Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-l-lactic acid fibers

Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.     

The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation

Peripheral nerve regeneration can be enhanced by the stimulation of formation of bands of Büngner prior to implantation. Aligned electrospun poly(epsilon-caprolactone) (PCL) fibers were fabricated to test their potential to provide contact guidance to human Schwann cells. After 7 days of culture, cell cytoskeleton and nuclei were observed to align and elongate along the fiber axes, emulating the structure of bands of Büngner. Microarray analysis revealed a general down-regulation in expression of neurotrophin and neurotrophic receptors in aligned cells as compared to cells seeded on two-dimensional PCL film. Real-time-PCR analyses confirmed the up-regulation of early myelination marker, myelin-associated glycoprotein (MAG), and the down-regulation of NCAM-1, a marker of immature Schwann cells. Similar gene expression changes were also observed on cells cultured on randomly oriented PCL electrospun fibers. However, up-regulation of the myelin-specific gene, P0, was observed only on aligned electrospun fibers, suggesting the propensity of aligned fibers in promoting Schwann cell maturation.     

Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration

Aligned, electrospun fibers have shown great promise in facilitating directed neurite outgrowth within cell and animal models. While electrospun fiber diameter does influence cellular behavior, it is not known how aligned, electrospun fiber scaffolds of differing diameter influence neurite outgrowth and Schwann cell (SC) migration. Thus, the goal of this study was to first create highly aligned, electrospun fiber scaffolds of varying diameter and then assess neurite and SC behavior from dorsal root ganglia (DRG) explants. Three groups of highly aligned, electrospun poly-l-lactic acid (PLLA) fibers were created (1325 + 383 nm, large diameter fibers; 759 + 179 nm, intermediate diameter fibers; and 293 + 65 nm, small diameter fibers). Embryonic stage nine (E9) chick DRG were cultured on fiber substrates for 5 days and then the explants were stained against neurofilament and S100. DAPI stain was used to assess SC migration. Neurite length and SC migration distance were determined. In general, the direction of neurite extension and SC migration were guided along the aligned fibers. On the small diameter fiber substrate, the neurite length was 42% and 36% shorter than those on the intermediate and large fiber substrates, respectively. Interestingly, SC migration did not correlate with that of neurite extension in all situations. SCs migrated equivalently with extending neurites in both the small and large diameter scaffolds, but lagged behind neurites on the intermediate diameter scaffolds. Thus, in some situations, topography alone is sufficient to guide neurites without the leading support of SCs. Scanning electron microscopy images show that neurites cover the fibers and do not reside exclusively between fibers. Further, at the interface between fibers and neurites, filopodial extensions grab and attach to nearby fibers as they extend down the fiber substrate. Overall, the results and observations suggest that fiber diameter is an important parameter to consider when constructing aligned, electrospun fibers for nerve regeneration applications.     

Osteogenic Differentiation of Marrow Stromal Cells on Random and Aligned Electrospun Poly(l-lactide) Nanofibers

The fibrillar structure and sub-micron diameter of electrospun nanofibers can be used to reproduce the morphology and structure of the natural extracellular matrix (ECM). The objective of this work was to investigate the effect of fiber alignment on osteogenic differentiation of bone marrow stromal (BMS) cells. Random and aligned poly(l-lactide) (PLLA) nanofibers were produced by collecting the spun fibers on a stationary plate and a rotating wheel, respectively, as the ground electrode. Morphology and alignment of the BMS cells seeded on the fibers were characterized by SEM. The effect of fiber orientation on osteogenic differentiation of BMS cells was determined by measuring alkaline phosphatase (ALPase) activity, calcium content, and mRNA expression levels of osteogenic markers. There was a strong correlation between the fiber and cell distributions for the random (p = 0.16) and aligned (p = 0.81) fibers. Percent deviation from ideal randomness (PDIR) values indicated that cells seeded on the random fibers (PDIR = 6.5%) were likely to be distributed randomly in all directions while cells seeded on the aligned fibers (PDIR = 86%) were highly likely to be aligned with the direction of fibers. BMS cell seeded on random and aligned fibers had similar cell count and ALPase activity with incubation time, but the calcium content on aligned fibers was significantly higher after 21 days compared to that of random fibers (p = 0.003). Osteopontin (OP) and osteocalcin (OC) expression levels of BMS cells on fibers increased with incubation time. However, there was no difference between the expression levels of OP and OC on aligned vs. random fibers. The results indicate that BMS cells aligned in the direction of PLLA fibers to form long cell extensions, and fiber orientation affected the extent of mineralization, but it had no effect on cell proliferation or mRNA expression of osteogenic markers.     

Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth

Nerve injury, a significant cause of disability, may be treated more effectively using nerve guidance channels containing longitudinally aligned fibers. Aligned, electrospun nanofibers direct the neurite growth of immortalized neural stem cells, demonstrating potential for directing regenerating neurites. However, no study of neurite guidance on these fibers has yet been performed with primary neurons. Here, we examined neurites from dorsal root ganglia explants on electrospun poly-L-lactate nanofibers of high, intermediate, and random alignment. On aligned fibers, neurites grew radially outward from the ganglia and turned to follow the fibers upon contact. Neurite guidance was robust, with neurites never leaving the fibers to grow on the surrounding cover slip. To compare the alignment of neurites to that of the nanofiber substrates, Fourier methods were used to quantify the alignment. Neurite alignment, however striking, was inferior to fiber alignment on all but the randomly aligned fibers. Neurites on highly aligned substrates were 20 and 16% longer than neurites on random and intermediate fibers, respectively. Schwann cells on fibers assumed a very narrow morphology compared to those on the surrounding coverslip. The robust neurite guidance demonstrated here is a significant step toward the use of aligned, electrospun nanofibers for nerve regeneration. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.     

Salicylic acid-derived poly(anhydride-ester) electrospun fibers designed for regenerating the peripheral nervous system

Continuous biomaterial advances and the regenerating potential of the adult human peripheral nervous system offer great promise for restoring full function to innervated tissue following traumatic injury via synthetic nerve guidance conduits (NGCs). To most effectively facilitate nerve regeneration, a tissue engineering scaffold within a conduit must be similar to the linear microenvironment of the healthy nerve. To mimic the native nerve structure, aligned poly(lactic-co-glycolic acid)/bioactive polyanhydride fibrous substrates were fabricated through optimized electrospinning parameters with diameters of 600 ± 200 nm. Scanning electron microscopy images show fibers with a high degree of alignment. Schwann cells and dissociated rat dorsal root ganglia demonstrated elongated and healthy proliferation in a direction parallel to orientated electrospun fibers with significantly longer Schwann cell process length and neurite outgrowth when compared to randomly orientated fibers. Results suggest that an aligned polyanhydride fiber mat holds tremendous promise as a supplement scaffold for the interior of a degradable polymer NGC. Bioactive salicylic acid-based polyanhydride fibers are not limited to nerve regeneration and offer exciting promise for a wide variety of biomedical applications.     

Electrospun Poly(Hydroxybutyrate-co-Hydroxyvalerate) Fibrous Membranes Consisting of Parallel-Aligned Fibers or Cross-Aligned Fibers: Characterization and Biological Evaluation

Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) fibrous membranes consisting of parallel-aligned fibers or cross-aligned fibers were fabricated through electrospinning with the help of a rotating cylinder as fiber collector and auxiliary electrodes, and their application as tissue-engineering scaffolds was assessed. First, these membranes were characterized in terms of fiber diameter, spacing between adjacent aligned fibers or interstitial pore diameter, degree of fiber alignment, wettability and tensile properties. Then, human osteoblast-like cells (SaOS-2) were seeded and cultured on these membranes for up to 14 days. The cell morphology and proliferation were evaluated at different cell culture times. Membranes consisting of random fibers or parallel-aligned fibers were obtained when the rotational speed of the cylinder was 500 rpm or 3000 rpm, respectively. A very high rotational speed of 15 000 rpm resulted in the formation of parallel-aligned fibers having low or no spacing between the aligned fibers. Membranes consisting of cross-aligned fibers were made at the rotational speed of 3000 rpm and micrometer-sized fiber spacing was observed in these membranes. The alignment of fibers led to enhanced wettability of fibrous membranes. Tensile testing revealed that the parallel-aligned fibrous membranes were strong in the longitudinal direction but weak in the transverse direction. The cross-aligned fibrous membranes did not exhibit particularly weak tensile properties in any direction. In vitro biological evaluation showed that SaOS-2 cells spread randomly on membranes of random fibers but elongated in membranes of aligned fibers. All membranes supported cell proliferation in spite of the differences in cell morphology.     

