Model porous surfaces for systematic studies of material-cell interactions

A model system for studying cell-surface interactions, based on microfabricated cell culture substrates, has been developed and is described here. Porous surfaces consisting of interconnecting channels with openings of subcellular dimensions are generated on flat, single crystal, silicon substrates. Channel size (width, depth), distribution, and surface coating can be varied independently and used for systematic investigation of how topographical, chemical, and elastic surface properties influence cell or tissue biological responses. Model porous surfaces have been produced by using two different microfabrication methods. Submicron-sized channels with very high depth-to-width aspect ratios (up to 30) have been made by using electron beam lithography and anisotropic reactive ion etching into single-crystal silicon. Another method uses thick-resist photolithography, which can be used to produce channels wider than 1 microm and with depth-to-width aspect ratios below 20 in an epoxy polymer. Preliminary cell culture tests show that fibroblasts bridge 0.8- to 1.8-microm-wide channels with very few exceptions (i.e., a continuous space below the cell-surface interface is created). It has also been shown that variation of channel periodicity significantly affects fibroblast morphology and attachment density. With this model system, it is possible to load the channels with bioactive substances intended to interact with cells at or near the surface in a time-dependent manner.     

Electrospray deposited fibronectin retains the ability to promote cell adhesion

Scaffolds for tissue engineering require the correct biochemical cues if the seeded cells are to migrate into the scaffold and proliferate. For complex tissues this would require precise patterning of the scaffold structure with the particular biochemical cue required at each location on the scaffold. Electrospray enables the deposition of a wide number of biomolecules onto surfaces and can be used for precise patterning. We assessed the functionality of a key cell-adhesion molecule, fibronectin, after depositing it onto a surface using the electrospray technique. The addition of polypropylene glycol allowed a stable spray to be obtained from solutions with a range of fibronectin concentrations. Immunoassay tests showed that the amount of fibronectin retained on the surface was proportional to that sprayed from the solution. Increasing the surface density of fibronectin deposited onto silicon surfaces enhanced fibroblast attachment. The fibronectin thus appears to have retained its cell attachment functionality after undergoing the electrospray process. Since recent advances allow electrospray to pattern material from solution with micrometre accuracy this may allow materials to be biologically functionalized on a similar scale.     

Attachment of astroglial cells to microfabricated pillar arrays of different geometries

We studied the attachment of astroglial cells on smooth silicon and arrays of silicon pillars and wells with various widths and separations. Standard semiconductor industry photolithographic techniques were used to fabricate pillar arrays and wells in single-crystal silicon. The resulting pillars varied in width from 0. 5 to 2.0 micrometer, had interpillar gaps of 1.0-5.0 micrometer, and were 1.0 micrometer in height. Arrays also contained 1.0-micromter-deep wells that were 0.5 micrometer in diameter and separated by 0.5-2.0 micrometer. Fluorescence, reflectance, and confocal light microscopies as well as scanning electron microscopy were used to quantify cell attachment, describe cell morphologies, and study the distribution of cytoskeletal proteins actin and vinculin on surfaces with pillars, wells, and smooth silicon. Seventy percent of LRM55 astroglial cells displayed a preference for pillars over smooth silicon, whereas only 40% preferred the wells to the smooth surfaces. Analysis of variance statistics performed on the data sets yielded values of p > approximately.5 for the comparison between pillar data sets and < approximately.0003 in the comparison between pillar and well data sets. Actin and vinculin distributions were highly polarized in cells found on pillar arrays. Scanning electron microscopy clearly demonstrated that cells made contact with the tops of the pillars and did not reach down into the spaces between pillars even when the interpillar gap was 5.0 microm. These experiments support the use of surface topography to direct the attachment, growth, and morphology of cells. These surfaces can be used to study fundamental cell properties such as cell attachment, proliferation, and gene expression. Such topography might also be used to modify implantable medical devices such as neural implants and lead to future developments in tissue engineering.     

