Biomaterials patterned with discontinuous microwalls for vascular smooth muscle cell culture: biodegradable small diameter vascular grafts and stable cell culture substrates

The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).     

Aligned 3D human aortic smooth muscle tissue via layer by layer technique inside microchannels with novel combination of collagen and oxidized alginate hydrogel

Tissue engineering of the small diameter blood vessel medial layer has been challenging. Recreation of the circumferentially aligned multilayer smooth muscle tissue has been one of the major technical difficulties. Some research has utilized cyclic stress to align smooth muscle cells (SMCs) but due to the long time conditioning needed, it was not possible to use primary human cells because of expeditious senescence occurred . We demonstrate rapid buildup of a homogeneous relatively thick (30-40 μm) aligned smooth muscle tissue via layer by layer (LBL) technique within microchannels and a soft cell-adhesive hydrogel. Using a microchannelled scaffold with gapped microwalls, two layers of primary human SMCs separated by an interlayer hydrogel were cultured to confluence within the microchannels. The SMCs aligned along the microchannels because of the physically constraining microwalls. A novel double layered gel consisting of a mixture of pristine and oxidized alginate hydrogel coated with collagen was designed to place between each layer of cells, leading to a thicker tissue in a shorter time. The SMCs penetrated the soft thin interlayer hydrogel within 6 days of seeding of the 2nd cell layer so that the entire construct became more or less homogeneously populated by the SMCs. The unique LBL technique applied within the micropatterned scaffold using a soft cell-adhesive gel interlayer allows rapid growth and confluence of SMCs on 2D surface but at the same time aligns the cells and builds up multiple layers into a 3D tissue. This pseudo-3D buildup method avoids the typical steric resistance of hydrogel embedding.     

Influence of Mechanical Stimulation in the Development of a Medial Equivalent Tissue-Engineered Vascular Construct using a Gelatin-g-Vinyl Acetate Co-Polymer Scaffold

Abstract

Vascular regeneration in the area of small diameter (<6 mm) vessels via the tissue-engineering approach has been in focus for some time now. In this study, we report the development and evaluation of a tissue-engineered medial equivalent using gelatin-g-vinyl acetate co-polymer (GeVAc) as the scaffold material. GeVAc was synthesized by co-polymerizing gelatin and vinyl acetate monomer in the presence of AIBN as the initiator and subjected to physico-chemical characterization. A porous 3-D scaffold with open interconnected pores was then produced from GeVAc. The scaffold is non-cytotoxic with good smooth muscle cell proliferative capacity and high cell viability. Influence of smooth muscle cell phenotype in response to these scaffolds has been studied under mechanical stimulation. It was found that the cell-seeded tubular GeVAc constructs under mechanical stimulation preferentially supported the contractile phenotype of smooth muscle cells, as evidenced by the elevated expression of contractile protein markers such as alpha-SMA, calponin and SM22α. The mechanical properties and the ECM secretion were also increased on applying the mechanical stimulation. Hence, the results showed the promising potential of the GeVAc scaffolds in the regeneration of the medial equivalent tissue-engineered vascular construct.     

Aligned biodegradable nanofibrous structure: a potential scaffold for blood vessel engineering

A unique biodegradable nanofibrous structure, aligned poly(L-lactid-co-epsilon-caprolactone) [P(LLA-CL)] (75:25) copolymer nanofibrous scaffold was produced by electrospinning. The diameter of the generated fibers was around 500 nm with an aligned topography which mimics the circumferential orientation of cells and fibrils found in the medial layer of a native artery. A favorable interaction between this scaffold with human coronary artery smooth muscle cells (SMCs) was demonstrated via MTS assay, phase contrast light microscopy, scanning electron microscopy, immunohistology assay and laser scanning confocal microscopy separately. Tissue culture polystyrene and plane solvent-cast P(LLA-CL) film were used as controls. The results showed that, the SMCs attached and migrated along the axis of the aligned nanofibers and expressed a spindle-like contractile phenotype; the distribution and organization of smooth muscle cytoskeleton proteins inside SMCs were parallel to the direction of the nanofibers; the adhesion and proliferation rate of SMCs on the aligned nanofibrous scaffold was significantly improved than on the plane polymer films. The above results strongly suggest that this synthetic aligned matrix combines with the advantages of synthetic biodegradable polymers, nanometer-scale dimension mimicking the natural ECM and a defined architecture replicating the in vivo-like vascular structure, may represent an ideal tissue engineering scaffold, especially for blood vessel engineering.     