The influence of electrospun aligned poly(epsilon-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes

Current treatment options for restoring large skeletal muscle tissue defects due to trauma or tumor ablation are limited by the host muscle tissue availability and donor site morbidity of muscle flap implantation. Creation of implantable functional muscle tissue that could restore muscle defects may bea possible solution. To engineer functional muscle tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(epsilon-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal muscle cells may provide implantable functional muscle tissues for patients with large muscle defects.     

Influence of the structure of poly (L-lactic acid) electrospun fibers on the bioactivity of endothelial cells: proliferation and inflammatory cytokines expression

Electrospinning has been used to fabricate random and aligned poly (L-lactic acid) (PLLA) fibers with three kinds of diameter under optimal conditions. The main purpose of this paper was to investigate the influence of the diameter and orientation of fibers on the bioactivity of endothelial cells, especially on the inflammatory cytokines expression. The morphology of electrospun fibers and the cells on the fibers after 3 and 6 days culture were observed by scanning electron microscopy. Also the cell proliferation activity and cell cycle were tested and the results showed that the random fibers were more favorable for endothelial cells growth. The effect of PLLA film (served as a control) and six kinds of PLLA fibers mats on the inflammatory cytokines expression after cells incubated for 2 and 4 days were investigated. It was concluded that there was more intense inflammatory cytokines expression by cells on flat PLLA film than that on electrospun fiber mats. Also the fiber diameter has greater effect on the activity and inflammatory cytokines expression of endothelial cells than the fiber orientation, in which fibers with smaller size has weaker inflammatory reaction.     

Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering

Efficacy of aligned poly(l-lactic acid) (PLLA) nano/micro fibrous scaffolds for neural tissue engineering is described and their performance with random PLLA scaffolds is compared as well in this study. Perfectly aligned PLLA fibrous scaffolds were fabricated by an electrospinning technique under optimum condition and the diameter of the electrospun fibers can easily be tailored by adjusting the concentration of polymer solution. As the structure of PLLA scaffold was intended for neural tissue engineering, its suitability was evaluated in vitro using neural stem cells (NSCs) as a model cell line. Cell morphology, differentiation and neurite outgrowth were studied by various microscopic techniques. The results show that the direction of NSC elongation and its neurite outgrowth is parallel to the direction of PLLA fibers for aligned scaffolds. No significant changes were observed on the cell orientation with respect to the fiber diameters. However, the rate of NSC differentiation was higher for PLLA nanofibers than that of micro fibers and it was independent of the fiber alignment. Based on the experimental results, the aligned nanofibrous PLLA scaffold could be used as a potential cell carrier in neural tissue engineering.     