Evaluation of mammalian cell adhesion on surface-modified porous silicon

Porous silicon is a promising biomaterial that is non-toxic and biodegradable. Surface modification can offer control over the degradation rate and can also impart properties that promote cell adhesion. In this study, we modified the surface of porous silicon surface by ozone oxidation, silanisation or coating with collagen or serum. For each surface, topography was characterised using atomic force microscopy, wettability by water contact angle measurements, degradation in aqueous buffer by interferometric reflectance spectroscopy and surface chemistry by Fourier-transform infrared spectroscopy. The adhesion of rat pheochromocytoma (PC12) and human lens epithelial cells to these surfaces was investigated. Cells were incubated on the surfaces for 4 and 24 h, and adhesion characteristics were determined by using a fluorescent vital stain and cell counts. Collagen coated and amino silanised porous silicon promoted cell attachment for both cell lines whereas cells attached poorly to ozone oxidised and polyethylene glycol silanised surfaces. We showed that the two cell lines had different adhesion characteristics on the various surfaces at different time points. The use of the vitality assays Alamar Blue (redox based assay) and neutral red (active cellular uptake assay) with porous silicon was also investigated. We reveal incompatibilities between certain resazurin (Alamar Blue), lysosomal incorporation assays (neutral red) and porous silicon.     

Surface immobilization of neural adhesion molecule L1 for improving the biocompatibility of chronic neural probes: In vitro characterization

Silicon-based implantable neural electrode arrays are known to experience failure during long-term recording, partially due to host tissue responses. Surface modification and immobilization of biomolecules may provide a means to improve their biocompatibility and integration within the host brain tissue. Previously, the laminin biomolecule or laminin fragments have been used to modify the neural probe's silicon surface to promote neuronal attachment and growth. Here we report the successful immobilization of the L1 biomolecule on a silicon surface. L1 is a neuronal adhesion molecule that can specifically promote neurite outgrowth and neuronal survival. Silane chemistry and the heterobifunctional coupling agent 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) were used to covalently bind these two biomolecules onto the surface of silicon dioxide wafers, which mimic the surface of silicon-based implantable neural probes. After covalent binding of the biomolecules, polyethylene glycol (PEG)-NH(2) was used to cap the unreacted GMBS groups. Surface immobilization was verified by goniometry, dual polarization interferometry, and immunostaining techniques. Primary murine neurons or astrocytes were used to evaluate the modified silicon surfaces. Both L1- and laminin-modified surfaces promoted neuronal attachment, while the L1-modified surface demonstrated significantly enhanced levels of neurite outgrowth (p<0.05). In addition, the laminin-modified surface promoted astrocyte attachment, while the L1-modified surface showed significantly reduced levels of astrocyte attachment relative to the laminin-modified surface and other controls (p<0.05). These results demonstrate the ability of the L1-immobilized surface to specifically promote neuronal growth and neurite extension, while inhibiting the attachment of astrocytes, one of the main cellular components of the glial sheath. Such unique properties present vast potentials to improve the biocompatibility and chronic recording performance of neural probes.     

Fibroblast adhesion to micro- and nano-heterogeneous topography using diblock copolymers and homopolymers

Polymeric substrates of different surface chemistry and length scales were found to have profound influence on cell adhesion. The adhesion of fibroblasts on surfaces of oxidized polystyrene (PS), on surfaces modified with random copolymers of PS and poly(methyl methacrylate) [P(S-r-MMA)] with topographic features, and chemically patterned surfaces that varied in lateral length scales from nanometers to microns were studied. Surfaces with heterogeneous topographies were generated from thin film mixtures of a block copolymer, PS-b-MMA, with homopolymers of PS and PMMA. The two homopolymers macroscopically phase separated and, with the addition of diblock copolymer, the size scales of the phases decreased to nanometer dimensions. Cell spreading area analysis showed that a thin film of oxidized PS surface promoted adhesion whereas a thin film of P(S-r-MMA) surface did not. Fibroblast adhesion was examined on surfaces in which the lateral length scale varied from 60 nm to 6 microm. It was found that, as the lateral length scale between the oxidized PS surfaces decreased, cell spreading area and degree of actin stress fiber formation increased. In addition, scanning electron microscopy was used to evaluate the location of filopodia and lamellipodia. It was found that most of the filopodia and lamellipodia interacted with the oxidized PS surfaces. This can be attributed to both chemical and topographic surface interactions that prevent cells from interacting with the P(S-r-MMA) at the base of the topographic features.     

