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Fabrication of cell penetration enhanced poly (L-lactic acid-co-e�-caprolactone)/silk vascular scaffolds utilizing air-impedance electrospinning

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... However, it may not be available for some patients due to vascular disease, amputation and previous harvest. Moreover, the use of this kind of vein requires a secondary surgical procedure to obtain the vessel [2]. Currently, vascular grafts based on polyethylene terephthalate (Dacron), expanded polytetrafluoroethylene (ePTFE) and decellularized bovine ureters are used in clinics [3][4][5]. ...
... Currently, vascular grafts based on polyethylene terephthalate (Dacron), expanded polytetrafluoroethylene (ePTFE) and decellularized bovine ureters are used in clinics [3][4][5]. Dacron and ePTFE are synthesized polymeric materials those have limited success when used as small-diameter arterial substitutes whereas they have been successfully used as large-caliber arterial substitutes [2]. The decellularized tissues are increasingly attracting attention since these materials are naturally derived and inherently sustain cell behaviors [6,7]. ...
... To date, electrospinning technology has become an attractive method, which can control the composition, structure and mechanical properties of fibrous scaffolds. Moreover, the drugs, growth factors and other biomolecular signaling agents could be combined together in the polymeric solution for electrospinning [2]. A variety of biodegradable polymers, including natural materials such as collagen [15], elastin [16], silk fibroin [2] and synthetics like poly(lactic acid) [17], polycaprolactone (PCL) [18] and poly (L-lactide-e-caplacton) (PLLA-CL) [2,15], have been investigated via electrospinning for fabrication of vascular scaffolds. ...
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The construction of a smooth muscle layer for blood vessel through electrospinning method plays a key role in vascular tissue engineering. However, smooth muscle cells (SMCs) penetration into the electrospun graft to form a smooth muscle layer is limited due to the dense packing of fibers and lack of inducing factors. In this paper, silk fibroin/poly (L-lactide-ε-caplacton) (SF/PLLA-CL) vascular graft loaded with platelet-rich growth factor (PRGF) was fabricated by electrospinning. The in vitro results showed that SMCs cultured in the graft grew fast, and the incorporation of PRGF could induce deeper SMCs infiltrating compared to the SF/PLLA-CL graft alone. Mechanical properties measurement showed that PRGF-incorporated graft had proper tensile stress, suture retention strength, burst pressure and compliance which could match the demand of native blood vessel. The success in the fabrication of PRGF-incorporated SF/PLLA-CL graft to induce fast SMCs growth and their strong penetration into graft has important application for tissue-engineered blood vessels.
... PCL is an FDA approved polyester that has been used for fabrication of several tissue engineered scaffolds intended for skeletal muscle repair [35]. It biodegrades slowly in vivo, on the order of months to years, and is ideal where long-term mechanical or structural support is required [36,[37][38][39]. The goal of this study is to develop and characterize the electrospun scaffolds composed of D-ECM and evaluate the extent to which these scaffolds can promote myogenesis in primary satellite cells in vitro. ...
... Increasing the rotational speed and surface velocity of the mandrel allowed for consistent deposition of aligned nanofibers (figures 3-4) [19,52,53]. Nanofiber diameter and scaffold porosity are comparable to electrospun meshes in the literature (figure 5) [19,37], that were found to be conducive for cellular adhesion and infiltration through the scaffold's three-dimensional structure [38]. Fiber alignment had a noticeable effect on the mechanical strength of each scaffold composition (figure 6). ...
Article
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Volumetric muscle loss (VML) is a loss of over ~10% of muscle mass that results in functional impairment. Although skeletal muscle possesses the ability to repair and regenerate itself following minor injuries, VML injuries are irrecoverable. Currently, there are no successful clinical therapies for the treatment of VML. Previous studies have treated VML defects with decellularized extracellular matrix (D-ECM) scaffolds derived from either pig urinary bladder or small intestinal submucosa. These therapies were unsuccessful due to the poor mechanical stability of D-ECM leading to quick degradation in vivo. To circumvent these issues, in this manuscript aligned nanofibers of D-ECM were created using electrospinning that mimicked native muscle architecture and provided topographical cues to primary satellite cells. Additionally, combining D-ECM with polycaprolactone (PCL) improved the tensile mechanical properties of the electrospun scaffold. In vitro testing shows that the electrospun scaffold with aligned nanofibers of PCL and D-ECM supports satellite cell growth, myogenic protein expression, and myokine production.
... This airflow-treated PCL scaffold showed similar compliance with the conventional electrospun PCL scaffold [52]. This method has been employed to electrospin several synthetic and natural polymers, including PCL [52], regenerative SF [53], polydioxanone (PDO) [54] and P(LLA-CL)/SF [55], yielding a number of porous scaffolds. Enhanced cell infiltration of various cells including human dermal fibroblasts [52], human breast epithelial cells [53], mouse bone marrow-derived macrophages [54] and human aortic smooth muscle cells [55] could be found in those airflow-treated scaffolds, implying a great potential of such scaffolds for a range of tissue engineering applications. ...
... This method has been employed to electrospin several synthetic and natural polymers, including PCL [52], regenerative SF [53], polydioxanone (PDO) [54] and P(LLA-CL)/SF [55], yielding a number of porous scaffolds. Enhanced cell infiltration of various cells including human dermal fibroblasts [52], human breast epithelial cells [53], mouse bone marrow-derived macrophages [54] and human aortic smooth muscle cells [55] could be found in those airflow-treated scaffolds, implying a great potential of such scaffolds for a range of tissue engineering applications. ...
Article
Electrospinning is one of the most effective approaches to fabricate tissue-engineered scaffolds composed of nano-to sub-microscale fibers that simulate a native extracellular matrix. However, one major concern about electrospun scaffolds for tissue repair and regeneration is that their small pores defined by densely compacted fibers markedly hinder cell infiltration and tissue ingrowth. To address this problem, researchers have developed and investigated various methods of manipulating scaffold structures to increase pore size or loosen the scaffold. These methods involve the use of physical treatments, such as salt leaching, gas foaming and custom-made collectors, and combined techniques to obtain electrospun scaffolds with loose fibrous structures and large pores. This article provides a summary of these motivating electrospinning techniques to enhance cell infiltration of electrospun scaffolds, which may inspire new electrospinning techniques and their new biomedical applications.
... Using a novel electrospinning technique, Yin et al. [116] used a porous mandrel through which pressurized air was pumped. Termed air-impedance electrospinning, the pressurized air impeded the deposition of PCLC/SF nanofibers resulting in larger pore sizes. ...
Article
Due to the prevalence of cardiovascular diseases, there is a large need for small diameter vascular grafts that cannot be fulfilled using autologous vessels. Although medium to large diameter synthetic vessels are in use, no suitable small diameter vascular graft has been developed due to the unique dynamic environment that exists in small vessels. To achieve long term patency, a successful tissue engineered vascular graft would need to closely match the mechanical properties of native tissue, be non-thrombotic and non-immunogenic, and elicit the proper healing response and undergo remodeling to incorporate into the native vasculature. Electrospinning presents a promising approach to the development of a suitable tissue engineered vascular graft. This review provides a comprehensive overview of the different polymers, techniques, and functionalization approaches that have been used to develop an electrospun tissue engineered vascular graft.
... Based on the suitability of PLCL and silk as biomaterials, the rationale for their association within the novel silk/PLCL composite scaffold reported in the present contribution was to conserve their satisfying biological properties and remedy both the slow degeneration rate of silk as well as the possible mechanical weakness of PLCL, resulting in a biocompatible and biodegradable scaffold for ligament tissue engineering. Composite scaffolds made of silk and PLCL have already been reported for vascular tissue engineering [23], peripheral nerve regeneration [24] and bone regeneration [25] applications. In these studies, silk and PLCL were both fabricated as nanofibers by electrospinning, and cell affinity of PLCL-based scaffolds was improved by the combination with silk. ...
Article
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(1) Background: A suitable scaffold with adapted mechanical and biological properties for ligament tissue engineering is still missing. (2) Methods: Different scaffold configurations were characterized in terms of morphology and a mechanical response, and their interactions with two types of stem cells (Wharton's jelly mesenchymal stromal cells (WJ-MSCs) and bone marrow mesenchymal stromal cells (BM-MSCs)) were assessed. The scaffold configurations consisted of multilayer braids with various number of silk layers (n = 1, 2, 3), and a novel composite scaffold made of a layer of copoly(lactic acid-co-(e-caprolactone)) (PLCL) embedded between two layers of silk. (3) Results: The insertion of a PLCL layer resulted in a higher porosity and better mechanical behavior compared with pure silk scaffold. The metabolic activities of both WJ-MSCs and BM-MSCs increased from day 1 to day 7 except for the three-layer silk scaffold (S3), probably due to its lower porosity. Collagen I (Col I), collagen III (Col III) and tenascin-c (TNC) were expressed by both MSCs on all scaffolds, and expression of Col I was higher than Col III and TNC. (4) Conclusions: the silk/PLCL composite scaffolds constituted the most suitable tested configuration to support MSCs migration, proliferation and tissue synthesis towards ligament tissue engineering.