Biomaterial guides for lymphatic endothelial cell alignment and migration

Axillary dissection during breast cancer surgery produces extensive lymphatic vessel damage that often leads to lifelong secondary lymphedema of the arm. We have developed a biodegradable material conduit for lymphatic vessel reconstruction where fibers electrospun along the conduit lumen promote endothelial cell alignment and migration in vitro. The diameter and density of the electrospun fibers were optimized for cell migration and direction on two-dimensional substrates by seeding human lymphatic endothelial cells (LECs) onto aligned fibers of varying diameters and densities, randomly oriented fibers, and film substrates with no fibers. We found that LECs became aligned in the fiber direction, with cells seeded on the randomly oriented fibers becoming oriented in random directions, whereas cells seeded on the highly aligned fibers became highly aligned. Cell migration was dependent upon fiber alignment and density, with optimal migration found on 1300 nm diameter aligned fibers of low density. Blood endothelial cells seeded on the fibers exhibited similar behavior as the LECs. Fiber alignment was preserved upon rolling the two-dimensional substrate into the tubular geometry of a lymphatic vessel. The data suggest that aligned electrospun fibers may promote endothelial migration across the conduit in a manner that is independent of lymphatic growth factors.     

Electrospun nanofiber fabrication as synthetic extracellular matrix and its potential for vascular tissue engineering

Substantial effort is being invested by the bioengineering community to develop biodegradable polymer scaffolds suitable for tissue-engineering applications. An ideal scaffold should mimic the structural and purposeful profile of materials found in the natural extracellular matrix (ECM) architecture. To accomplish this goal, poly (L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (75:25) copolymer with a novel architecture produced by an electrospinning process has been developed for tissue-engineering applications. The diameter of this electrospun P(LLA-CL) fiber ranges from 400 to 800 nm, which mimicks the nanoscale dimension of native ECM. The mechanical properties of this structure are comparable to those of human coronary artery. To evaluate the feasibility of using this nanofibrous scaffold as a synthetic extracellular matrix for culturing human smooth muscle cells and endothelial cells, these two types of cells were seeded on the scaffold for 7 days. The data from scanning electron microscopy, immunohistochemical examination, laser scanning confocal microscopy, and a cell proliferation assay suggested that this electrospun nanofibrous scaffold is capable of supporting cell attachment and proliferation. Smooth muscle cells and endothelial cells seeded on this scaffold tend to maintain their phenotypic shape. They were also found to integrate with the nanofibers to form a three-dimensional cellular network. These results indicate a favorable interaction between this synthetic nanofibrous scaffold with the two types of cells and suggest its potential application in tissue engineering a blood vessel substitute.     

Structural Organization of Hepatic Portal Vein in Rat with Special Reference to Musculature,Intimal Folds,and Endothelial Cell Alignment

Structural organization of hepatic portal vein (HPV) was examined in adult rats by means of light and electron microscopy. Three characteristic features were found in the wall structure of rat HPV. (1) Tunica media consisted of two kinds of smooth muscle. The inner circular smooth muscle (CSM) was composed with one or two layer of smooth muscle cells, and was found in the entire length of the HPV and its tributaries. The outer longitudinal smooth muscle (LSM) was limited to a specific region of HPV; in particular it was well‐developed at distal half of HPV. CSM counteracts luminal hydrostatic pressure to prevent circumferential hyperextension of venous wall, whereas LSM is likely to counteract a tractive force produced by gravity and movement of small intestine. (2) Intima of HPV showed a unique feature, intimal folds, which protruded into the lumen and were aligned almost circumferentially. Intimal folds were found only at the same region where the LSM was well‐developed. Thus, LSM is presumably involved in the formation of intimal folds. (3) The endothelial cells between intimal folds were circumferentially aligned along the folds, although those in the other regions of HPV were arrayed along the longitudinal axis of HPV or the direction of blood flow as reported in other kinds of blood vessel. This finding implied that the circumferential blood flow locally occurs on the surface of intima between the intimal folds. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

In vitro study of human vascular endothelial cell function on materials with various surface roughness

In recent years, creating a biodegradable polymer scaffold with an endothelialized surface has become an attractive concept for replacement of small-diameter blood vessels. Toward this end, a better understanding of the interaction between endothelial cells and biodegradable polymer substrates is particularly important. Surface roughness of biomaterials is one of the important parameters that affect cell behavior. In this study, human vascular endothelial cells were cultured on electrospun and solvent-cast poly(L-lactic acid) substrates with different surface roughness. Cell responses were evaluated via both qualitative examinations of cell morphology changes as well as quantitative assessment of cell adhesion and proliferation rate on the different substrates. The results proved that endothelial cell function was enhanced on the smooth solvent-cast surface rather than on the rough electrospun surface of poly(L-lactic acid). Together with our previous findings that electrospun substrates favor vascular smooth muscle cell behavior, it is possible to design a unique three-dimensional scaffold for application of tissue-engineered small-diameter vessel replacement by combining the fabrication technique of solvent casting and electrospinning.     

具有周向微槽结构管状血管支架制备及诱导平滑肌细胞的取向生长

背景：对小口径血管组织工程化而言,平滑肌细胞的周向排列要求彻底改变以前支架的简单多孔结构,代之以能够诱导血管平滑肌细胞三维周向取向和排列新型微观结构。 目的：观察微槽结构对平滑肌细胞体外定向诱导的影响。 方法：用静电纺丝、熔融纺丝并利用溶剂/非溶剂和热压的方式制得了具有两层管壁、外壁具有周向微沟槽结构的仿生管状血管支架,用胶原蛋白固定改性后,在其上种植人脐静脉血管平滑肌细胞。扫描电镜和荧光显微镜观察支架不同缠绕角度对平滑肌细胞定向诱导能力的影响。 结果与结论：①选择比例为5∶95的氯仿/乙醇溶液,浸润时间为5 s,可以使乳酸-羟基乙酸共聚物电纺纤维和乳 酸-ε-己内酯共聚物熔纺纤维之间形成很好的粘连,形成支架。②通过碱降解使支架表面含有羧基,以1-(二甲基胺丙基)-3-乙基碳化二亚胺为缩合剂在支架表面接枝胶原。X射线光电子能谱证实了支架表面胶原大分子的存在。③当纤维之间的编织角度为30°即网孔尺寸适当时,细胞能在支架内部和表面大面积生长。④具有两层管壁结构的仿生管状血管支架具有良好的细胞相容性,其表面周向微槽结构对平滑肌细胞的取向排列具有明显的诱导作用。提示在电纺层外面再熔纺缠绕降解聚合物是制备管状仿生血管支架的可行方法。血管平滑肌细胞能沿着纤维暨微沟槽方向一致取向排列。     

The effect of scaffold macroporosity on angiogenesis and cell survival in tissue-engineered smooth muscle

Angiogenesis and survival of cells within thick scaffolds is a major concern in tissue engineering. The purpose of this study is to increase the survival of intestinal smooth muscle cells (SMCs) in implanted tissue-engineered constructs. We incorporated 250-μm pores in multi-layered, electrospun scaffolds with a macroporosity ranging from 15% to 25% to facilitate angiogenesis. The survival of green fluorescent protein (GFP)-expressing SMCs was evaluated after 2 weeks of implantation. Whereas host cellular infiltration was similar in scaffolds with different macroporosities, blood vessel development increased with increasing macroporosity. Scaffolds with 25% macropores had the most GFP-expressing SMCs, which correlated with the highest degree of angiogenesis over 1 mm away from the outermost layer. The 25% macroporous group exceeded a critical threshold of macropore connectivity, accelerating angiogenesis and improving implanted cell survival in a tissue-engineered smooth muscle construct.     

