed that more neurons adhered to the replicas than to the flat substrates, and that neurite growth on the replicas followed the underlying SC pattern. Neuronal alignment was dependent on cell density. Live SCs derived from the DRG also grew along the replica SC pattern. These results suggest that the combination of micro- and nanoscale topographical cues provided by SCs can influence nerve growth and point toward design parameters for future nerve guidance channels.     

Interactions of human embryonic stem cell-derived neural progenitors with an electrospun nanofibrillar surface in vitro

Stem cell technology combined with nano-scaffold surfaces provides a new tool for better induction involved in cell lineage differentiations and therefore for central nervous system repair. This study was undertaken to investigate appropriate neural cell-substrate interactions. Neural progenitors (NPs) were established from human embryonic stem cells (hESCs), as a first step, using an adherent system and a defined medium supplemented with a combination of factors. Next, the behavior of hESC-derived NPs (hESC-NPs) was evaluated on a synthetic, randomly oriented, three-dimensional nanofibrillar matrix composed of electrospun polyamide nanofibers (Ultra-Web?) using a variety of experimental approaches. We have demonstrated that homogenous, expandable, and self-renewable NPs can be easily generated from hESCs; they can express related markers Nestin, Sox1, and Pax6; and they can undergo multipotency differentiation to neurons and glials. Functionally, NPs cultured on nanofibers demonstrated an increase in the rate of migration, proliferation, morphology, and neurite length when compared with NPs cultured on two-dimensional culture surfaces. The results suggest that topographical features of the extracellular matrix of the cell environment have paved the way for a better understanding of human neuronal development, thus allowing for future clinical applications.     

Patterned growth of neuronal cells on modified diamond-like carbon substrates

Diamond-like carbon (DLC) has been explored as a biomaterial with potential use for coating implantable devices and surgical instruments. In this study the interaction of DLC with mammalian neuronal cells has been studied along with its modifications to improve its function as a biomaterial. We describe the use of DLC, oxidised DLC and phosphorus-doped DLC to support the growth and survival of primary central nervous system neurones and neuroblastoma cells. None of these substrates were cytotoxic and primary neurones adhered better to phosphorus-doped DLC than unmodified DLC. This property was used to culture cortical neurones in a predetermined micropattern. This raises the potential of DLC as a biomaterial for central nervous system (CNS) implantation. Furthermore, patterned DLC and phosphorus-doped DLC can direct neuronal growth, generating a powerful tool to study neuronal networks in a spatially distinct way. This study reports the generation of nerve cell patterns via patterned deposition of DLC.     

Orexin peptides enhance median preoptic nucleus neuronal excitability via postsynaptic membrane depolarization and enhancement of glutamatergic afferents

Subpopulations of neurons in the median preoptic nucleus (MnPO) located within the lamina terminalis contribute to thermoregulatory, cardiovascular and hydromineral homeostasis, and sleep-promotion. MnPO is innervated by lateral hypothalamic neurons that synthesize and secrete the arousal-promoting and excitatory orexin (hypocretin) neuropeptides. To evaluate the hypothesis that orexins modulate the excitability of MnPO neurons, we used patch-clamp recording techniques applied in rat brain slice preparations to assess the effects of exogenously applied orexin A and orexin B peptides on their intrinsic and synaptic properties. Whole cell recordings under current-clamp mode revealed that 11/15 tested MnPO neurons responded similarly to either orexin A or B (500-1000 nM) with a slowly rising, prolonged (10-15 min) and reversible membrane depolarization. Under voltage-clamp mode, orexin applications induced a tetrodotoxin-resistant inward current of -7.2+/-1.6 pA, indicating a direct (postsynaptic) activation, with a time course similar to the observed membrane depolarization. The orexin-induced responses in 4/7 neurons were associated with a significant decrease in membrane conductance and the net orexin-induced current that reversed at -99+/-5 mV, suggesting closure of potassium channels. Orexins did not attenuate the properties of excitatory (n=4) or inhibitory (n=7) postsynaptic currents evoked by subfornical organ stimulation. By contrast, orexins applications induce a significant increase in both frequency and amplitude of spontaneous glutamatergic postsynaptic currents (5/7 cells) but had no influence on spontaneous GABAergic currents (6/6 cells). Thus, in addition to a direct postsynaptic receptor-mediated excitation, orexins can also increase the excitability of MnPO neurons via increasing their excitatory inputs, presumably through an orexin receptor-mediated excitation of local glutamatergic neurons whose axons project to MnPO neurons.     