... In another interesting approach, McClure et al. replaced the conventional solid rotating mandrel with porous mandrel and purged pressurized air so as to impede fiber deposition in order to create macroporous PCL scaffold [74]. This setup was also used by Yin et al. to fabricate Poly-lactide-co-caprolactone/RSF scaffolds for vascular tissue engineering applications [75]. Vaquette et al. described the use of several patterned collectors such as wire collectors (stainless steel wire meshes having a mesh size of 0.5, 3.3, and 5 mm), round collectors (stainless steel plates in having holes of 0.75, 2, and 3 mm diameter), star collector, ladder collector, and round collectors ( Figure 12) [76]. ...
Article
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Tissue engineering aims to develop artificial human tissues by culturing cells on a scaffold in the presence of biochemical cues. Properties of scaffold such as architecture and composition highly influence the overall cell response. Electrospinning has emerged as one of the most affordable, versatile, and successful approaches to develop nonwoven nano/microscale fibrous scaffolds whose structural features resemble that of the native extracellular matrix. However, dense packing of the fibers leads to small-sized pores which obstruct cell infiltration and therefore is a major limitation for their use in tissue engineering applications. To this end, a variety of approaches have been investigated to enhance the pore properties of the electrospun scaffolds. In this review, we collect state-of-the-art modification methods and summarize them into six classes as follows: approaches focused on optimization of packing density by (a) conventional setup, (b) sequential or co-electrospinning setups, (c) involving sacrificial elements, (d) using special collectors, (e) post-production processing, and (f) other specialized methods. Overall, this review covers historical as well as latest methodologies in the field and therefore acts as a quick reference for those interested in electrospinning matrices for tissue engineering and beyond.
... The fibrous tubular scaffolds possessed a mean pore diameter of 1.1 AE 0.5 mm and an average fiber diameter of 409 AE 120 nm. Yin et al. (2014) also utilized an air-impedance electrospinning method to fabricate porous PLCL/silk vascular scaffolds. The vascular scaffolds presented good performance in mechanical properties and SMC infiltration. ...
... In consideration of these properties, silk was processed into various forms of vascular scaffolds, such as tubes, films, and patches [39][40][41]. Specifically, studies that employed SF as an artificial vessel confirmed it to have excellent hemocompatibility, cell viability, tissue integrity, and mechanical strength [42][43][44]. SF was also incorporated into cardiovascular patches purposed for vascular surgery [41,45]. Additionally, an in vivo study confirmed SF to have favorable biological and physical properties, and that it can be effectively used in vessel reconstruction surgery [41]. ...
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The aim of this study was to evaluate and compare the efficacy of 4-hexylresorcinol (4-HR)-incorporated silk as a vascular patch scaffold to that of the commercial polytetrafluoroethylene (PTFE) vascular patch (GORE® ACUSEAL). The expression of the vascular endothelial cell growth factor-A (VEGF-A) after application of 4-HR was studied in RAW264.7 and HUVEC cells. In the animal study, a carotid artery defect was modeled in Sprague Dawley rats (n = 30). The defect was directly closed in the control group (n = 10), or repaired with the PTFE or 4-HR silk patch in the experimental groups (n = 10 per group). Following patch angioplasty, angiography was performed and the peak systolic velocity (PSV) was measured to evaluate the artery patency. The application of 4-HR was shown to increase the expression of VEGF-A in RAW264.7 and HUVEC cells. The successful artery patency rate was 80% for the 4-HR silk group, 30% for the PTFE group, and 60% for the control group. The PSV of the 4-HR silk group was significantly different from that of the control group at one week and three weeks post-angioplasty (p = 0.005 and 0.024). Histological examination revealed new regeneration of the arterial wall, and that the arterial diameter was well maintained in the 4-HR silk group in the absence of an immune reaction. In contrast, an overgrowth of endothelium was observed in the PTFE group. In this study, the 4-HR silk patch was successfully used as a vascular patch, and achieved a higher vessel patency rate and lower PSV than the PTFE patch.
... In air impedance electrospinning, highly pressurized air flows through defined pores in a hollow electrospinning mandrel to disrupt fiber deposition and compaction, but only in the mandrel pores ( Figure 11) [125][126][127]. The lumen of the mandrel pores increase cell penetration up to the point where the pressure of the fibers overcome the air pressure; both the outside of the lumen and between the mandrel perforations show very little cell infiltration similar to that of a solid mandrel [126,128]. This method is effective in increasing the pore size in the resulting scaffold but requiring little sacrifice in overall mechanical strength, possibly due to the dense fiber regions providing stability and strucural support. ...
Article
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Electrospinning has been widely accepted for several decades by the tissue engineering and regenerative medicine community as a technique for nanofiber production. Owing to the inherent flexibility of the electrospinning process, a number of techniques can be easily implemented to control fiber deposition (i.e. electric/ magnetic field manipulation, use of alternating current, or air-based fiber focusing) and/or porosity (i.e. air impedance, sacrificial porogen/sacrificial fiber incorporation, cryo-electrospinning, or alternative techniques). The purpose of this review is to highlight some of the recent work using these techniques to create electrospun scaffolds appropriate for mimicking the structure of the native extracellular matrix, and to enhance the applicability of advanced electrospinning techniques in the field of tissue engineering.
... Outside of the perforations, the scaffold is similar to the solid mandrel. These results were also reported from a study by Yin et al., using P(LLA-CL)/SF blended composite scaffolds [183]. ...
Chapter
The treatment of dermal wounds has evolved significantly throughout the history of mankind, from simplistic dressings and sutures to full regeneration of the skin with the aid of cellularized dressings and dermal regeneration templates. The skin is the largest organ in the body and primarily functions as a barrier to external agents. While the skin has impressive inherent self-renewal/repair abilities, assisted skin regeneration is vital for closure in a number of aberrant wound healing conditions (i.e., burns, pressure ulcers, diabetic ulcers, etc.) and in the reduction of scar formation. Currently, there are several types of nonelectrospun skin grafts on the market, but each possesses inherent disadvantages. Electrospinning is an ideal fabrication method for the construction of the skin due to its nonwoven fibrillar structures, potential for cellular infiltration, attachment, and overall structural support. Electrospun scaffolds also allow for fine control over pore sizes and fiber diameters. With regard to skin applications, both synthetic and natural polymers have been utilized in electrospinning. Synthetic materials have controllable degradation rates and mechanical properties, whereas natural materials are biodegradable, biocompatible, and possess cell attachment sites. The electrospinning process also allows for a number of biomolecules to be readily included within the fibrous network to increase scaffold bioactivity. Additives such as Manuka honey or an assorted cytokine and chemokine milieu can be incorporated into these electrospun scaffolds to promote bacterial inhibition and modify the local inflammatory response, respectively. This review provides an overview of various electrospun scaffold fabrication techniques, scaffold compositions, and dopants to enhance wound healing and regeneration.
... Porosity and pore size can readily be increased by increasing the diameters of the fibers within the scaffold 3,5 ; however, larger fiber diameters accompany reduced cell adhesion characteristics compared to small diameter fibers 5,6 and have been shown to correlate with higher thrombin formation and platelet adherence when placed in blood contact. 7 Recently methods of increasing porosity independent of scaffold fiber diameter have been developed which, amongst others, include: modified collector designs (patterned, 2,8-10 low temperature 11 and air-flow impedance 12 type collectors), ultrasonication (post spinning), 13 laser ablation, 14 surface conductivity modification 15 or inclusion of electrospun sacrificial particles 16,17 and/or fibers. 18,19 Baker et al (2008) spun poly(E-caprolactone) (PCL) simultaneously, in a dual spinneret electrospinning configuration, with poly(ethylene oxide) (PEO) which was subsequently removed to create additional void space within the scaffold boundaries. ...
Article
Porosity, pore size and pore interconnectivity are critical factors for cellular infiltration into electrospun scaffolds. This study utilized dual electrospinning with sacrificial fiber extraction to produce scaffolds with engineered porosity and mechanical properties. Subsequently, scaffolds were covalently grafted with heparin, a known anti-coagulant with growth-factor binding properties. We hypothesized that the tissue ingrowth would correlate positively with the porosity of the scaffolds. Pellethane® (PU) was spun simultaneously with poly(ethylene oxide) (PEO, subsequently extracted). Low, medium and high porosity scaffolds and heparinized versions of each were characterized and implanted in vivo for evaluation of cellular infiltration and inflammation subcutaneously in male Wistar rats (7,14 and 28 days, n=6). Average pore-size for low (76±0.2%), medium (83±0.5%) and high (90±1.0%) porosity scaffolds was 4.0±2.3 μm, 9.9±4.2 μm and 11.1±5.5 μm (p<0.0001). Heparinization resulted in increased fiber diameter (3.6±1.1 μm vs. 1.8±0.8 μm, p<0.0001) but influenced neither pore-size (p=0.67) nor porosity (p=0.27). Cellular infiltration for low, medium and high porosity scaffolds reached 33±7%, 77±20% and 98±1% of scaffold width, respectively, by day 28 of implantation (p<0001); heparinization did not affect infiltration (p=0.89). The ultimate tensile strength (UTS) and Young's modulus (Ey) of the constructs increased linearly with increasing PU fiber fraction (UTS: r2=0.97, p<0.0001, Ey: r2= 0.76, p<0.0001) and heparinization resulted in decreased strength but increased stiffness compared to non-heparinized scaffolds. Increased PEO to PU fraction in the scaffold resulted in predictable losses to mechanical strength and improvements to cellular infiltration, which could make PEO to PU fraction a useful optimization parameter for small diameter vascular grafts.
... Fiber alignment analysis via FFT and Image J. Fast Fourier Transfer (FFT) approach is applied to measure the alignment rates of the nanofibers, and ImageJ, image analysis software was used to analyze the FFT results 21,22 . The FFT analysis of the SEM images of the nanofiber samples was used to characterize the anisotropy of the scaffolds to digitize the alignment level of the nanofibers. ...
Article
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Scaffolds made of aligned nanofibers are favorable for nerve regeneration due to their superior nerve cell attachment and proliferation. However, it is challenging not only to produce a neat mat or a conduit form with aligned nanofibers but also to use these for surgical applications as a nerve guide conduit due to their insufficient mechanical strength. Furthermore, no studies have been reported on the fabrication of aligned nanofibers and randomly-oriented nanofibers on the same mat. In this study, we have successfully produced a mat with both aligned and randomly-oriented nanofibers by using a novel electrospinning set up. A new conduit with a highly-aligned electrospun mat is produced with this modified electrospinning method, and this proposed conduit with favorable features, such as selective permeability, hydrophilicity and nerve growth directional steering, were fabricated as nerve guide conduits (NGCs). The inner surface of the nerve conduit is covered with highly aligned electrospun nanofibers and is able to enhance the proliferation of neural cells. The central part of the tube is double-coated with randomly-oriented nanofibers over the aligned nanofibers, strengthening the weak mechanical strength of the aligned nanofibers.
... It has been reported that smooth surfaces with few but large pores were suitable for SMC penetration. 37 Shalumon et al. 38 and Yin et al. 39 found that interconnected large pores with a multiscale structure or with the assistance of air pressure could promote SMC penetration into the inner part of the scaffolds. The TIPS method created an interconnected porous structure by phase separation. ...
Article
Fabrication of small diameter vascular scaffolds has been a challenge in recent years, especially scaffolds with multiple layers. In this study, two approaches were proposed to fabricate triple-layered vascular scaffolds based on the electrospinning method and the thermally induced phase separation (TIPS) method. It was found that the electrospun fibers had a compact fibrous structure that provided good mechanical properties. The porous TIPS layer had high porosity and pore interconnectivity to facilitate cell penetration; however, this structure alone could not ensure sufficient mechanical properties for surgical applications. The triple-layered scaffolds, which consisted of electrospun TPU, TIPS TPU, and electrospun PPC layers, showed the highest mechanical properties and best structure and dimensions for vascular graft applications. Preliminary endothelial cell culture results found that the cells could attach to and proliferate on the inside surface of the scaffolds with over 95% viability.
... Electrospining is considered as a promising way to fabricate polymer nanofibers, which have been widely used as drug carriers and tissue engineering scaffolds [10][11][12][13]. Electrospun nanofibers have unique advantages in biomedical fields by providing porous structures which are quite similar to the natural extracellular matrix [14,15]. ...
... Potential substrate for endothelial keratoplasty and proliferation, indicating their potential application in vascular tissue engineering and nerve regeneration. 27,28 Polymer nanofibers seem to be good materials for endothelial cells. [29][30][31] Some studies of the use of SF or P(LLA-CL) in cornea tissue engineering have been reported, [32][33][34] but none about the use of blended SF/P(LLA-CL) nanofiber membranes in regeneration of the corneal endothelium. ...
Article
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Cornea transplant technology has progressed markedly in recent decades, allowing surgeons to replace diseased corneal endothelium by a thin lamellar structure. A thin, transparent, biocompatible, tissue-engineered substratum with corneal endothelial cells for endothelial keratoplasty is currently of interest. Electrospinning a nanofibrous structure can simulate the extracellular matrix and have beneficial effects for cell culture. Silk fibroin (SF) has good biocompatibility but poor mechanical properties, while poly(l-lactic acid-co-ε-caprolactone) (P(LLA-CL)) has good mechanical properties but poor biocompatibility. Blending SF with P(LLA-CL) can maintain the advantages of both these materials and overcome their disadvantages. Blended electrospun nanofibrous membranes may be suitable for regeneration of the corneal endothelium. The aim of this study was to produce a tissue-engineered construct suitable for endothelial keratoplasty. Five scaffolds containing different SF:P(LLA-CL) blended ratios (100:0, 75:25, 50:50, 25:75, 0:100) were manufactured. A human corneal endothelial (B4G12) cell line was cultured on the membranes. Light transmission, speed of cell adherence, cell viability (live-dead test), cell proliferation (Ki-67, BrdU staining), and cell monolayer formation were detected on membranes with the different blended ratios, and expression of some functional genes was also detected by real-time polymerase chain reaction. Different blended ratios of scaffolds had different light transmittance properties. The 25:75 blended ratio membrane had the best transmittance among these scaffolds. All electrospun nanofibrous membranes showed improved speed of cell adherence when compared with the control group, especially when the P(LLA-CL) ratio increased. The 25:75 blended ratio membranes also had the highest cell proliferation. B4G12 cells could form a monolayer on all scaffolds, and most functional genes were also stably expressed on all scaffolds. Only two genes showed changes in expression. All blended ratios of SF:P(LLA-CL) scaffolds were evaluated and showed good biocompatibility for cell adherence and monolayer formation. Among them, the 25:75 blended ratio SF:P(LLA-CL) scaffold had the best transmittance and the highest cell proliferation. These attributes further the potential application of the SF:P(LLA-CL) scaffold for corneal endothelial transplantation.
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The role of periosteum rich in a variety of bone cells and growth factors in the treatment of bone defects has gradually been discovered. However, due to the limited number of healthy transplantable periosteum, there are still major challenges in the clinical treatment of critical-size bone defects. Various techniques for preparing biomimetic periosteal scaffolds that are similar in composition and structure to natural periosteal scaffold have gradually emerged. This article reviews the current preparation methods of biomimetic periosteal scaffolds based on various biomaterials, which are mainly divided into natural periosteal materials and various polymer biomaterials. Several preparation methods of biomimetic periosteal scaffolds with different principles are listed, their strengths and weaknesses are also discussed. It aims to provide a more systematic perspective for the preparation of biomimetic periosteal scaffolds in the future.
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Vascular tissue engineering is a rapidly growing field of regenerative medicine, which strives to find innovative solutions for vascular reconstruction. Considering the limited success of synthetic grafts, research impetus in the field is now shifted towards finding biologically active vascular substitutes bestowing in situ growth potential. In this regard, silk biomaterials have shown remarkable potential owing to their favorable inherent biological and mechanical properties. This review provides a comprehensive overview of the progressive development of silk-based small diameter (<6mm) tissue-engineered vascular grafts (TEVGs), emphasizing their pre-clinical implications. Herein, we first discuss the molecular structure of various mulberry and non-mulberry silkworm silk and identify their favorable properties at the onset of vascular regeneration. The emergence of various state-of-the-art fabrication methodologies for the advancement of silk TEVGs is rationally appraised in terms of their in vivo performance considering the following parameters: ease of handling, long-term patency, resistance to acute thrombosis, stenosis and aneurysm formation, immune reaction, neo-tissue formation, and overall remodeling. Finally, we provide an update on the pre-clinical status of silk-based TEVGs, followed by current challenges and future prospects. Statement of Significance : Limited availability of healthy autologous blood vessels to replace their diseased counterpart is concerning and demands other artificial substitutes. Currently available synthetic grafts are not suitable for small diameter blood vessels owing to frequent blockage. Tissue-engineered biological grafts tend to integrate well with the native tissue via remodeling and have lately witnessed remarkable success. Silk fibroin is a natural biomaterial, which has long been used as medical sutures. This review aims to identify several favorable properties of silk enabling vascular regeneration. Furthermore, various methodologies to fabricate tubular grafts are discussed and highlight their performance in animal models. An overview of our understanding to rationally improve the biological activity fostering the clinical success of silk-based grafts is finally discussed.
Article
In tissue engineering, the structure of nanofibrous scaffolds and optimization of their properties play important role in the enhancement of cell growth and proliferation. Therefore, the basic idea of the current study is to find a proper method for tuning the extent of porosity of the scaffold, study the effect of porosity on the cell growth, and optimize the extent of porosity with the aim of achieving the maximum cell growth. To tune the scaffold's porosity, four types of metal mesh with different mesh sizes were employed as collectors. For this purpose, the structural properties of polycaprolactone nanofibrous layers which were electrospun on collectors, and the level of neural A-172 cell growth on layers were investigated, and the results were compared with the results attained for the fabricated nanofibrous layer on a flat aluminum collector. It was found that upon changing the porosity of the metal mesh as collector, the fibers' diameter would be inevitably changed, albeit insignificantly, and following no specific trends. However, changing the mesh size has shown a significant effect on the thickness and porosity of nanofibrous layer. According to the MTT assay results, the optimum neural cell growth was observed for the electrospun nanofibrous scaffold with the porosity of 96% and pore size of (0.42-23 µm) which has been fabricated on the type-4 collector having a mesh size of 10. The fabricated scaffold using this mesh with the optimum extent of porosity (58%) resulted in 44% enhancement in the cell growth as compared with the fabricated layer on the flat collector.
Article
Fibers produced from electrospinning are well-known to be extremely fine with diameters ranging from tens of nanometers to a few microns. Such ultrafine fibers not only allow for engineering scaffolds resembling the ultrastructure of the native extracellular matrix, but also offer possibility to explore the remodeling behavior of cells in vitro, due to their mechanically ‘adequate’ softness endowed by their ultrafine fineness. However, the remodeling effect of cells on the biomimicking fibrous substrates remains to be understood, because the crisscrossing and entangling among nanofibers in those tightly packed fibrous mats ultimately lead to merely a topological phenomenon, similar to that of the nanofiber-like topography embossed on the surface of a solid matter. In this study, the effect of nanofiber density on cellular response behavior was investigated by reducing the density of electrospun fiber networks. Using polycaprolactone (PCL) as a model polymer, randomly oriented fiber networks with various densities, namely, 37.7 ± 16.3 μg/cm² (D1), 103.8 ± 16.3 μg/cm² (D2), 198.2 ± 40.0 μg/cm² (D3), and 471.8 ± 32.7 μg/cm² (D4), were prepared by electrospinning for varied collection durations (10 s, 50 s, 100 s, and 10 min, respectively). By examining the responsive behavior of the human induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSCs) cultured on these nanofibrous networks, we showed that the fiber network with a moderate density (D2) is beneficial to the cell attachment, spreading, actin polymerization, contractility and migration. There also showed an increased tendency in nuclear localization of the Yes-associated protein (YAP) and subsequent activation of YAP responsive gene transcription, and cell proliferation and collagen synthesis were also enhanced on the D2. However, further increasing the fiber density (D3, D4) gave rise to weakened induction effect of fibers on the cellular responses. These results enrich our understanding on the effect of fiber density on cell behavior, and disclose the dependence of cellular responses on fiber density. This study paves the way to precisely design biomimetic fibrous scaffolds for achieving enhanced cell-scaffold interaction and tissue regeneration.
Article
The fabrication of a functional small-diameter vascular graft with good biocompatibility, in particular hemocompatibility, has become an urgent clinical necessity. We fabricated a native bilayer, small-diameter vascular graft using PEGylated chitosan (PEG-CS) and poly (L-lactic acid-co-ε-caprolactone; PLCL). To stabilize the inner layer, a PEG-CS blend with PLCL at ratio of 1:6 was casted on a round metal bar by a drip feed, and the outer layer, a PLCL blend with water-soluble PEG that acted as a sacrificial part to enhance pore size, was fabricated by electrospinning. The results showed excellent hemocompatibility and strong mechanical properties. In vitro, the degradation of the graft was evaluated by measuring the graft structure, mass loss rate, and changes in molecular weight. The results indicated that the graft had adequate support for the regeneration of blood vessels before collapse. An in vivo study was performed in a canine femoral artery model for up to 24 weeks, which demonstrated that the PEGylated bilayer grafts possessed excellent structural integrity, high compatibility with blood, good endothelial cell (EC) and smooth muscle cell (SMC) growth, and high expression levels of angiogenesis-related proteins, features that are highly similar to autologous blood vessels. Moreover, the results showed almost negligible calcification within 24 weeks. These findings confirm that the bilayer graft mimics native cells, thereby effectively improving vascular remodeling.
Article
Rapid endothelialization and prevention of restenosis are two vital challenges for the preparation of small-diameter vascular graft (SDVG); while, postoperative infection after implantation is often neglected. In the present study, carboxymethyl chitosan (CMC) and chitosan (CS) were chosen as the anti-thrombotic and anti-bacterial components, respectively; and then an asymmetric vascular graft was fabricated by co-electrospinning of poly (ε-caprolactone) (PCL)/CMC and PCL/CS. The mechanical property of the asymmetric vascular graft was much better than that of native vessels. In vitro blood compatibility tests indicated that the inner layer of the graft could inhibit thrombosis effectively. The outer layer of the graft had certain anti-bacterial effect owing to the addition of chitosan. Besides, the inner layer of the graft could greatly promote the growth of endothelial cells. It is believed that the asymmetric SDVG with anti-thrombotic and anti-bacterial functions could contribute to the future clinical implantation of tissue engineered vascular grafts.
Article
In this study, we fabricated native tissue mimicking a bilayered small-diameter vascular graft based on PEGylated chitosan (PEG–CS) and poly(l-lactic acid-co-ε-caprolactone) (PLCL). PEG–CS possesses good hemocompatibility, and as the inner layer of a vascular graft, it is more likely to swell and deform to debris, causing a blood clot, so the PLCL was added to stabilize it. In order to optimize the performance of the inner layer, PEG–CS blends with different ratios of PLCL (P/C:PLCL) casting films were fabricated, and endothelial cell compatibility and hemocompatibility were tested. The outer layer of the graft was fabricated via electrospinning with PLCL blends with different ratios of water-soluble polyethylene glycol. After water treatment, we tested smooth muscle cell penetration. Designing a two-layer vascular graft structure with an inner layer of P/C:PLCL = 1:6 and an outer layer being composed of a PLCL blend with 12% polyethylene glycol offers very good hemocompatibility and fast cell growth and also reserves well mechanical properties with good cell infiltration. Therefore, this kind of graft has a high potential to be used as an artificial blood vessel.
Article
One of the major problems associated with the electrospun scaffolds is their small pore size, which limits the cellular infiltration for bone tissue engineering. In this study, the effect of increasing the pore size on cellular infiltration was studied in poly‌caprolactone (PCL)/nanohydroxyapatite (nHA) electrospun scaffolds, which were modified using ultrasonication, co-electrospinning with poly (ethylene oxide) (PEO), and a combination of both. Ultrasonic process was optimized by central composite design (CCD). The ultrasonic output power and time of the process were considered as the effective parameters. The pore size of the scaffolds was evaluated by scanning electron microscope. The optimum conditions, according to the pore area and mechanical properties of the scaffolds were selected, and finally the groups that had the highest pore size and mechanical strength were selected for the combined method. Increasing the pore size enhanced the cellular proliferation, extension and infiltration, as well as the osteodifferentiation of stem cells. At the optimum condition, the average cellular infiltration was 36.51 µm compared to the control group with no cellular infiltration. In addition, alkaline phosphatase activity (ALP activity) and the expression of osteocalcin) OCN (and collagen I (COL I) were respectively 1.86, 2.54, and 2.16 fold compared to the control group on day 14. This article is protected by copyright. All rights reserved.
Article
Small diameter vascular grafts possessing desirable biocompatibility and suitable mechanical properties have become an urgent clinic demand. Herein, heparin loaded fibrous grafts of collagen/chitosan/poly(L-lactic acid-co-ε-caprolactone) (PLCL) were successfully fabricated via coaxial electrospinning. By controlling the concentration of heparin and the ratio of collagen/chitosan/PLCL, most grafts had the heparin encapsulation efficiency higher than 70%, and the heparin presented sustained release for more than 45 days. Particularly, such multicomponent grafts had relative low initial burst release, and after heparin releasing for 3 weeks, the grafts still showed good anti-platelet adhesion ability. In addition, along with the excellent cell biocompatibility, the fabricated grafts possessed suitable mechanical properties including good tensile strength, suture retention strength, burst pressure and compliance which could well match the native blood vessels. Thus, the optimized graft properties could be properly addressed for vascular tissue application via coaxial electrospinning.
Chapter
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Intensive studies have been done to a wide range of natural and synthetic polymeric scaffolds which have been done for the use of implantable and temporal devices in tissue engineering. Biodegradable and biocompatible scaffolds having a highly open porous structure with compatible mechanical strength are needed to provide an optimal microenvironment for cell proliferation, migration, differentiation, and guidance for cellular in growth at host tissue. One of the most abundantly available biopolymer chitins and its deacetylated derivatives is chitosan which is non-toxic and biodegradable. It has potential biomedical applications such as tissue engineering scaffolds, wound dressings, separation membranes, antibacterial coatings, stent coatings, and biosensors. Recent literature shows the use of chitin and chitosan in electrospinning to produce scaffolds with improved cytocompatibility, which could mimic the native extra-cellular matrix (ECM). Similarly, silk from the Bombyx mori silkworm, a protein-based natural fiber, having superior machinability, biocompatibility, biodegradation, and bioresorbability, has evolved as an important candidate for biomedical porous material. This chapter focuses on recent advancements made in chitin, chitosan, and silk fibroin-based electrospun nanofibrous scaffolds, emphasizing on tissue engineering for regenerative medicine.
Article
Cardiovascular disease is the primary cause of morbidity and mortality in today's world. Due to the lack of healthy autologous vessels, more tissue-engineered blood vessels are needed to repair or replace the damaged arteries. Biomaterials play an indispensable role in creating a living neovessel with biological responses. Silk fibroin produced by silkworms possesses good cytocompatibility, tailorable biodegradability, suitable mechanical properties, and minimal inflammatory reactions. In addition, regenerated silk fibroin solutions can be processed into various forms of scaffolds such as films, fibers, tubes, and porous sponges. These features make silk fibroin a promising biomaterial for small-diameter vascular grafts. The present article focuses on the applications of silk fibroin for vascular regeneration. A brief overview of the properties of silk fibroin is provided, following which the current research status and future directions of the main types of silk fibroin scaffolds for vascular regeneration are reviewed and discussed. Microsc. Res. Tech., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
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To investigate the structure of silk and its degradation properties, we have monitored the structure of silk using scanning electron microscopy and frozen sections. Raw silk and degummed raw silk were immersed in four types of degradation solutions for 156 d to observe their degradation properties. The subcutaneous implants in rats were removed after 7, 14, 56, 84, 129, and 145 d for frozen sectioning and subsequent staining with hematoxylin and eosin (H.E.), DAPI, Beta-actin and Collagen I immunofluorescence staining. The in vitro weight loss ratio of raw silk and degummed raw silk in water, PBS, DMEM and DMEM containing 10% FBS (F-DMEM) were, respectively, 14%/11%, 12.5%/12.9%, 11.1%/14.3%, 8.8%/11.6%. Silk began to degrade after 7 d subcutaneous implantation and after 145 d non-degraded silk was still observed. These findings suggest the immunogenicity of fibroin and sericin had no essential difference. In the process of in vitro degradation of silk, the role of the enzyme is not significant. The in vivo degradation of silk is related to phagocytotic activity and fibroblasts may be involved in this process to secrete collagen. This study also shows the developing process of cocoons and raw silk. Copyright © 2015. Published by Elsevier B.V.
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Electrospinning is a fabrication technique, which can be used to create nanofibrous non‐wovens from a variety of starting materials. The structure, chemical and mechanical stability, functionality, and other properties of the mats can be modified to match end applications. In this review, an introduction to biopolymers and the electrospinning process, as well as an overview of applications of nanofibrous biopolymer mats created by the electrospinning process will be discussed. Biopolymers will include polysaccharides (cellulose, chitin, chitosan, dextrose), proteins (collagen, gelatin, silk, etc.), DNA, as well as some biopolymer derivatives and composites.
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Vascular disease results in the decreased utility and decreased availability of autologus vascular tissue for small diameter (< 6 mm) vessel replacements. While synthetic polymer alternatives to date have failed to meet the performance of autogenous conduits, tissue-engineered replacement vessels represent an ideal solution to this clinical problem. Ongoing progress requires combined approaches from biomaterials science, cell biology, and translational medicine to develop feasible solutions with the requisite mechanical support, a non-fouling surface for blood flow, and tissue regeneration. Over the past two decades interest in blood vessel tissue engineering has soared on a global scale, resulting in the first clinical implants of multiple technologies, steady progress with several other systems, and critical lessons-learned. This review will highlight the current inadequacies of autologus and synthetic grafts, the engineering requirements for implantation of tissue-engineered grafts, and the current status of tissue-engineered blood vessel research.
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There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle-specific alpha-actin-positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individual's own cells.
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There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77+/-20% vs. 66+/-6% at day 0) while CD34 expression was reduced (19+/-12% vs. 61+/-10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127+/-900 mm Hg and suture retention strength was 1.3+/-0.3N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.
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Significant challenges must be overcome before the true benefit and economic impact of vascular tissue engineering can be fully realized. Toward that end, we have pioneered the electrospinning of micro- and nano-fibrous scaffoldings from the natural polymers collagen and elastin and applied these to development of biomimicking vascular tissue engineered constructs. The vascular wall composition and structure is highly intricate and imparts unique biomechanical properties that challenge the development of a living tissue engineered vascular replacement that can withstand the high pressure and pulsatile environment of the bloodstream. The potential of the novel scaffold presented here for the development of a viable vascular prosthetic meets these stringent requirements in that it can replicate the complex architecture of the blood vessel wall. This replication potential creates an "ideal" environment for subsequent in vitro development of a vascular replacement. The research presented herein provides preliminary data toward the development of electrospun collagen and elastin tissue engineering scaffolds for the development of a three layer vascular construct.
Article
1. Abstract2. Introduction3. Methods3.1. Electrospinning3.2. Scaffold Characterization3.3. Cell Lines and Cell Culture3.4. Scaffolding Crosslinking, Disinfection, Seeding, and Culture3.5. Preliminary Fabrication of a Three Layered Vascular Construct3.6. Histology4. Results4.1. Electrospinning Collagen4.2. Electrospinning Elastin4.3. Electrospinning Collagen/Elastin Blend4.4. Preliminary Fabrication of a Three Layered Vascular Construct5. Discussion6. Acknowledgements7. References
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Pore size and porosity control the rate and depth of cellular migration in electrospun vascular fabrics and thus have a strong impact on long-term graft success. In this study we investigated the effect of graft porosity on cell migration in vitro and in vivo. Polyurethane (PU) grafts were fabricated by electrospinning as fine-mesh, low-porosity grafts (void fraction (VF) 53%) and coarse-mesh, high-porosity grafts (VF 80%). The fabricated grafts were evaluated in vitro for endothelial cell attachment and proliferation. Prostheses were investigated in a rat model for either 7 days, 1, 3 or 6 months (n = 7 per time point) and analyzed after retrieval by biomechanical analysis and various histological techniques. Cell migration was calculated by computer-assisted morphometry. In vitro, fine-pore mesh favored early cell attachment. In vivo, coarse mesh grafts revealed significantly higher cell populations at all time points in all areas of the conduit wall. Biomechanical tests indicated sufficient compliance, tensile and suture retention strength before and after implantation. Increased porosity improves host cell ingrowth and survival in electrospun conduits. These conduits show successful natural host vessel reconstitution without limitation of biomechanical properties.
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Silk fibroin (SF)/Poly(L-lactide-co-caprolactone) P(LLA-CL) nanoyarn scaffolds were prepared by dynamic liquid electrospinning. The scaffold morphology was observed by scanning electron microscopy (SEM) and mechanical properties of the scaffold were examined. L929 mouse fibroblasts were cultured on the nanoyarn scaffolds. Cell morphology, infiltration and proliferation on the scaffolds were investigated by SEM, hematoxylin-eosin (H&E) staining and methylthiazol tetrazolium (MTT) assay, respectively. The results indicated that cells showed an organized morphology along the nanoyarns and considerable infiltration into the nanoyarn scaffolds. It was also observed that the nanoyarn scaffold significantly facilitated cell proliferation. Therefore, this work provides a promising approach to fabricate scaffolds for tissue engineering applications.
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Nanofibrous scaffolds of silk fibroin (SF) and poly(l-lactic acid-co-ɛ-caprolactone) (P(LLA-CL)) blends fabricated via electrospinning possessed good mechanical property and biocompatibility, as demonstrated by a previous study in vitro. However, the degradation behavior of the scaffolds, which may significantly influence tissue repair and regeneration, needs further exploration. In this study, in vitro degradation of pure SF, P(LLA-CL) and SF/P(LLA-CL) blended nanofibrous scaffolds were performed in phosphate-buffered saline (PBS, pH 7.4 ± 0.1) at 37 °C for 6 months. A series of analyses and characterizations (including morphologic changes, loss weight, pH changes of PBS solutions, DSC, XRD and FTIR-ATR) were conducted to the nanofibrous scaffolds after degradation and the results showed that the pure SF nanofibrous scaffolds were not completely degradable in PBS while pure P(LLA-CL) nanofibrous scaffolds had the fastest degradation rate. Moreover, the addition of SF reduced the degradation rate of P(LLA-CL) in SF/P(LLA-CL) blended nanofibrous scaffolds. This was probably caused by the intermolecular interactions between SF and P(LLA-CL), which hindered the movement of P(LLA-CL) molecular chains.
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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(ɛ-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.
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The growth of suitable tissue to replace natural blood vessels requires a degradable scaffold material that is processable into porous structures with appropriate mechanical and cell growth properties. This study investigates the fabrication of degradable, crosslinkable prepolymers of l-lactide-co-trimethylene carbonate into porous scaffolds by electrospinning. After crosslinking by γ-radiation, dimensionally stable scaffolds were obtained with up to 56% trimethylene carbonate incorporation. The fibrous mats showed Young's moduli closely matching human arteries (0.4-0.8MPa). Repeated cyclic extension yielded negligible change in mechanical properties, demonstrating the potential for use under dynamic physiological conditions. The scaffolds remained elastic and resilient at 30% strain after 84days of degradation in phosphate buffer, while the modulus and ultimate stress and strain progressively decreased. The electrospun mats are mechanically superior to solid films of the same materials. In vitro, human mesenchymal stem cells adhered to and readily proliferated on the three-dimensional fiber network, demonstrating that these polymers may find use in growing artificial blood vessels in vivo.
Article
For blood vessel tissue engineering, an ideal vascular graft should possess excellent biocompatibility and mechanical properties. For this study, a elastic material of poly (L-lactic acid-co-ϵ-caprolactone) (P(LLA-CL)), collagen and chitosan blended scaffold at different ratios were fabricated by electrospinning. Upon fabrication, the scaffolds were evaluated to determine the tensile strength, burst pressure, and dynamic compliance. In addition, the contact angle and endothelial cell proliferation on the scaffolds were evaluated to demonstrate the structures potential to serve as a vascular prosthetic capable of in situ regeneration. The collagen/chitosan/P(LLA-CL) scaffold with the ratio of 20:5:75 reached the highest tensile strength with the value of 16.9 MPa, and it was elastic with strain at break values of ∼112%, elastic modulus of 10.3 MPa. The burst pressure strength of the scaffold was greater than 3365 mmHg and compliance value was 0.7%/100 mmHg. Endothelial cells proliferation was significantly increased on the blended scaffolds versus the P(LLA-CL). Meanwhile, the endothelial cells were more adherent based on the increase in the degree of cell spreading on the surface of collagen/chitosan/P(LLA-CL) scaffolds. Such blended scaffold especially with the ratio of 20:5:75 thus has the potential for vascular graft applications. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.
Article
The influences of surfactants and medical drugs on the diameter size and uniformity of electrospun poly(L-lactic acid) (PLLA) fibers were examined by adding various surfactants (cationic, anionic, and nonionic) and typical drugs into the PLLA solution. Significant diameter reduction and uniformity improvement were observed. It was shown that the drugs were capsulated inside of the fibers and the drug release in the presence of proteinase K followed nearly zero-order kinetics due to the degradation of the PLLA fibers. Such ultrafine fiber mats containing drugs may find clinical applications in the future.
Article
We developed a novel technique involving knitting and electrospinning to fabricate a composite scaffold for ligament tissue engineering. Knitted structures were coated with poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a rotating cylinder and a PLCL solution was electrospun onto the structure. Highly aligned 2-μm-diameter microfibers covered the space between the stitches and adhered to the knitted scaffolds. The stress–strain tensile curves exhibited an initial toe region similar to the tensile behavior of ligaments. Composite scaffolds had an elastic modulus (150 ± 14 MPa) similar to the modulus of human ligaments. Biological evaluation showed that cells proliferated on the composite scaffolds and they spontaneously orientated along the direction of microfiber alignment. The microfiber architecture also induced a high level of extracellular matrix secretion, which was characterized by immunostaining. We found that cells produced collagen type I and type III, two main components found in ligaments. After 14 days of culture, collagen type III started to form a fibrous network. We fabricated a composite scaffold having the mechanical properties of the knitted structure and the morphological properties of the aligned microfibers. It is difficult to seed a highly macroporous structure with cells, however the technique we developed enabled an easy cell seeding due to presence of the microfiber layer. Therefore, these scaffolds presented attractive properties for a future use in bioreactors for ligament tissue engineering. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.
Article
Each year, the American Heart Association (AHA), in conjunction with the Centers for Disease Control and Prevention, the National Institutes of Health, and other government agencies, brings together the most up-to-date statistics on heart disease, stroke, other vascular diseases, and their risk factors and presents them in its Heart Disease and Stroke Statistical Update. The Statistical Update is a valuable resource for researchers, clinicians, healthcare policy makers, media professionals, the lay public, and many others who seek the best national data available on disease morbidity and mortality and the risks, quality of care, medical procedures and operations, and costs associated with the management of these diseases in a single document. Indeed, since 1999, the Statistical Update has been cited more than 8700 times in the literature (including citations of all annual versions). In 2010 alone, the various Statistical Updates were cited 1600 times (data from ISI Web of Science). In recent years, the Statistical Update has undergone some major changes with the addition of new chapters and major updates across multiple areas. For this year's edition, the Statistics Committee, which produces the document for the AHA, updated all of the current chapters with the most recent nationally representative data and inclusion of relevant articles from the literature over the past year and added a new chapter detailing various disorders of heart rhythm. Also, the 2012 Statistical Update is a major source for monitoring both cardiovascular health and disease in the population, with a focus on progress toward achievement of the AHA's 2020 Impact Goals. Below are a few highlights from this year's Update.
Article
Electrospun non-woven structures have the potential to form bioresorbable vascular grafts that promote tissue regeneration in situ as they degrade and are replaced by autologous tissue. Current bioresorbable grafts lack appropriate regeneration potential since they do not have optimal architecture, and their fabrication must be altered by the manipulation of process parameters, especially enhancing porosity. We describe here an air-impedance process where the solid mandrel is replaced with a porous mandrel that has pressurized air exiting the pores to impede fiber deposition. The mandrel design, in terms of air-flow rate, pore size, and pore distribution, allows for control over fiber deposition and scaffold porosity, giving greater cell penetration without a detrimental loss of mechanical properties or structural integrity.
Article
To improve water-resistant ability and mechanical properties of silk fibroin (SF)/hydroxybutyl chitosan (HBC) nanofibrous scaffolds for tissue-engineering applications, genipin, glutaraldehyde (GTA), and ethanol were used to crosslink electrospun nanofibers, respectively. The mechanical properties of nanofibrous scaffolds were obviously improved after 24 h of crosslinking with genipin and were superior to those crosslinked with GTA and ethanol for 24 h. SEM indicated that crosslinked nanofibers with genipin and GTA vapor had good water-resistant ability. Characterization of the microstructure (porosity and pore structure) demonstrated crosslinked nanofibrous scaffolds with genipin and GTA vapor had lager porosities and mean diameters than those with ethanol. Characterization of FTIR-ATR and (13)C NMR clarified both genipin and GTA acted as crosslinking agents for SF and HBC. Furthermore, genipin could induce SF conformation from random coil or α-helix to β-sheet. Although GTA could also successfully crosslink SF/HBC nanofibrous scaffolds, in long run, genipin maybe a better method due to lower cytotoxicity than GTA. Cell viability studies and wound-healing test in rats clarified that the genipin-crosslinked SF/HBC nanofibrous scaffolds had a good biocompatibility both in vitro and in vivo. These results suggested that genipin-crosslinked SF/HBC nanofibrous scaffolds might be potential candidates for wound dressing and tissue-engineering scaffolds.
Article
Small-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (threefold vs. control) and proliferation by 54.7 ± 1.1% (3 days vs. control). Tropoelastin, when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications, had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time, was reduced for tropoelastin (60.4 ± 8.2% vs. control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model.
Article
Thermoplastic polyurethanes are used in a variety of medical devices and experimental tissue engineering scaffolds. Despite advances in polymer composition to improve their stability, the correct balance between chemical and mechanical properties is not always achieved. A model compound (MC) simulating the structure of a widely used medical polyurethane (Pellethane) was synthesized and reacted with aliphatic and olefinic acyl chlorides to study the reaction site and conditions. After adopting the conditions to the olefinic modification of Pellethane, processing into flat sheets, and crosslinking by thermal initiation or ultraviolet radiation, mechanical properties were determined. The modified polyurethane was additionally electrospun under ultraviolet light to produce a crosslinked tubular vascular graft prototype. Model compound studies showed reaction at the carbamide nitrogen, and the modification of Pellethane with pentenoyl chloride could be accurately controlled to up to 20% (correlation: rho=0.99). Successful crosslinking was confirmed by insolubility of the materials. Initiator concentrations were optimized and the crosslink densities shown to increase with increasing modification. Crosslinking of Pellethane containing an increasing number of pentenoyl groups resulted in decreases (up to 42%, p<0.01) in the hysteresis and 44% in creep (p<0.05), and in a significant improvement in degradation resistance in vitro. Modified Pellethane was successfully electrospun into tubular grafts and crosslinked using UV irradiation during and after spinning to render them insoluble. Prototype grafts had sufficient burst pressure (>550 mm Hg), and compliances of 12.1+/-0.8 and 6.2+/-0.3%/100 mm Hg for uncrosslinked and crosslinked samples, respectively. It is concluded that the viscoelastic properties of a standard thermoplastic polyurethane can be improved by modification and subsequent crosslinking, and that the modified material may be electrospun and initiated to yield crosslinked scaffolds. Such materials hold promise for the production of vascular and other porous scaffolds, where decreased hysteresis and creep may be required to prevent aneurismal dilation.
Article
Electrospinning using natural proteins and synthetic polymers offers an attractive technique for producing fibrous scaffolds with potential for tissue regeneration and repair. Nanofibrous scaffolds of silk fibroin (SF) and poly(L-lactic acid-co-epsilon-caprolactone) (P(LLA-CL)) blends were fabricated using 1,1,1,3,3,3-hexafluoro-2-propanol as a solvent via electrospinning. The average nanofibrous diameter increased with increasing polymer concentration and decreasing the blend ratio of SF to P(LLA-CL). Characterizations of XPS and (13)C NMR clarified the presence of SF on their surfaces and no obvious chemical bond reaction between SF with P(LLA-CL) and SF in SF/P(LLA-CL) nanofibers was present in a random coil conformation, SF conformation transformed from random coil to beta-sheet when treated with water vapor. Whereas water contact angle measurements conformed greater hydrophilicity than P(LLA-CL). Both the tensile strength and elongation at break increased with the content increasing of P(LLA-CL). Cell viability studies with pig iliac endothelial cells demonstrated that SF/P(LLA-CL) blended nanofibrous scaffolds significantly promoted cell growth in comparison with P(LLA-CL), especially when the weight ratio of SF to P(LLA-CL) was 25:75. These results suggested that SF/P(LLA-CL) blended nanofibrous scaffolds might be potential candidates for vascular tissue engineering.
Article
As a contribution to the functionality of scaffolds in tissue engineering, here we report on advanced scaffold design through introduction and evaluation of topographical, mechanical and chemical cues. For scaffolding, we used silk fibroin (SF), a well-established biomaterial. Biomimetic alignment of fibers was achieved as a function of the rotational speed of the cylindrical target during electrospinning of a SF solution blended with polyethylene oxide. Seeding fibrous SF scaffolds with human mesenchymal stem cells (hMSCs) demonstrated that fiber alignment could guide hMSC morphology and orientation demonstrating the impact of scaffold topography on the engineering of oriented tissues. Beyond currently established methodologies to measure bulk properties, we assessed the mechanical properties of the fibers by conducting extension at breakage experiments on the level of single fibers. Chemical modification of the scaffolds was tested using donor/acceptor fluorophore labeled fibronectin. Fluorescence resonance energy transfer imaging allowed to assess the conformation of fibronectin when adsorbed on the SF scaffolds, and demonstrated an intermediate extension level of its subunits. Biological assays based on hMSCs showed enhanced cellular adhesion and spreading as a result of fibronectin adsorbed on the scaffolds. Our studies demonstrate the versatility of SF as a biomaterial to engineer modified fibrous scaffolds and underscore the use of biofunctionally relevant analytical assays to optimize fibrous biomaterial scaffolds.
Article
Establishing thrombosis-resistant surface is crucial to develop tissue-engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anti-coagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Cellular components of porcine carotid arteries were completely removed with chemical and physical means. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulphate salt and heparin-EDC. Bound heparin contents were measured by quantitative colorimetric assay of toluidine blue staining. The average content of heparin in treated vessels was 35.6 +/- 11.6 mg/cm(2) tissue, which represented 6.21 +/- 2.03 UPS heparin/cm(2) tissue. The stability of heparin linkage was tested by incubating the heparin-linked vessels either in PBS at 37 degrees C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Anti-coagulation property of bound heparin was determined with a clotting time assay using fresh dog blood. Standardized small pieces of non-heparin-bound vessels were clotted in fresh dog blood within 10 min., whereas all heparin-bound vessels did not form clot during 1-hr observation. In vivo platelet deposition of the vessel was determined with a baboon model of the femoral arteriovenous external shunt and (111)Indium labelling of platelets. There were 1.38 +/- 0.07 x 10(9) and 0.64 +/- 0.11 x 10(9) baboon platelets deposited on the control and heparin-linked vessels, respectively, at 60 min. These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue-engineered biological vascular grafts, which could be used for human coronary or low extremity artery bypasses.
Article
To avoid complications of prosthetic vascular grafts, engineered vascular constructs have been investigated as an alternative for vascular reconstruction. The scaffolds for vascular tissue engineering remain a cornerstone of these efforts and yet many currently available options are limited by issues of inconsistency, poor adherence of vascular cells, or inadequate biomechanical properties. In this study, we investigated whether PCL/collagen scaffolds could support cell growth and withstand physiologic conditions while maintaining patency in a rabbit aortoiliac bypass model. Our results indicate that electrospun scaffolds support adherence and growth of vascular cells under physiologic conditions and that endothelialized grafts resisted adherence of platelets when exposed to blood. When implanted in vivo, these scaffolds were able to retain their structural integrity over 1 month of implantation as demonstrated by serial ultrasonography. Further, at retrieval, these scaffolds continued to maintain biomechanical strength that was comparable to native artery. This study suggests that electrospun scaffolds combined with vascular cells may become an alternative to prosthetic vascular grafts for vascular reconstruction.
Article
Tissue engineering of small diameter (<5 mm) blood vessels is a promising approach to develop viable alternatives for autologous vascular grafts. Development of a functional, adherent, shear resisting endothelial cell (EC) layer is one of the major issues limiting the successful application of these tissue engineered grafts. The goal of the present study was to create a confluent EC layer on a rectangular 3D cardiovascular construct using human venous cells and to determine the influence of this layer on the extracellular matrix composition and mechanical properties of the constructs. Rectangular cardiovascular constructs were created by seeding myofibroblasts (MFs) on poly(glycolic acid) poly-4-hydroxybutyrate scaffolds using fibrin gel. After 3 or 4 weeks, ECs were seeded and co-cultured using EGM-2 medium for 2 or 1 week, respectively. A confluent EC layer could be created and maintained for up to 2 weeks. The EGM-2 medium lowered the collagen production by MFs, resulting in weaker constructs, especially in the 2 week cultured constructs. Co-culturing with ECs slightly reduced the collagen content, but had no additional affect on the mechanical performance. A confluent endothelial layer was created on 3D human cardiovascular constructs. The layer was co-cultured for 1 and 2 weeks. Although, the collagen production of the MFs was slightly lowered, co-culturing ECs for 1 week results in constructs with good mechanical properties and a confluent EC layer.
Article
Various types of natural and synthetic scaffolds with arterial tissue cells or differentiated stem cells have recently attracted interest as potential small-caliber vascular grafts. It was thought that the synthetic graft with the potential to promote autologous tissue regeneration without any seeding would be more practical than a seeded graft. In this study, we investigated in situ tissue regeneration in small-diameter arteries using a novel tissue-engineered biodegradable vascular graft that did not require ex vivo cell seeding. Small-caliber vascular grafts (4 mm in diameter) were fabricated by compounding a collagen microsponge with a biodegradable woven polymer tube that was constructed in a plain weave pattern with a double layer of polyglycolic acid (core) and poly-L-lactic acid (sheath) fibers. We implanted these tissue-engineered vascular grafts bilaterally into the carotid arteries of mongrel dogs (body weight, 20-25 kg). No anticoagulation regimen was used after implantation. We sacrificed the dogs 2, 4, 6, and 12 months (n = 4 in each group) after implantation and evaluated the explants histologically and biochemically. All of the tissue-engineered vascular grafts were patent with no signs of thrombosis or aneurysm at any time. Histologic and biochemical examinations showed excellent in situ tissue regeneration with an endothelial cell monolayer, smooth muscle cells, and a reconstructed vessel wall with elastin and collagen fibers. Our study indicated that this novel tissue-engineered vascular graft promoted in situ tissue regeneration and did not require ex vivo cell seeding, thereby conferring better patency on small-caliber vascular prostheses.
Article
Hypertension is known to decrease arterial distensibility and systemic compliance. However, the arterial tree is not homogeneous, and it has been shown that the medium-size radial artery does not behave like the proximal, elastic, large, common carotid artery. Indeed, radial artery compliance in hypertensive patients (HTs) has been shown to be paradoxically increased when compared with that in normotensive control subjects (NTs) at the same blood pressure level. To determine whether this increase was due to hypertension-related hypertrophy of the arterial wall, radial artery functional and geometric parameters from 22 NTs (mean +/- SD, 44 +/- 11 years) were compared with those from 25 age- and sex-matched never-treated essential HTs (48 +/- 12 years) by using a high-precision ultrasonic, echo-tracking system coupled to a photoplethysmograph (Finapres system), which allows simultaneous arterial internal diameter, intima-media thickness, and finger blood pressure measurements. When the values for HTs were compared with those of NTs at their respective mean arterial pressures, HTs had similar internal diameter (2.50 +/- 0.56 versus 2.53 +/- 0.32 mm, mean +/- SD) and greater intima-media thickness (0.40 +/- 0.06 versus 0.28 +/- 0.05 mm, P < .001) measurements and increased arterial wall cross-sectional areas (3.79 +/- 1.14 versus 2.45 +/- 0.57 mm2, P < .001). Circumferential wall stress was not significantly different between the two groups. Compliance calculated for a given blood pressure, ie, 100 mm Hg (C100), was greater in HTs than NTs (3.46 +/- 2.41 versus 2.10 +/- 1.55 m2.kPa-1 x 10(-8), P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.