Nanofibrous collagen nerve conduits for spinal cord repair

Nerve regeneration in an injured spinal cord is often restricted, contributing to the devastating outcome of neurologic impairment below the site of injury. Although implantation of tissue-engineered scaffolds has evolved as a potential treatment method, the outcomes remain sub-optimal. One possible reason may be the lack of topographical signals from these constructs to provide contact guidance to invading cells or regrowing axons. Nanofibers mimic the natural extracellular matrix architecturally and may therefore promote physiologically relevant cellular phenotypes. In this study, the potential application of electrospun collagen nanofibers (diameter=208.2±90.4 nm) for spinal cord injury (SCI) treatment was evaluated in vitro and in vivo. Primary rat astrocytes and dorsal root ganglias (DRGs) were seeded on collagen-coated glass cover slips (two-dimensional [2D] substrate controls), and randomly oriented or aligned collagen fibers to evaluate scaffold topographical effects on astrocyte behavior and neurite outgrowth, respectively. When cultured on collagen nanofibers, astrocyte proliferation and expression of glial fibrillary acidic protein (GFAP) were suppressed as compared to cells on 2D controls at days 3 (p<0.05) and 7 (p<0.01). Aligned fibers resulted in elongated astrocytes (elongation factor >4, p<0.01) and directed the orientation of neurite outgrowth from DRGs along fiber axes. In the contrast, neurites emanated radially on randomly oriented collagen fibers. By forming collagen scaffolds into spiral tubular structures, we demonstrated the feasibility of using electrospun nanofibers for the treatment of acute SCI using a rat hemi-section model. At days 10 and 30 postimplantation, extensive cellular penetration into the constructs was observed regardless of fiber orientation. However, scaffolds with aligned fibers appeared more structurally intact at day 30. ED1 immunofluorescent staining revealed macrophage invasion by day 10, which decreased significantly by day 30. Neural fiber sprouting as evaluated by neurofilament staining was observed as early as day 10. In addition, GFAP immunostained astrocytes were found only at the boundary of the lesion site, and no astrocyte accumulation was observed in the implantation area at any time point. These findings indicate the feasibility of fabricating 3D spiral constructs using electrospun collagen fibers and demonstrated the potential of these scaffolds for SCI repair.     

bFGF-containing electrospun gelatin scaffolds with controlled nano-architectural features for directed angiogenesis

Current therapeutic angiogenesis strategies are focused on the development of biologically responsive scaffolds that can deliver multiple angiogenic cytokines and/or cells in ischemic regions. Herein, we report on a novel electrospinning approach to fabricate cytokine-containing nanofibrous scaffolds with tunable architecture to promote angiogenesis. Fiber diameter and uniformity were controlled by varying the concentration of the polymeric (i.e. gelatin) solution, the feed rate, needle to collector distance, and electric field potential between the collector plate and injection needle. Scaffold fiber orientation (random vs. aligned) was achieved by alternating the polarity of two parallel electrodes placed on the collector plate thus dictating fiber deposition patterns. Basic fibroblast growth factor (bFGF) was physically immobilized within the gelatin scaffolds at variable concentrations and human umbilical vein endothelial cells (HUVEC) were seeded on the top of the scaffolds. Cell proliferation and migration was assessed as a function of growth factor loading and scaffold architecture. HUVECs successfully adhered onto gelatin B scaffolds and cell proliferation was directly proportional to the loading concentrations of the growth factor (0-100 bFGF ng/mL). Fiber orientation had a pronounced effect on cell morphology and orientation. Cells were spread along the fibers of the electrospun scaffolds with the aligned orientation and developed a spindle-like morphology parallel to the scaffold's fibers. In contrast, cells seeded onto the scaffolds with random fiber orientation, did not demonstrate any directionality and appeared to have a rounder shape. Capillary formation (i.e. sprouts length and number of sprouts per bead), assessed in a 3-D in vitro angiogenesis assay, was a function of bFGF loading concentration (0 ng, 50 ng and 100 ng per scaffold) for both types of electrospun scaffolds (i.e. with aligned or random fiber orientation).     

Orthogonally oriented scaffolds with aligned fibers for engineering intestinal smooth muscle

Controlling cellular alignment is critical in engineering intestines with desired structure and function. Although previous studies have examined the directional alignment of cells on the surface (x–y plane) of parallel fibers, quantitative analysis of the cellular alignment inside implanted scaffolds with oriented fibers has not been reported. This study examined the cellular alignment in the x–z and y–z planes of scaffolds made with two layers of orthogonally oriented fibers. The cellular orientation inside implanted scaffolds was evaluated with immunofluorescence. Quantitative analysis of coherency between cell orientation and fiber direction confirmed that cells aligned along the fibers not only on the surface (x–y plane) but also inside the scaffolds (x–z & y–z planes). Our study demonstrated that two layers of orthogonally aligned scaffolds can generate the histological organization of cells similar to that of intestinal circular and longitudinal smooth muscle.     