Bounds in the Sensitivity of BioMEMS Devices for Cell Detection

This paper presents an ongoing effort to characterize performance and reliability of micro electromechanical systems used for biomedical diagnostics (BioMEMS). In order to study the interactions of human osteosarcoma (HOS) cells with BioMEMS devices, cultures were performed on silicon (Si) surfaces as well as silicon surfaces coated with 50 nm of titanium (Ti). Cell spreading on the surfaces was observed over time for up to 2 hours. It was seen that titanium coated silicon surfaces have the potential to provide a better interface for BioMEMS devices, due to enhanced adherence and spreading of the cells on these surfaces. Atomic force microscope (AFM) cantilevers were used as cell detection sensors. These cantilevers were coated with 50nm of titanium metal to provide a cell friendly surface. Theoretical models were then developed for the prediction of the vibrational responses of the AFM cantilevers before and after cell attachment. The models were used to relate the experimentally observed changes in frequency to the number of cells that are attached on the cantilever. The bounds in the possible frequency changes were determined within a theoretical framework. From experimentally calculated values for the mass of cells, random number simulations were carried out to determine the probability of cell attachment as a function of the change in resonance frequency of the cantilever sensor. The implications of the results are then discussed for the future reliability modeling of the sensor.     

Effect of nanofiber-coated surfaces on the proliferation and differentiation of osteoprogenitors in vitro

The osteoconductive property of titanium (Ti) surfaces is important in orthopedic and dental implant devices. Surface modifications of Ti have been proposed to further improve osseointegration. In this study, three different materials, silicon (Si), silicon oxide (SiO(2)), and titanium oxide (TiO(2)), were used to construct nanofibers for surface coating of Ti alloy Ti-6Al-4 V (Ti alloy). MC3T3-E1 osteoprogenitor cells were seeded on nanofiber-coated discs and cultured for 42 days. DNA, alkaline phosphatase, osteocalcin, and mineralization nodules were measured using PicoGreen, enzyme-linked immunosorbent assay, and calcein blue staining to detect the attachment, proliferation, differentiation, and mineralization of MC3T3-E1 cells, respectively. The results demonstrated that the initial cell attachments on nanofiber-coated discs were significantly lower, although cell proliferation on Si and SiO(2) nanofiber-coated discs was better than on Ti alloy surfaces. TiO(2) nanofibers facilitated a higher cellular differentiation capacity than Ti alloy and tissue culture-treated polystyrene surfaces. Thus, surface modification using nanofibers of various materials can alter the attachment, proliferation, and differentiation of osteoprogenitor cells in vitro.     

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell’s capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.     

Combined chemical and topographical guidance cues for directing cytoarchitectural polarization in primary neurons

Chemical and topographical cues can be used to guide dissociated neurons into user-defined network geometries on artificial substrates, yet control of neuron polarity (differentiation into axons and dendrites) remains an elusive goal. We developed a dual guidance cue strategy for directing morphological maturity in neurons in vitro using combined chemical and topographical guidance cues on glass substrates. The surface chemistry provides chemical attraction and repulsion for controlling neuron placement and outgrowth, while the topography provides additional surface area for neuron attachment. Poly-l-lysine (PLL) was adsorbed into etched trenches in glass substrates, and an acetone liftoff process was used to produce bifunctional surfaces with a hydrophobic hexamethyldisilazane (HMDS) background and trench patterns of PLL. We examined the cytoarchitectural polarization of dissociated hippocampal pyramidal neurons on guidance cues designed to promote rapid outgrowth of neurites onto continuous line features and delayed neurite outgrowth onto interrupted line features. An optimum distance of approximately 5 μm between the cell body attachment node and the first interrupted line guidance cue led to specific cytoarchitectural polarization of ≥60% of neurons by 3 days of culture in vitro.     

Length-scale mediated adhesion and directed growth of neural cells by surface-patterned poly(ethylene glycol) hydrogels

We engineered surfaces that permit the adhesion and directed growth of neuronal cell processes but that prevent the adhesion of astrocytes. This effect was achieved based on the spatial distribution of sub-micron-sized cell-repulsive poly(ethylene glycol) [PEG] hydrogels patterned on an otherwise cell-adhesive substrate. Patterns were identified that promoted cellular responses ranging from complete non-attachment, selective attachment, and directed growth at both cellular and subcellular length scales. At the highest patterning density where the individual hydrogels almost overlapped, there was no cellular adhesion. As the spacing between individual hydrogels was increased, patterns were identified where neurites could grow on the adhesive surface between hydrogels while astrocytes were unable to adhere. Patterns such as lines or arrays were identified that could direct the growth of these subcellular neuronal processes. At higher hydrogel spacings, both neurons and astrocytes adhered and grew in a manner approaching that of unpatterned control surfaces. Patterned lines could once again direct growth at cellular length scales. Significantly, we have demonstrated that the patterning of sub-micron/nano scale cell-repulsive features at microscale lengths on an otherwise cell-adhesive surface can differently control the adhesion and growth of cells and cell processes based on the difference in their characteristic sizes. This concept could potentially be applied to an implantable nerve-guidance device that would selectively enable regrowing axons to bridge a spinal-cord injury without interference from the glial scar.     

Nanotopographical guidance of C6 glioma cell alignment and oriented growth

The surface properties of the extracellular matrix play vital roles in cellular behavior such as adhesion, spreading, migration, proliferation and differentiation. While cell attachment and adhesion onto surfaces are mainly mediated by surface molecular interaction, cell morphology and orientation are significantly affected by the topographical cues of the substrate. We reported here the alignment of C6 glioma cells on polystyrene (PS) substrate containing periodic nanotopography. The ridge/groove type structures (210 nm in periodicity, and 30-40 nm in depth) were generated on polystyrene surface using Nd:YAG polarized laser radiation at 266 nm. The cultured cells were shown to align strictly along the direction of the ridges/grooves. And there were distinctive features such as elongated morphology and asymmetrical cell surface extensions, revealed by confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and scanning electron microscopy (SEM). The results indicated that ordered and continuous nanostructures on substrates can pattern cell, and guide cell alignment and oriented growth along definite directions. The possible mechanism and significance of these observations were also discussed.     

Synergistic effects of UV photofunctionalization and micro-nano hybrid topography on the biological properties of titanium

Titanium surfaces with micro-nano hybrid topography (nanoscale nodules in microscale pits) have been recently demonstrated to show higher biological capability than those with microtopography alone. On the other hand, UV treatment of titanium surfaces, which is called UV photofunctionalization, has recently been introduced to substantially increase the biological capability and osteoconductivity of titanium surfaces. However, synergistic effects of these two advanced surface modification technologies and regulatory factors to potentially modulate the mutual effects have never been addressed. In this study, utilization of a recently discovered controllable self-assembly of TiO(2) nanonodules has enabled the exploration of the relative contribution of different sizes of nanostructures to determine the biological capability of titanium surfaces and their relative responsiveness to UV photofunctionalization. Rat bone marrow-derived osteoblasts were cultured on titanium disks with either micropits alone, micropits with 100-nm nodules, micropits with 300-nm nodules, or micropits with 500-nm nodules, with or without UV treatment. Although UV treatment increased the attachment, spread, proliferation, and mineralization of these cells on all titanium surfaces, these effects were more accentuated (3-5 times) on nanonodular surfaces than on surfaces with micropits alone and were disproportionate depending on nanonodule sizes. For instance, on UV-treated micro-nano hybrid surfaces, cell attachment correlated with nanonodule sizes in a quadratic approximation with its peak for 300-nm nodules followed by a decline for 500-nm nodules, while cell attachment exponentially correlated with surface roughness with its plateau achieved for 300-nm nodules without a subsequent decline. Moreover, cell attachment increased in a linear correlation with the surface area, while no significant effect of the inter-irregularities space or degree of hydrophilicity was observed on cell attachment. These results suggest that the effect of UV photofunctionalization can be multiplied on micro-nano hybrid titanium surfaces compared with the surfaces with micropits alone. This multiplication is disproportionately regulated by a selected set of topographical parameters of the titanium surfaces. Among the nanonodules tested in this study, 300-nm nodules seemed to create the most effective morphological environment for responding to UV photofunctionalization. The data provide a systematic platform to effectively optimize nanostructures on titanium surfaces in order to enhance their biological capability as well as their susceptibility to UV photofunctionalization.     