Article
Small-intestine submucosa (SIS) is cell-free collagen, 100 mu thick, derived from the small intestine. It has been used as a vascular graft and has the highly desirable property of remodeling itself to become host tissue. To date there has been limited reporting on its preimplantation mechanical properties as a vascular graft. In this study, compliance, elastic modulus, and burst pressure have been measured on 5- and 8-mm SIS grafts. The compliance (percent of diameter increase for a pressure rise from 80 to 120 mmHg) was 4.6% av (range 2.9 to 8.6%) for the 5-mm grafts. For the 8-mm graft, the increase in diameter for the same pressure rise was 8.7% av (range 7.2 to 9.5%). The modulus of elasticity (E) increased exponentially with increasing pressure according to E = E(o)e(alphaP), where Eo is the zero-pressure modulus and alpha is the exponent that describes the rate of increase in E with pressure; the units for E, Eo, and P are g/cm2. The mean value for Eo was 4106 (g/cm2 range 1348-5601). The mean value for alpha was 0.0059 (range 0.0028-0.0125). At 100 mmHg, the mean value for E was 8.91 x 10(3) g/cm2 (range 1.02-8.80 x 10(3)). The mean burst pressure for 5.5-mm grafts was 3517 mm Hg (range 2069-4654). In terms of preimplant compliance, the small-diameter SIS graft is about (1/2) as compliant as the dog carotid artery, about four times more compliant than a typical vein graft, and more than an order of magnitude more compliant than synthetic vascular grafts.
Article
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.
Article
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.
Article
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.
Article
Chitosan-based nanofibers with an average fiber diameter controllable from a few microns down to approximately 40 nm and a narrow size distribution were fabricated by electrospinning solutions containing chitosan, polyethylene oxide (PEO), and Triton X-100. Rheological study showed a strong dependence of spinnability and fiber morphology on solution viscosity and thus on chitosan-to-PEO ratio. The nanofibers can be deposited either as a nonwoven mat or as a highly aligned bundle of controllable size. Potential use of this nanofibrous matrix for tissue engineering was studied by examining its integrity in water and cellular compatibility. It was found that the matrix with a chitosan/PEO ratio of 90/10 retained excellent integrity of the fibrous structure in water. Experimental results from cell stain assay and SEM imaging showed that the nanofibrous structure promoted the attachment of human osteoblasts and chondrocytes and maintained characteristic cell morphology and viability throughout the period of study. This nanofibrous matrix is of particular interest in tissue engineering for controlled drug release and tissue remodeling.
Article
Although the need for a functional arterial replacement is clear, the lower blood flow velocities of small-diameter arteries like the coronary artery have led to the failure of synthetic materials that are successful for large-diameter grafts. Although autologous vessels remain the standard for small diameter grafts, many patients do not have a vessel suitable for use because of vascular disease, amputation, or previous harvest. As a result, tissue engineering has emerged as a promising approach to address the shortcomings of current therapies. Investigators have explored the use of arterial tissue cells or differentiated stem cells combined with various types of natural and synthetic scaffolds to make tubular constructs and subject them to chemical and/or mechanical stimulation in an attempt to develop a functional small-diameter arterial replacement graft with varying degrees of success. Here, we review the progress in all these major facets of the field.
Article
Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.
Article
Polydioxanone (PDS) is a colorless, crystalline, bioabsorbable polymer that was first developed specifically for wound closure sutures. The compatibility, degradation rate, and mechanical properties (including shape memory) of PDS are of interest when considering the design of tissue engineering scaffolds. This research presents the electrospinning of PDS to fabricate unique nanofibrous structures for a variety of biomedical applications. Electrospinning is a polymer processing technique that utilizes an electric field to form fibers from a polymer solution or melt and allows the fabrication of nanofibrous non-woven structures. Results demonstrate the ability to control the fiber diameter of PDS as a function of solution concentrations and the fiber orientation with our prototype electrospinning apparatus. The results also show dependence between the fiber orientation and the elastic modulus, peak stress, and strain to failure of PDS in a uniaxial model.
Article
We describe the use of the fast Fourier transform (FFT) in the measurement of anisotropy in electrospun scaffolds of gelatin as a function of the starting conditions. In electrospinning, fiber alignment and overall scaffold anisotropy can be manipulated by controlling the motion of the collecting mandrel with respect to the source electrospinning solution. By using FFT to assign relative alignment values to an electrospun matrix it is possible to systematically evaluate how different processing variables impact the structure and material properties of a scaffold. Gelatin was suspended at varying concentrations (80, 100, 130, 150 mg/ml) and electrospun from 2,2,2 trifluoroethanol onto rotating mandrels (200-7000 RPM). At each starting concentration, fiber diameter remained constant over a wide range of mandrel RPM. Scaffold anisotropy developed as a function of fiber diameter and mandrel RPM. The induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. The FFT is a rapid method for evaluating fiber alignment in tissue-engineering materials.
Article
Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.
Article
In humans, prosthetic vascular grafts remain largely without an endothelium, even after decades of implantation. While this shortcoming does not affect the clinical performance of large bore prostheses in aortic or iliac position, it contributes significantly to the high failure rate of small- to medium-sized grafts (SMGs). For decades intensive but largely futile research efforts have been under way to address this issue. In spite of the abundance of previous studies, a broad analysis of biological events dominating the incorporation of vascular grafts was hitherto lacking. By focusing on the three main contemporary graft types, expanded polytetrafluoroethylene (ePTFE), Dacron and Polyurethane (PU), accumulated clinical and experimental experience of almost half a century was available. The main outcome of this broad analysis-supported by our own experience in a senescent non-human primate model-was twofold: Firstly, inappropriate animal models, which addressed scientific questions that missed the point of clinical relevance, were largely used. This led to a situation where the vast majority of investigators unintentionally studied transanastomotic rather than transmural or blood-borne endothelialization. Given the fact that in patients transanastomotic endothelialization (TAE) covers only the immediate perianastomotic region of sometimes very long prostheses, TAE is rather irrelevant in the clinical context. Secondly, transmural endothelialization seems to have a time window of opportunity before a build-up of an adverse microenvironment. In selecting animal models that prematurely terminate this build-up through the early presence of an endothelium, the most significant 'impairment factor' for physiological tissue regeneration in vascular grafts remained ignored. By providing insight into mechanisms and experimental designs which obscured the purpose and scope of several decades of vascular graft studies, future research may better address clinical relevance.
Article
This study characterizes the cross-linking of electrospun elastin and the mechanical properties of suture-reinforced 1.5mm internal diameter electrospun tubes composed of blended polydioxanone (PDO) and soluble elastin. Several tube configurations were tested to assess the effects of reinforcement on tube mechanical properties. Between the electrospun layers of each double-layered prosthetic, zero, one or two 6-0 sutures were wound, maintaining 1mm spacing with a pitch of 9 degrees . Single-layered tubes without suture were also examined. Samples were cross-linked and tested for compliance and burst strength. Compliance decreased significantly (p <0.05) and burst strength significantly increased (p <0.01) with reinforcement. Uncross-linked tubes were also tested to determine the effects of cross-linking. Results demonstrated that cross-linking significantly decreases burst strength (p <0.01), while decreases in compliance for cross-linked tubes were not significant. Cross-linked suture-reinforced PDO-elastin tubes had burst pressures more than 10 times greater than normal systolic pressures and exhibited a range of compliance values, including those matching native artery. These tubes display many characteristics of the "ideal" small-diameter graft, having mechanical properties that can be tailored to match those desired in vascular replacement applications.
Article
Aligned electrospun scaffolds are promising tools for engineering fibrous musculoskeletal tissues, as they reproduce the mechanical anisotropy of these tissues and can direct ordered neo-tissue formation. However, these scaffolds suffer from a slow cellular infiltration rate, likely due in part to their dense fiber packing. We hypothesized that cell ingress could be expedited in scaffolds by increasing porosity, while at the same time preserving overall scaffold anisotropy. To test this hypothesis, poly(epsilon-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds. Adjusting fabrication parameters produced aligned scaffolds with a full range of sacrificial (PEO) fiber contents. Tensile properties of scaffolds were functions of the ratio of PCL to PEO in the composite scaffolds, and were altered in a predictable fashion with removal of the PEO component. When seeded with mesenchymal stem cells (MSCs), increases in the starting sacrificial fraction (and porosity) improved cell infiltration and distribution after three weeks in culture. In pure PCL scaffolds, cells lined the scaffold periphery, while scaffolds containing >50% sacrificial PEO content had cells present throughout the scaffold. These findings indicate that cell infiltration can be expedited in dense fibrous assemblies with the removal of sacrificial fibers. This strategy may enhance in vitro and in vivo formation and maturation of functional constructs for fibrous tissue engineering.
Article
Nondegradable synthetic polymer vascular grafts used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. Tissue engineering of vascular grafts with autologous stem cells and biodegradable polymeric materials could solve these problems. The present study is aimed to develop a tissue-engineered vascular graft (TEVG) with functional endothelium using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-ε-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. TEVGs (internal diameter: 10 mm, length: 40 mm) were fabricated by seeding vascular cells differentiated from BMCs onto PGA/PLCL scaffolds and implanted into the abdominal aorta of bone marrow donor dogs (n = 7). Eight weeks after implantation of the TEVGs, the vascular grafts remained patent. Histological and immunohistochemical analyses of the vascular grafts retrieved at 8 weeks revealed the regeneration of endothelium and smooth muscle and the presence of collagen. Western blot analysis showed that endothelial nitric oxide synthase (eNOS) was expressed in TEVGs comparable to native abdominal aortas. This study demonstrates that vascular grafts with significant eNOS activity can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2008
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