Tissue engineering of skeletal muscle using polymer fiber arrays

The purpose of this study was to assess a new scaffold design for muscle tissue engineering: arrays of parallel-oriented polymer microfibers. First, C2C12 skeletal myoblasts were seeded onto single, laminin-coated polypropylene fibers and their growth and alignment were characterized. With the aim of creating skeletal muscle sheets, it was then investigated whether cell layers of single fibers merged when in close proximity to neighboring fibers. The optimal fiber spacing needed to achieve cell alignment with the lowest possible content of scaffold material was established. Further, it was assessed whether such a cell sheet became contractile and whether it survived in vitro for extended periods of time. C2C12 cells, cultured on fibers 10 to 15 microm in diameter, formed up to 50-microm-thick layers of longitudinally aligned cells. Four different groups based on fiber spacing (30 to 35, 50 to 55, 70 to 75, and 90 to 95 microm) were evaluated. Complete cell sheets formed between fibers that were spaced 55 microm apart or less; larger spacing led to no or incomplete sheets. C2C12 cells, seeded onto a 10 x 20 mm fiber array, formed a contractile cell sheet that was maintained in vitro for 70 days. Larger, three-dimensional structures might be created by arranging fibers in several layers or by stacking cellular sheets.     

Guided orientation of cardiomyocytes on electrospun aligned nanofibers for cardiac tissue engineering

Cardiac tissue engineering (TE) is one of the most promising strategies to reconstruct the infarct myocardium and the major challenge involves producing a bioactive scaffold with anisotropic properties that assist in cell guidance to mimic the heart tissue. In this study, random and aligned poly(ε-caprolactone)/gelatin (PG) composite nanofibrous scaffolds were electrospun to structurally mimic the oriented extracellular matrix (ECM). Morphological, chemical and mechanical properties of the electrospun PG nanofibers were evaluated by scanning electron microscopy (SEM), water contact angle, attenuated total reflectance Fourier transform infrared spectroscopy and tensile measurements. Results indicated that PG nanofibrous scaffolds possessed smaller fiber diameters (239 ± 37 nm for random fibers and 269 ± 33 nm for aligned fibers), increased hydrophilicity, and lower stiffness compared to electrospun PCL nanofibers. The aligned PG nanofibers showed anisotropic wetting characteristics and mechanical properties, which closely match the requirements of native cardiac anisotropy. Rabbit cardiomyocytes were cultured on electrospun random and aligned nanofibers to assess the biocompatibility of scaffolds, together with its potential for cell guidance. The SEM and immunocytochemical analysis showed that the aligned PG scaffold greatly promoted cell attachment and alignment because of the biological components and ordered topography of the scaffolds. Moreover, we concluded that the aligned PG nanofibrous scaffolds could be more promising substrates suitable for the regeneration of infarct myocardium and other cardiac defects.     

Aligned hybrid silk scaffold for enhanced differentiation of mesenchymal stem cells into ligament fibroblasts

The concept of contact guidance utilizes the phenomenon of anchorage dependence of cells on the topography of seeded surfaces. It has been shown in previous studies that cells were guided to align along the topographical alignment of the seeding substrate and produced enhanced amounts of oriented extracellular matrix (ECM). In this study, we aimed to apply this concept to a three-dimensional full silk fibroin (SF) hybrid scaffold system, which comprised of knitted SF and aligned SF electrospun fibers (SFEFs), for ligament tissue engineering applications. Specifically, knitted SF, which contributed to the mechanical robustness of the system, was integrated with highly aligned SFEF mesh, which acted as the initial ECM to provide environmental cues for positive cellular response. Mesenchymal stem cells seeded on the aligned hybrid scaffolds were shown to be proliferative and aligned along the integrated aligned SFEF, forming oriented spindle-shaped morphology and produced an aligned ECM network. Expression and production of ligament-related proteins were also increased as compared to hybrid SF scaffolds with randomly arranged SFEFs, indicating differentiative cues for ligament fibroblasts present in the aligned hybrid SF scaffolds. Consequently, the tensile properties of cultured aligned constructs were significantly improved and superior to the counterpart with randomly arranged SFEF. These results thus show that the aligned hybrid scaffold system is promising for enhancing cell proliferation, differentiation, and function for ligament tissue engineering applications.     