Induction of myofibroblastic differentiation in vitro by covalently immobilized transforming growth factor-beta(1)

Growth factors are an important tool in tissue engineering. Bone morphogenetic protein-2 and transforming growth factor-beta(1) (TGF-beta(1)) are used to provide bioactivity to surgical implants and tissue substitute materials. Mostly growth factors are used in soluble or adsorbed form. However, simple adsorption of proteins to surfaces is always accompanied by reduced stability and undefined pharmacokinetics. This study aims to prove that TGF-beta(1) can be covalently immobilized to functionalized surfaces, maintaining its ability to induce myofibroblastic differentiation of normal human dermal fibroblasts. In vivo, fibroblasts differentiate to myofibroblasts (MFs) during soft tissue healing by the action of TGF-beta(1). As surfaces for our experiments, we used slides bearing aldehyde, epoxy, or amino groups. For our in vitro cell culture experiments, we used the expression of alpha-smooth muscle actin as a marker for MFs after immunochemical staining. Using the aldehyde and the epoxy slides, we were able to demonstrate the activity of immobilized TGF-beta(1) through a significant increase in MF differentiation rate. A simple immunological test was established to detect TGF-beta(1) on the surfaces. This technology enables the creation of molecular "landscapes" consisting of several factors arranged in a distinct spatial pattern and immobilized on appropriate surfaces.     

Endothelial cell adhesion and proliferation to PEGylated polymers with covalently linked RGD peptides

A nonfouling peptide grafted polymer was synthesized that can promote endothelial cell (EC) binding. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate, and CGRGDS peptide. The peptide was incorporated into the polymer system either by a chain transfer reaction or by coupling to an acrylate-PEG-N-hydroxysuccinimide (NHS) comonomer. The introduction of PEG chains minimizes protein adsorption. Human umbilical vein ECs and endothelial colony forming cells were cultured on these surfaces in short term and long-term studies. A difference in number and morphology of ECs was observed depending on the method of peptide incorporation. Both cell types adhered better to polymer films containing NHS coupled RGD peptide after 2 h even in the presence of albumin but significant cell detachment occurred after 4 days. Polymer solutions were electrospun into fibrous scaffolds. Both nonfouling and peptide binding characteristics were retained after processing.     

血管内皮细胞生长因子表位肽体外抗肿瘤作用研究

目的:研究筛选血管内皮细胞生长因子(VEGF)不同表位肽抗肿瘤及抗血管新生效用。方法:构建4种不同的pCANTAB5-E/VEGF表位肽载体,将VEGF表位肽通过噬菌体展示技术展示于噬菌体表面,MTT法检测VEGF噬菌体表位肽对黑色素瘤细胞(B16)、人脐静脉血管内皮细胞(HUVEC)增殖的影响,Transwell小室检测其对B16F10细胞迁移的影响,ECMgel检测其对HUVEC成管的影响。结果:筛选出VEGF的4种(VSB、VNB、VEB、VTB)表位肽中,VNB对HUVEC细胞增殖的抑制率达到64.8%,VEB、VTB对B16的增殖抑制率分别为75.7%和66.5%。4株表位肽对高转移细胞B16F10迁移抑制率均超过80%。结论:筛选得到的4株VEGF表位肽对黑色素瘤细胞、血管内皮细胞的增殖、迁移和成管有抑制作用,通过分析其表位区段后可制备成小分子多肽,为今后抗肿瘤多肽药物和疫苗的研发奠定基础。     

The phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently immobilized VEGF

Growth factors are a class of signaling proteins that direct cell fate through interaction with cell-surface receptors. Although a myriad of possible cell fates stems from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor – soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm² and 100 pg/cm², respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature.