Guide Cell Adhesion Research Articles

3D printing of PEGDA/bioceramic for guiding cell adhesion and migration

The adhesion and migration characteristics of osteocytes on the surface of bone materials are crucial for bone tissue regeneration, which is conducive to the directional repair of bone defects. Herein, the adhesion and migration characteristics of MC3T3-E1 cells on the surface of SLA-3D printed PEGDA/bioceramic materials were analyzed in detail. The results indicated that, in comparison to the cured PEGDA matrix, the pseudopods of cells preferred to adhere to ceramic particles, demonstrating the characteristics of hard drive migration. Furthermore, on a wavy surface with a surface roughness (Ra=2 μm) fabricated by the laser of the SLA-3D printer, MC3T3-E1 cells could grow directionally along the rough channel via contact guidance. Therefore, by controlling the distribution of ceramic particles and adjusting the surface morphology of the printed PEGDA/bioceramic materials, the migration direction of cells can be dominated, which is of great significance for the targeted repair of bone tissue.

Effects of Nanomaterials on Synthesis and Degradation of the Extracellular Matrix.

Extracellular matrix (ECM) remodeling is accompanied by the continuous synthesis and degradation of the ECM components. This dynamic process plays an important role in guiding cell adhesion, migration, proliferation, and differentiation, as well as in tissue development, body repair, and maintenance of homeostasis. Nanomaterials, due to their photoelectric and catalytic properties and special structure, have garnered much attention in biomedical fields for use in processes such as tissue engineering and disease treatment. Nanomaterials can reshape the cell microenvironment by changing the synthesis and degradation of ECM-related proteins, thereby indirectly changing the behavior of the surrounding cells. This review focuses on the regulatory role of nanomaterials in the process of cell synthesis of different ECM-related proteins and extracellular protease. We discuss influencing factors and possible related mechanisms of nanomaterials in ECM remodeling, which may provide different insights into the design and development of nanomaterials for the treatment of ECM disorder-related diseases.

Tuning the Topography of Dynamic 3D Scaffolds through Functional Protein Wrinkled Coatings.

Surface wrinkling provides an approach to fabricate micron and sub-micron-level biomaterial topographies that can mimic features of the dynamic, in vivo cell environment and guide cell adhesion, alignment, and differentiation. Most wrinkling research to date has used planar, two-dimensional (2D) substrates, and wrinkling work on three-dimensional (3D) structures has been limited. To enable wrinkle formation on architecturally complex, biomimetic 3D structures, here, we report a simple, low-cost experimental wrinkling approach that combines natural silk fibroin films with a recently developed advanced manufacturing technique for programming strain in complex 3D shape-memory polymer (SMP) scaffolds. By systematically investigating the influence of SMP programmed strain magnitude, silk film thickness, and aqueous media on wrinkle morphology and stability, we reveal how to generate and tune silk wrinkles on the micron and sub-micron scale. We find that increasing SMP programmed strain magnitude increases wavelength and decreases amplitudes of silk wrinkled topographies, while increasing silk film thickness increases wavelength and amplitude. Silk wrinkles persist after 24 h in cell culture medium. Wrinkled topographies demonstrate high cell viability and attachment. These findings suggest the potential for fabricating biomimetic cellular microenvironments that can advance understanding and control of cell-material interactions in engineering tissue constructs.

Photolithographically Patterned Cell-Repellent PEG-b-PTHPMA Diblock Copolymer for Guided Cell Adhesion and Growth.

Surfaces for guided cell adhesion and growth are indispensable in several diagnostic and therapeutic applications. Towards this direction, four diblock copolymers comprising polyethylene glycol (PEG) and poly(2-tetrahydropyranyl methacrylate) (PTHPMA) are synthesized employing PEG macroinitiators of different chain lengths. The copolymer with a 5000Da PEG block and a PEG-PTHPMA comonomers weight ratio of 43-57 provides a film with the highest stability in the culture medium and the strongest cell repellent properties. This copolymer is used to develop a positive photolithographic material and create stripe patterns onto silicon substrates. The highest selectivity regarding smooth muscle cell adhesion and growth and the highest fidelity of adhered cells for up to 3 days in culture is achieved for stripe patterns with widths between 25 and 27.5µm. Smooth muscle cells cultured on such patterned substrates exhibit a decrease in their proliferation rate and nucleus area and an increase in their major axis length, compared to the cells cultured onto non-patterned substrates. These alterations are indicative of the adoption of a contractile rather than a synthetic phenotype of the smooth muscle cells grown onto the patterned substrates and demonstrate the potential of the novel photolithographic material and patterning method for guided cell adhesion and growth.

Fabrication and Characterization of Layer-by-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration.

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-β1) via bioaffinity. The JBNm was assembled at a TGF-β1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-β1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-β1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-β1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

Blood-Vessel-Inspired Hierarchical Trilayer Scaffolds: PCL/Gelatin-Driven Protein Adsorption and Cellular Interaction.

Fabrication of scaffolds with hierarchical structures exhibiting the blood vessel topological and biochemical features of the native extracellular matrix that maintain long-term patency remains a major challenge. Within this context, scaffold assembly using biodegradable synthetic polymers (BSPs) via electrospinning had led to soft-tissue-resembling microstructures that allow cell infiltration. However, BSPs fail to exhibit the sufficient surface reactivity, limiting protein adsorption and/or cell adhesion and jeopardizing the overall graft performance. Here, we present a methodology for the fabrication of three-layered polycaprolactone (PCL)-based tubular structures with biochemical cues to improve protein adsorption and cell adhesion. For this purpose, PCL was backbone-oxidized (O-PCL) and cast over a photolithography-manufactured microgrooved mold to obtain a bioactive surface as demonstrated using a protein adsorption assay (BSA), Fourier transform infrared spectroscopy (FTIR) and calorimetric analyses. Then, two layers of PCL:gelatin (75:25 and 95:5 w/w), obtained using a novel single-desolvation method, were electrospun over the casted O-PCL to mimic a vascular wall with a physicochemical gradient to guide cell adhesion. Furthermore, tensile properties were shown to withstand the physiological mechanical stresses and strains. In vitro characterization, using L929 mouse fibroblasts, demonstrated that the multilayered scaffold is a suitable platform for cell infiltration and proliferation from the innermost to the outermost layer as is needed for vascular wall regeneration. Our work holds promise as a strategy for the low-cost manufacture of next-generation polymer-based hierarchical scaffolds with high bioactivity and resemblance of ECM’s microstructure to accurately guide cell attachment and proliferation.

Small extracellular vesicles with nanomorphology memory promote osteogenesis.

Nanotopographical cues endow biomaterials the ability to guide cell adhesion, proliferation, and differentiation. Cellular mechanical memory can maintain the cell status by retaining cellular information obtained from past mechanical microenvironments. Here, we propose a new concept “morphology memory of small extracellular vesicles (sEV)” for bone regeneration. We performed nanotopography on titanium plates through alkali and heat (Ti8) treatment to promote human mesenchymal stem cell (hMSC) differentiation. Next, we extracted the sEVs from the hMSC, which were cultured on the nanotopographical Ti plates for 21 days (Ti8-21-sEV). We demonstrated that Ti8-21-sEV had superior pro-osteogenesis ability in vitro and in vivo. RNA sequencing further confirmed that Ti8-21-sEV promote bone regeneration through osteogenic-related pathways, including the PI3K-AKT signaling pathway, MAPK signaling pathway, focal adhesion, and extracellular matrix-receptor interaction. Finally, we decorated the Ti8-21-sEV on a 3D printed porous polyetheretherketone scaffold. The femoral condyle defect model of rabbits was used to demonstrate that Ti8-21-sEV had the best bone ingrowth. In summary, our study demonstrated that the Ti8-21-sEV have memory function by copying the pro-osteogenesis information from the nanotopography. We expect that our study will encourage the discovery of other sEV with morphology memory for tissue regeneration.

Guiding cell adhesion and motility by modulating cross-linking and topographic properties of microgel arrays.

Biomaterial-driven modulation of cell adhesion and migration is a challenging aspect of tissue engineering. Here, we investigated the impact of surface-bound microgel arrays with variable geometry and adjustable cross-linking properties on cell adhesion and migration. We show that cell migration is inversely correlated with microgel array spacing, whereas directionality increases as array spacing increases. Focal adhesion dynamics is also modulated by microgel topography resulting in less dynamic focal adhesions on surface-bound microgels. Microgels also modulate the motility and adhesion of Sertoli cells used as a model for cell migration and adhesion. Both focal adhesion dynamics and speed are reduced on microgels. Interestingly, Gas2L1, a component of the cytoskeleton that mediates the interaction between microtubules and microfilaments, is dispensable for the regulation of cell adhesion and migration on microgels. Finally, increasing microgel cross-linking causes a clear reduction of focal adhesion turnover in Sertoli cells. These findings not only show that spacing and rigidity of surface-grafted microgels arrays can be effectively used to modulate cell adhesion and motility of diverse cellular systems, but they also form the basis for future developments in the fields of medicine and tissue engineering.

Musculoskeletal Tissue Engineering Using Fibrous Biomaterials.

In tissue engineering, scaffolds should provide the topological and physical cues as native tissues to guide cell adhesion, growth, migration, and differentiation. Fibrous structure is commonly present in human musculoskeletal tissues, including muscles, tendons, ligaments, and cartilage. Biomimetic fibrous scaffolds are thus critical for musculoskeletal tissue engineering. Electrospinning is a versatile technique for fabricating nanofibers from a variety of biomaterials. However, conventional electrospinning can only generate 2D nanofiber mats. Postprocessing methods are often needed to create 3D electrospun nanofiber scaffolds. In this chapter, we present two novel electrospinning-based scaffold fabrication techniques, which can generate 3D nanofiber scaffolds in one-station process: divergence electrospinning and hybrid 3D printing with parallel electrospinning. These techniques can be applied for engineering tissues with aligned fiber structures.

Development of a Piezoelectric PVDF-TrFE Fibrous Scaffold to Guide Cell Adhesion, Proliferation, and Alignment.

Severe peripheral nervous system injuries currently hold limited therapeutic solutions. Existing clinical techniques such as autografts, allografts, and newer nerve guidance conduits have shown variable outcomes in functional recovery, adverse immune responses, and in some cases low or minimal availability. This can be attributed in part to the lack of chemical, physical, and electrical cues directing both nerve guidance and regeneration. To address this pressing clinical issue, electrospun nanofibers and microfibers composed of piezoelectric polyvinylidene flouride-triflouroethylene (PVDF-TrFE) have been introduced as an alternative template for tissue engineered biomaterials, specifically as it pertains to their relevance in soft tissue and nerve repair. Here, biocompatible scaffolds of PVDF-TrFE are fabricated and their ability to generate an electrical response to mechanical deformations and produce a suitable regenerative microenvironment is examined. It is determined that 20% (w/v) PVDF-TrFE in (6:4) dimethyl formamide (DMF):acetone solvent maintains a desirable piezoelectric coefficient and the proper physical and electrical characteristics for tissue regeneration. Further, it is concluded that scaffolds of varying thickness promoted the adhesion and alignment of Schwann cells and fibroblasts. This work offers a prelude to further advancements in nanofibrous technology and a promising outlook for alternative, autologous remedies to peripheral nerve damage.

Impact of Various Sterilization and Disinfection Techniques on Electrospun Poly-ε-caprolactone.

Electrospun materials made from biodegradable polycaprolactone are used widely in various tissue engineering and regenerative medicine applications because of their morphological similarity to the extracellular matrix. However, the main prerequisite for the use of such materials in clinical practice consists of the selection of the appropriate sterilization technique. This study is devoted to the study of the impact of traditional sterilization and disinfection methods on a nanofibrous polycaprolactone layer constructed by means of the needleless electrospinning technique. It was determined that hydrogen peroxide plasma treatment led to the loss of fibrous morphology and the creation of a foil. However, certain sterilization (ethylene oxide, gamma irradiation, and peracetic acid) and disinfection techniques (ethanol and UV irradiation) were found not to lead to a change in morphology; thus, the study investigates their impact on thermal properties, molecular weight, and interactions with a fibroblast cell line. It was determined that the surface properties that guide cell adhesion and proliferation were affected more than the bulk properties. The highest proliferation rate of fibroblasts seeded on nanofibrous scaffolds was observed with respect to gamma-irradiated polycaprolactone, while the lowest proliferation rate was observed following ethylene oxide sterilization.

Amphiphilic cationic cyclodextrin nanovesicles: a versatile cue for guiding cell adhesion.

It is well known that amphiphilic cationic β-cyclodextrins (amβCDs) form nanovesicles able to release their cargo in aqueous solution upon applying different stimuli. In addition they can be selectively positioned onto substrates by unconventional soft lithography. This makes them a powerful tool for designing environments where different cues can be externally supplied to the cells helping to achieve good control of their fate. Lithographically controlled wetting (LCW) of amβCD nanovesicles loaded with fluorescein isothiocyanate (FITC), amβCD/FITC, has been used here to fabricate geometrically functionalized surfaces, thus achieving multiscale control of the cell environment. The amβCD functionalization was strongly influenced by the surface energy of the underlying substrates that, according to their hydrophobicity, orient the amβCD in a different way, thus “offering” different portions to the cells. The structure of the pattern was characterized both over large scales exploiting the FITC fluorescence and at the nanoscale by atomic force microscopy. Cell guidance and aCD/FITC cell internalization were demonstrated in human neuroblastoma SHSY5Y cells.

Guided cell adhesion, orientation, morphology and differentiation on silicon substrates photolithographically micropatterned with a cell-repellent cross-linked poly(vinyl alcohol) film

In this work, silicon substrates with poly(vinyl alcohol) (PVA) patterns created by a simple, low-cost and high-fidelity photolithographic procedure were evaluated with respect to cell adhesion and alignment, viability, metabolic activity, proliferation and cell cycle progression using the human glioblastoma cell-line U87MG and human skin fibroblasts. In addition, rat adrenal pheochromocytoma cells (PC-12) were employed to evaluate a modified photolithographic protocol appropriate for adhesion of cells requiring extracellular matrix components to adhere on the surface and to demonstrate that the proposed patterned substrates could provide unhindered cell differentiation. Regarding U87MG cells and skin fibroblasts, it was found that as the stripes width increased from 10 to 50 μm, the percentage of cells attached to Si versus the total area (Si + PVA) increased from 78% and 72% to 98.5% and 94.5% (p < 0.05), for U87MG cells and skin fibroblasts, respectively, with optimum cell alignment (≥95% of adherent cells with fidelity between 0.90 and 1.0; p < 0.05) for stripes width ranging between 20 and 22.5 μm. Concerning the viability, metabolic activity and proliferation of adherent cells, no statistically significant differences were observed compared to cells cultured onto non-patterned surfaces. Regarding PC-12 cells, a modification of the patterning procedure was followed involving coating of the substrate with type IV collagen prior to the photolithographic procedure, since they could not adhere on plain Si substrates. It was found that PC-12 cells adhere selectively (>95%) to collagen-coated Si stripes when the pattern width was equal to or wider than 10 μm. Following treatment with nerve growth factor, approximately 80% (p < 0.05) of the adherent cells differentiated to neuron-like cells extending neurites exclusively within the pattern. Given that the proposed patterning procedure allows highly selective cell adhesion without affecting cell proliferation, metabolic activity, and differentiation it could serve as a useful tool in various fields including tissue engineering, cell-based sensors and analytical microsystems.

How do immobilised cell-adhesive Arg–Gly–Asp-containing peptides behave at the PAA brush surface?

ABSTRACTBio-engineered surfaces that aim to induce normal cell behaviour in vitro need to ‘mimic’ the extracellular matrix in a way that allows cell adhesion. In this computational work, several model cell-binding peptides with a minimal cell-adhesive Arg–Gly–Asp sequence are investigated in the bulk as well as immobilised on a soft surface. For this reason, a combination of density functional theory and all-atom MD simulations is applied. The major goal of the modelling is to characterise the accessibility of the cell-recognition motif on the functionalised soft polymer surface. As a reference system, the behaviour of three peptide sequences is preliminarily studied in explicit water simulations. From the analysis of the MD trajectories, the solvent accessible surface area, the distribution of water molecules around peptide groups, the secondary structure and the thermodynamics of hydration are evaluated. Furthermore, each peptide is immobilised on the surface of a homopolymer poly(acrylic acid) brush. During MD simulations, all three peptides approach closely toward PAA brush, and their surface accessibility is characterised. Although the peptides are adsorbed onto the brush, they are not hidden by the polymer strands, with RGD unit accessible on the surface and available for guided cell adhesion.

CaMKK2 Regulates Mechanosensitive Assembly of Contractile Actin Stress Fibers

Stress fibers are contractile actomyosin bundles that guide cell adhesion, migration, and morphogenesis. Their assembly and alignment are under precise mechanosensitive control. Thus, stress fiber networks undergo rapid modification in response to changes in biophysical properties of the cell's surroundings. Stress fiber maturation requires mechanosensitive activation of 5'AMP-activated protein kinase (AMPK), which phosphorylates vasodilator-stimulated phosphoprotein (VASP) to inhibit actin polymerization at focal adhesions. Here, we identify Ca2+-calmodulin-dependent kinase kinase 2 (CaMKK2) as a critical upstream factor controlling mechanosensitive AMPK activation. CaMKK2 and Ca2+ influxes were enriched around focal adhesions at the ends of contractile stress fibers. Inhibition of either CaMKK2 or mechanosensitive Ca2+ channels led to defects in phosphorylation of AMPK and VASP, resulting in a loss of contractile bundles and a decrease in cell-exerted forces. These data provide evidence that Ca2+, CaMKK2, AMPK, and VASP form a mechanosensitive signaling cascade at focal adhesions that is critical for stress fiber assembly.

The correlation between osteopontin adsorption and cell adhesion to mixed self-assembled monolayers of varying charges and wettability.

Osteopontin (OPN) is a key mediator of cell interactions with biomaterials. However, few studies have been dedicated to studying cell adhesion on OPN-adsorbed substrates with controlled charge and wettability. Here, amino-carboxyl (NH2/COOH) and hydroxyl-methyl (OH/CH3) mixed self-assembled monolayers (SAMs) of varying charges and wettability, respectively, were used as controllable model surfaces to study OPN adsorption and subsequent mesenchymal stem cell (MSC) adhesion. The amount of OPN adsorbed onto the NH2/COOH mixed SAMs appeared to monotonically depend on the surface charge, whereas only a moderately hydrophilic surface was conducive to OPN adsorption on OH/CH3 mixed SAMs. The results correlated well with cell spreading on OPN-coated surfaces in a serum-free medium culture. In addition, the OH/CH3 mixed SAMs with moderate wettability tended to promote β1, β3, αv and α5 integrins, indicating that wettability may guide cell adhesion by mediating the integrins signaling pathway. This work will have reference value for designing biologically responsive substrate surfaces.

Nanoscale Surface Modifications of Medical Implants for Cartilage Tissue Repair and Regeneration.

Background: Natural cartilage regeneration is limited after trauma or degenerative processes. Due to the clinical challenge of reconstruction of articular cartilage, research into developing biomaterials to support cartilage regeneration have evolved. The structural architecture of composition of the cartilage extracellular matrix (ECM) is vital in guiding cell adhesion, migration and formation of cartilage. Current technologies have tried to mimic the cell’s nanoscale microenvironment to improve implants to improve cartilage tissue repair.Methods: This review evaluates nanoscale techniques used to modify the implant surface for cartilage regeneration.Results: The surface of biomaterial is a vital parameter to guide cell adhesion and consequently allow for the formation of ECM and allow for tissue repair. By providing nanosized cues on the surface in the form of a nanotopography or nanosized molecules, allows for better control of cell behaviour and regeneration of cartilage. Chemical, physical and lithography techniques have all been explored for modifying the nanoscale surface of implants to promote chondrocyte adhesion and ECM formation.Conclusion: Future studies are needed to further establish the optimal nanoscale modification of implants for cartilage tissue regeneration.

An update on the Application of Nanotechnology in Bone Tissue Engineering.

Background: Natural bone is a complex and hierarchical structure. Bone possesses an extracellular matrix that has a precise nano-sized environment to encourage osteoblasts to lay down bone by directing them through physical and chemical cues. For bone tissue regeneration, it is crucial for the scaffolds to mimic the native bone structure. Nanomaterials, with features on the nanoscale have shown the ability to provide the appropriate matrix environment to guide cell adhesion, migration and differentiation.Methods:This review summarises the new developments in bone tissue engineering using nanobiomaterials. The design and selection of fabrication methods and biomaterial types for bone tissue engineering will be reviewed. The interactions of cells with different nanostructured scaffolds will be discussed including nanocomposites, nanofibres and nanoparticles.Results: Several composite nanomaterials have been able to mimic the architecture of natural bone. Bioceramics biomaterials have shown to be very useful biomaterials for bone tissue engineering as they have osteoconductive and osteoinductive properties. Nanofibrous scaffolds have the ability to provide the appropriate matrix environment as they can mimic the extracellular matrix structure of bone. Nanoparticles have been used to deliver bioactive molecules and label and track stem cells.Conclusion:Future studies to improve the application of nanomaterials for bone tissue engineering are needed.

Biohybrid Janus Motors Driven by Escherichia coli

There has been a significant interest in the development of microswimmers for medical drug and cargo delivery, but the majority of current micromotors rely on toxic fuel sources and materials in their design making them irrelevant for biomedical applications. Bacteria represent an excellent motor alternative, as they are powered using their surrounding biological fluids. For a motile, biohybrid swimmer, Escherichia coli (E. coli) are integrated onto metal capped, polystyrene (PS) Janus particles. Fabrication of the biohybrid is rapid and simple for a microswimmer capable of magnetic guidance and ferrying an anticancer agent. Cell adhesion is regulated as E. coli adheres only to the particle's metal caps allowing the PS surface to be utilized for drug attachment, creating a multifunctional system. E. coli adhesion is investigated on multiple metal caps (Pt, Fe, Ti, or Au) and displays a strong preference to attach to Pt surfaces over other metals. Surface hydrophobicity and surface charge are examined to interpret the cell specific adhesion on the Janus particles. The dual capability of the biohybrid to have guided cell adhesion and localized drug attachment allows the swimmer to have multiple applications for biomedical microswimmers, future bacteria‐interface systems, and micro‐biorobots.

Static and cyclic mechanical loading of mesenchymal stem cells on elastomeric, electrospun polyurethane meshes.

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 μm) and small (0.60 μm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.