Engineering of an elastic large muscular vessel wall with pulsatile stimulation in bioreactor

Tissue engineering offers a new approach for the construction of vascular substitutes in vitro with proper mechanical properties. Although success has been made in the engineering of small blood vessels (<6mm in diameter), it remains a challenge to engineer large vessels (>6mm in diameter) due to their insufficient biomechanical property. In the current study, an elastic large vessel wall (6mm in diameter) was engineered by loading a polyglycolic acid (PGA) unwoven fiber scaffold seeded with smooth muscle cells (SMCs) on a vessel reactor designed with dynamic culture conditions. SMCs were isolated from canine carotid artery and expanded before seeding on a PGA fiber mesh. The cell-seeded PGA mesh was then loaded on a vessel reactor and subjected to pulsatile stimuli. Grossly, an elastic vessel wall was formed after 8 weeks of dynamic engineering. Histological examination showed well-orientated smooth muscle cells and collagenous fibers in the group with dynamic culture. In addition, the phenotype of SMCs was confirmed by positive staining of smooth muscle alpha-actin and calponin. On the contrary, disorganized smooth muscle cells and collagenous fibers were observed in the group under static culture without stimuli. Furthermore, the engineered vessels under dynamic culture exhibited significant improvements on biomechanical property over the one from static culture. Our results indicate that the approach developed in the current work is efficient for large vessel engineering. This approach may also be suitable for the engineering of other tissues with muscular tubular structure.     

Mechanical loading modulates the differentiation state of vascular smooth muscle cells

The cause underlying the onset of stenosis after vascular reconstruction is not well understood. In the present study, we evaluated the effect of mechanical unloading on the differentiation state of human vascular smooth muscle cells (hVSMCs) using a tissue-engineered vascular media (TEVM). hVSMCs cultured in a mechanically loaded three-dimensional environment, known as a living tissue sheet, had a higher differentiated state than cells grown on plastic. When the living tissue sheet was detached from its support, the release of the residual stress resulted in a mechanical unloading and cells within the extracellular matrix (ECM) dedifferentiated as shown by downregulation of differentiation markers. The relaxed living tissue sheet can be rolled onto a tubular mandrel to form a TEVM. The rolling procedure resulted in the reintroduction of a mechanical load leading to a cohesive compacted tissue. During this period, cells gradually redifferentiated and aligned circumferentially to the tubular support. Our results suggest that differentiation of hVSMCs can be driven by mechanical loading and may occur simultaneously in the absence of other cell types. The extrapolation of our results to the clinical context suggests the hypothesis that hVSMCs may adopt a proliferative phenotype resulting from the mechanical unloading of explanted blood vessels during vascular reconstruction. Therefore, we propose that this mechanical unloading may play an important role in the onset of vascular graft stenosis.     

Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering

Intestinal tissue engineering is an emerging field due to a growing demand for intestinal lengthening and replacement procedures secondary to massive resections of the bowel. Here, we demonstrate the potential use of a chitosan/collagen scaffold as a 3D matrix to support the bioengineered circular muscle constructs maintain their physiological functionality. We investigated the biocompatibility of chitosan by growing rabbit colonic circular smooth muscle cells (RCSMCs) on chitosan-coated plates. The cells maintained their spindle-like morphology and preserved their smooth muscle phenotypic markers. We manufactured tubular scaffolds with central openings composed of chitosan and collagen in a 1:1 ratio. Concentrically aligned 3D circular muscle constructs were bioengineered using fibrin-based hydrogel seeded with RCSMCs. The constructs were placed around the scaffold for 2 weeks, after which they were taken off and tested for their physiological functionality. The muscle constructs contracted in response to acetylcholine (Ach) and potassium chloride (KCl) and they relaxed in response to vasoactive intestinal peptide (VIP). These results demonstrate that chitosan is a biomaterial possibly suitable for intestinal tissue engineering applications.     

Circumferentially aligned fibers guided functional neoartery regeneration in vivo

An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application.     

Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel

The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.     

Biomimetic and synthetic esophageal tissue engineering

Background/PurposeA tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit.MethodsDonor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted.ResultsIsolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds.ConclusionsWe demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue.     