Rapid Fabrication and Chemical Patterning of Polymer Microstructures and their Applications as a Platform for Cell Cultures

Much of the current knowledge regarding biological processes has been obtained through in-vitro studies in bulk aqueous solutions or in conventional Petri-dishes, with neither methodology accurately duplicating the actual in-vivo biological processes. Recently, a number of innovative approaches have attempted to address these shortcomings by providing substrates with controlled features. In particular, tunable surface chemistries and topographical micro and nanostructures have been used as model systems to study the complex biological processes. We herein report a versatile and rapid fabrication method to produce a variety of microstructured polymer substrates with precise control and tailoring of their surface chemistries. A poly(dimethylsiloxane) (PDMS) substrate, produced by replication over a master mold with specific microstructures, is modified by a fluoro siloxane derivative to enhance its anti-adhesion characteristics and used as a secondary replication mold. A curable material, deposited by spin coating on various substrates, is stamped with the secondary mold and crosslinked. The removal of the secondary mold produces a microstructured surface with the same topographical features as the initial master mold. The facile chemical patterning of the microstructured substrates is demonstrated through the use of microcontact printing methods and these materials are tested as a platform to guide cell attachment, growth and proliferation. The master mold and flexible fluorinated PDMS stamps can be used in a repeated manner without any degradation of the anti-adhesion characteristics opening the way to the development of high-throughput fabrication methods that can yield reliable and inexpensive microstructured and chemically patterned substrates.     

Increasing cell adhesion on plasma deposited fluorocarbon coatings by changing the surface topography

In designing new biomaterials, it is of outstanding importance to consider how cells respond to specific chemical and topographical features on the material surface. The behavior of most cell types in vivo is strictly related to specific chemical and topographical cues that characterize the extra cellular environment. In particular, during their lives cells react to topographical patterns such as those of the extracellular matrix (ECM), of micro and/or nanometric dimensions. The production of micrometric and/or nanometric features on artificial materials usually involves expensive and time-consuming methods of manufacturing, such as electron beam and colloidal lithography. In this article, different "Teflon-like" structured surfaces were deposited from tetrafluoroethylene (C(2)F(4))-fed plasmas, for the study of cell adhesion and growth. The reaction of different cell lines to different topographical features was evaluated and compared with cell behavior on flat samples with the same chemical composition. Cell adhesion was calculated from area covered by cells at different time of culture. Beside this, cell proliferation was determined with the MTT test. Cell morphology and filopodia interaction with the nanofeatures were also estimated by optical and scanning electron microscopy. A dramatic difference both in adhesion and growth was found between cells seeded on flat and rough surfaces with the density and spreading of adhered cells varying as a function of the roughness of coatings.     

The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their suitability as cell growth support surfaces

Nanocrystalline diamond (NCD) films and nanoparticulate diamond powder (DP) are the two main representatives of diamond at the nanoscale. This study was designed to investigate the suitability of these biomaterials as cell growth supports and to determine surface characteristic properties best suited to cell attachment and proliferation. Surface topography, chemical termination and wetting properties of NCD- and DP-coated borosilicate glass substrates were correlated to attachment, proliferation and differentially regulated gene expression of human renal epithelial cells (HK-2 cell line) cultured on these surfaces. Hydrogen-terminated NCD (NCD-H) surfaces were shown to inhibit cell attachment, which indicates that the lack of functional polar groups prevents adherent cells from settling on a surface, whether nanostructured or not. In contrast to NCD-H, oxygen-terminated NCD (NCD-O) as well as DP surfaces demonstrated improved cell attachment, as compared to borosilicate glass, which is a commonly used material for cell growth supports. NCD-O not only revealed an increased cell attachment, but also a markedly increased proliferation rate. Finally, none of the investigated surface modifications appeared to cause adverse cellular reactions or markedly alter cellular phenotype.     

Nanoscaled periodic surface structures of medical stainless steel and their effect on osteoblast cells

Nanoscaled lamellar surface structures have been prepared on medical stainless steel AISI 316LVM surfaces by chemical etching of the decomposed phases. The effect of this structure on osteoblastic cells has been investigated. Long filopodia were developed by the cells perpendicular to the lamellar structure while almost no or only short filopodia were formed parallel to the lamellae. These results are explained in terms of a topographical influence of the nanostructure. During the growth process of the filopodia a nearly flat surface was recognized parallel to the lamellae while a topographical change was sensed perpendicular to the structure, which was preferred by the cells.     