Alignment of human vascular smooth muscle cells on parallel electrospun synthetic elastin fibers

We generated parallel elastic fibers from synthetic elastin (SE) as a model of the arterial media and assessed the alignment of smooth muscle cells (SMCs). SE utilized crosslinked electrospun human tropoelastin to form aligned fibers that mimicked the topography and elastin-rich content of the medial extracellular matrix. Bundled parallel fibers were anisotropically more elastic than randomly arranged scaffolds (111 ± 25 kPa vs. 265 ± 17 kPa) in the direction of the fibers. Aligned and random fiber scaffolds each supported SMC growth. Following attachment, SMCs proliferated longitudinally on the parallel fibers and expressed native α-smooth muscle actin.     

Combining cell sheet technology and electrospun scaffolding for engineered tubular,aligned, and contractile blood vessels

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL.     

Positive changes in bone marrow-derived cells in response to culture on an aligned bioscaffold

Previous studies have shown that cultured cells align with the local topography of their substrate following a concept called "contact guidance." Additionally, if the topography is highly aligned, the cells produce newly synthesized matrix that is also aligned. The objective of this study was to elucidate the positive effect of cell seeding on an elongated porcine small intestinal submucosa (SIS), which has been shown to improve ligament and tendon healing, by measuring the cellular response as a result of the changes in alignment. Because elongation is known to align the fibers of SIS through recruitment along the direction of elongation, we hypothesized that rabbit bone marrow-derived cells (BMDCs) seeded on SIS with improved fiber alignment would increase the expression and production of collagen following the concept of contact guidance. Using the small-angle light-scattering technique, it was found that a 15% elongation together with BMDC seeding on SIS (elongated, seeded group) improved its alignment of collagen fibers up to 16 times more than no elongation and no BMDC seeding (non-elongated, non-seeded group). Furthermore, BMDCs were also aligned along the direction of elongation and showed 200% greater collagen type I gene expression in the elongated, seeded group than in Petri dish controls. More importantly, the production of collagen was also 24% greater. The results of this study demonstrate that alignment of a bioscaffold can result in positive changes in cellular response, making the bioscaffold more attractive for functional tissue engineering to potentially enhance healing of ligaments and tendons.     

A biomaterial model of tumor stromal microenvironment promotes mesenchymal morphology but not epithelial to mesenchymal transition in epithelial cells

The stromal tissue surrounding most carcinomas is comprised of an extracellular matrix densely packed with collagen-I fibers, which are often highly aligned in metastatic disease. Here we developed an in vitro model to test the effect of an aligned fibrous environment on cancer cell morphology and behavior, independent of collagen ligand presentation. We grew cells on a biomimetic surface of aligned electrospun poly-l-lactic acid (PLLA) fibers and then examined the effect of this environment on growth rate, morphology, cytoskeletal organization, biochemical and genetic markers of epithelial to mesenchymal transition (EMT), cell surface adhesion, and cell migration. We grew a phenotypically normal breast epithelial cell line (MCF10A) and an invasive breast cancer cell line (MDA-MB-231) on three different substrates: typical flat culture surface (glass or plastic), flat PLLA (glass coated with PLLA) or electrospun PLLA fibers. Cells of both types adopted a more mesenchymal morphology when grown on PLLA fibers, and this effect was exaggerated in the more metastatic-like MDA-MB-231 cells. However, neither cell type underwent the changes in gene expression indicative of EMT despite the changes in cell shape, nor did they exhibit the decreased adhesive strength or increased migration typical of metastatic cells. These results suggest that changes in cell morphology alone do not promote a more mesenchymal phenotype and consequently that the aligned fibrous environment surrounding epithelial cancers may not promote EMT solely through topographical cues.