骨髓间充质干细胞复合蛛丝蛋白血管支架构建小直径组织工程血管的研究

应用动态培养的方式,将骨髓间充质干细胞和蛛丝蛋白血管支架复合培养构建小直径组织工程血管,为心血管疾病修复提供新的血管移植物来源。将间充质干细胞种植到管状的血管支架内腔,应用动态培养的方法构建小直径组织工程血管,并根据扫描电子显微镜观察、HE染色、缝合强度测试、DNA含量检测、羟脯氨酸测定等指标评价组织工程血管的形成程度。复合培养7 d后,细胞在纳米纤维支架表面充分铺展,并且可以迁移到纤维内部生长,细胞与血管支架表现出较好的相容性。组织工程血管的缝合强度经测试为(0.95±0.12) N/针,是天然血管的29.6%。随着时间的持续,组织工程血管中的羟脯氨酸含量和DNA含量不断增加,羟脯氨酸含量在第14和第28 d分别达到0.16和0.2 μg/mg, 且与对照组相比在统计学上有显著性差异。成功构建了一种小直径组织工程血管,各指标较为优良,为其临床应用奠定了基础。     

Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies

The purpose of this study was to produce a well-characterised electrospun polystyrene scaffold which could be used routinely for three-dimensional (3D) cell culture experimentation. A linear relationship (p<0.01) between three principal process variables (applied voltage, working distance and polymer concentration) and fibre diameter was reliably established enabling a mathematical model to be developed to standardise the electrospinning process. Surface chemistry and bulk architecture were manipulated to increase wetting and handling characteristics, respectively. X-ray photoelectron spectroscopy (XPS) confirmed the presence of oxygen-containing groups after argon plasma treatment, resulting in a similar surface chemistry to treated tissue culture plastic. The bulk architecture of the scaffolds was characterised by scanning electron microscopy (SEM) to assess the alignment of both random and aligned electrospun fibres, which were calculated to be 0.15 and 0.66, respectively. This compared to 0.51 for collagen fibres associated with native tissue. Tensile strength and strain of approximately of 0.15 MPa and 2.5%, respectively, allowed the scaffolds to be routinely handled for tissue culture purposes. The efficiency of attachment of smooth muscle cells to electrospun scaffolds was assessed using a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay and cell morphology was assessed by phalloidin-FITC staining of F-actin. Argon plasma treatment of electrospun polystyrene scaffold resulted in significantly increased cell attachment (p<0.05). The alignment factors of the actin filaments were 0.19 and 0.74 for the random and aligned scaffold respectively, compared to 0.51 for the native tissue. The data suggests that electrospinning of polystyrene generates 3D scaffolds which complement polystyrene used in 2D cell culture systems.     

Effect of substrate composition and alignment on corneal cell phenotype

Corneal blindness is a significant problem treated primarily by corneal transplants. Donor tissue supply is low, creating a growing need for an alternative. A tissue-engineered cornea made from patient-derived cells and biopolymer scaffold materials would be widely accessible to all patients and would alleviate the need for donor sources. Previous work in this lab led to a method for electrospinning type I collagen scaffolds for culturing corneal fibroblasts ex vivo that mimics the microenvironment in the native cornea. This electrospun scaffold is composed of small-diameter, aligned collagen fibers. In this study, we investigate the effect of scaffold nanostructure and composition on the phenotype of corneal stromal cells. Rabbit-derived corneal fibroblasts were cultured on aligned and unaligned collagen type I fibers ranging from 50 to 300?nm in diameter and assessed for expression of α-smooth muscle actin, a protein marker upregulated in hazy corneas. In addition, the optical properties of the cell-matrix constructs were assessed using optical coherence microscopy. Cells grown on collagen scaffolds had reduced myofibroblast phenotype expression compared to cells grown on tissue culture plates. Cells grown on aligned collagen type I fibers downregulated α-smooth muscle actin protein expression significantly more than unaligned collagen scaffolds, and also exhibited reduced overall light scattering by the tissue construct. These results suggest that aligned collagen type I fibrous scaffolds are viable platforms for engineering corneal replacement tissue.     

Electrospun P(LLA-CL) nanofiber: a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation

Poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] with L-lactide to epsilon-caprolactone ratio of 75 to 25 has been electrospun into nanofibers. The relationship between electrospinning parameters and fiber diameter has been investigated. The fiber diameter decreased with decreasing polymer concentration and with increasing electrospinning voltage. The X-ray diffractometer and differential scanning colorimeter results suggested that the electrospun nanofibers developed highly oriented structure in CL-unit sequences during the electrospinning process. The biocompatibility of the nanofiber scaffold has been investigated by culturing cells on the nanofiber scaffold. Both smooth muscle cell and endothelial cell adhered and proliferated well on the P(LLA-CL) nanofiber scaffolds.