Comparison of proliferation and growth of human keratinocytes on plasma copolymers of acrylic acid/1,7-octadiene and self-assembled monolayers.

Human keratinocytes were cultured on plasma copolymers (PCPs), self-assembled monolayers (SAMs), and tissue culture poly(styrene) (TCPS). Plasma copolymerization was used to deposit films with controlled concentrations of carboxylic acid functional groups (<5%). Human keratinocytes were cultured onto these PCP surfaces, TCPS, and collagen I. A hydrocarbon plasma polymer surface was used as the negative control. Keratinocyte attachment was measured at 24 h and cell proliferation and growth at 3 and 7 days using optical microscopy and DNA concentrations. The PCP surfaces were compared with two SAM systems comprising pure acid and pure hydrocarbon functionalities, and pure gold was used as a control surface. PCP surfaces containing carboxylic acid functionalities promoted keratinocyte attachment. The level of attachment on these surfaces was comparable to that seen on collagen I, a preferred substratum for the culturing of keratinocytes. After several days in culture the cells were well attached and proliferative, forming confluent sheets of keratinocytes. This result was confirmed by DNA assays that suggested the acid PCP surfaces were performing as well as collagen I. Keratinocytes attached well to gold and acid-terminated SAMs but attached poorly to methyl-terminated SAMs. The acid functionality also promoted proliferation and growth of keratinocytes after several days in culture. DNA assays revealed that keratinocyte growth on the acid surface was higher than on collagen I.     

The Effects of Collagen Type I Topography on Myoblasts In Vitro

Cells respond to a variety of cues from their environment, which can include chemical, mechanical, and topographical signals. The differentiation of myoblasts requires a combination of signals. Myoblast fusion is strongly influenced by the chemical nature of the surrounding matrix and can be affected by mechanical stimulation. Studies also have shown that a large variety of cell types also are influenced by details of surface topography of a substrate as small as 44 nm. Cells grown on a collagen-coated surface differentiate more readily than those grown in the absence of the extracellular matrix protein. It is not known whether the effects of myoblast interaction with collagen are due solely to chemical interactions or if myoblasts also respond to the topography of collagen type I fibers. To determine the importance of collagen-generated topographical signals on myoblast development, cells were cultured and differentiated in vitro on surfaces that had been coated with either soluble collagen type I or fibrous collagen type I. Both surfaces present the same chemical interactions, but the additional topographical signals lead to differences in cell morphology, adhesion, spreading rates and, proliferation. Cells on the fibrous form of collagen are more stellate, form more adhesion plaques, spread faster, and proliferate at a faster, rate than cells on a surface of soluble collagen. Our data indicate that topographical signals play a role in early muscle development, but that other or additional signaling pathways regulate differentiation.     

The effects of collagen type I topography on myoblasts in vitro

Cells respond to a variety of cues from their environment, which can include chemical, mechanical, and topographical signals. The differentiation of myoblasts requires a combination of signals. Myoblast fusion is strongly influenced by the chemical nature of the surrounding matrix and can be affected by mechanical stimulation. Studies also have shown that a large variety of cell types also are influenced by details of surface topography of a substrate as small as 44 nm. Cells grown on a collagen-coated surface differentiate more readily than those grown in the absence of the extracellular matrix protein. It is not known whether the effects of myoblast interaction with collagen are due solely to chemical interactions or if myoblasts also respond to the topography of collagen type I fibers. To determine the importance of collagen-generated topographical signals on myoblast development, cells were cultured and differentiated in vitro on surfaces that had been coated with either soluble collagen type I or fibrous collagen type I. Both surfaces present the same chemical interactions, but the additional topographical signals lead to differences in cell morphology, adhesion, spreading rates and, proliferation. Cells on the fibrous form of collagen are more stellate, form more adhesion plaques, spread faster, and proliferate at a faster, rate than cells on a surface of soluble collagen. Our data indicate that topographical signals play a role in early muscle development, but that other or additional signaling pathways regulate differentiation.