Fabrication of scaffold‐free contractile skeletal muscle tissue using magnetite‐incorporated myogenic C2C12 cells

We have fabricated a functional skeletal muscle tissue using magnetite‐incorporated myogenic cell line C2C12 and a magnetic field. Magnetite‐incorporated C2C12 cells were patterned linearly on a monolayer of fibroblast NIH3T3 cells, using a magnetic field concentrator. After induction of differentiation, the C2C12 cells fused and formed multi‐nucleated myotubes. The 3T3 layer became detached in a sheet‐like manner after cultivation in differentiation medium for 5–8 days. When two separate collagen films were placed on a culture dish as tendon structures, a cylindrical construct was formed. Histological observation of the fabricated cylindrical tissue revealed the presence of multinucleate cells within it. Immunofluorescence staining of the construct showed the presence of sarcomere structures within the construct. Western blot analysis showed that muscle proteins were expressed in the construct. When the construct was stimulated with electric pulses, it exhibited active tension of approximately 1 µ N. These results demonstrate that functional skeletal muscle tissue was formed through magnetic force‐based tissue engineering. This is the first report of fabrication of skeletal muscle tissue with active tension‐generating capability using magnetic force‐based tissue engineering. The scaffold‐free skeletal muscle tissue engineering technique presented in this study will be useful for regenerative medicine, drug screening or use as a bio‐actuator. Copyright © 2010 John Wiley & Sons, Ltd.     

The potential of tissue engineering for developing alternatives to animal experiments: a systematic review

An underexposed ethical issue raised by tissue engineering is the use of laboratory animals in tissue engineering research. Even though this research results in suffering and loss of life in animals, tissue engineering also has great potential for the development of alternatives to animal experiments. With the objective of promoting a joint effort of tissue engineers and alternative experts to fully realise this potential, this study provides the first comprehensive overview of the possibilities of using tissue‐engineered constructs as a replacement of laboratory animals. Through searches in two large biomedical databases (PubMed, Embase) and several specialised 3R databases, 244 relevant primary scientific articles, published between 1991 and 2011, were identified. By far most articles reviewed related to the use of tissue‐engineered skin/epidermis for toxicological applications such as testing for skin irritation. This review article demonstrates, however, that the potential for the development of alternatives also extends to other tissues such as other epithelia and the liver, as well as to other fields of application such as drug screening and basic physiology. This review discusses which impediments need to be overcome to maximise the contributions that the field of tissue engineering can make, through the development of alternative methods, to the reduction of the use and suffering of laboratory animals. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of heat stimulation and l‐ascorbic acid 2‐phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs

Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue‐engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l ‐ascorbic acid derivative, l ‐ascorbic acid 2‐phosphate (AscP), on myoblast differentiation and physical force generation of tissue‐engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue‐engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue‐engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd.     

骨骼肌卫星细胞的体外培养与生物学特性(英文)

目的:建立理想的骨骼肌卫星细胞体外培养方法,探讨其生物学特性,以达到应用于组织工程和基因治疗的目的。方法:采用Ⅰ型胶原酶和胰蛋白酶二步消化法获取大鼠骨骼肌卫星细胞,进行体外原代和传代培养。观察细胞的形态,通过生长曲线、细胞融合率研究骨骼肌卫星细胞的增殖与分化能力,利用免疫细胞化学染色对所获得的细胞进行鉴定。结果:二步消化法适用于骨骼肌卫星细胞的获取。在生长培养基作用下,细胞增殖旺盛;在分化培养基条件下,细胞分化良好,可融合成肌管。α-sarcometric肌动蛋白和肌球蛋白细胞免疫化学染色,骨骼肌卫星细胞弱阳性,肌管强阳性。结论:体外培养的骨骼肌卫星细胞具有良好的增殖与分化能力,适用于组织工程和基因治疗。     

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor‐A expression and downregulation of miRNA‐1, miRNA‐206 and angiopoietin‐1

Vascularization is a major hurdle for growing three‐dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR‐1 and miR‐206 (p < 0.05) and angiopoietin‐1 (p < 0.05) with upregulation of vascular endothelial growth factor‐A (VEGF‐A; p < 0.05). The miR‐1 and angiopoietin‐1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR‐206 in L6 myoblasts caused a significant increase in VEGF‐A expression (p < 0.05), further establishing that changes in VEGF‐A expression are influenced by miR‐206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel? into isolated bilateral tissue engineering chambers incorporating a flow‐through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin‐1, miR‐1 and miR‐206. The relatively simple strategy of hypoxic preconditioning of implanted cells ‐ including non‐stem cell types – has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.     

An in vitro expansion score for tissue‐engineering applications with human bone marrow‐derived mesenchymal stem cells

Human bone marrow‐derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue‐engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony‐forming unit (CFU) assay, population doubling time (PDT), senescence‐associated β‐galactosidase (SA‐β‐Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA‐β‐Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late‐passage cells than in early‐passage cells. Early‐passage cells underwent efficient osteogenic differentiation, with mid‐passage cells performing best in chondrogenic differentiation. Late‐passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.     

Initial evaluation of vascular ingrowth into superporous hydrogels

There is a need for new materials and architectures for tissue engineering and regenerative medicine. Based upon our recent results developing novel scaffold architecture, we hypothesized that this new architecture would foster vascularization, a particular need for tissue engineering. We report on the potential of superporous hydrogel (SPH) scaffolds for in vivo cellular infiltration and vascularization. Poly(ethylene glycol) diacrylate (PEGDA) SPH scaffolds were implanted in the dorsum of severe combined immunodeficient (SCID) mice and harvested after 4 weeks of in vivo implantation. The SPHs were visibly red and vascularized, as apparent when compared to the non‐porous hydrogel controls, which were macroscopically avascular. Host cell infiltration was observed throughout the SPHs. Blood cells and vascular structures, confirmed through staining for CD34 and smooth muscle α‐actin, were observed throughout the scaffolds. This novel soft material may be utilized for cell transplantation, tissue engineering and in combination with cell therapies. The neovasularization and limited fibrotic response suggest that the architecture may be conducive to cell survival and rapid vessel development. Copyright © 2009 John Wiley & Sons, Ltd.     

Adipose‐derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co‐culture with adipose‐derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co‐cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase‐14. Moreover, we showed OEC/ASC‐induced vessel‐like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co‐culture‐induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system. Copyright © 2012 John Wiley & Sons, Ltd.     

Compressed collagen gel: a novel scaffold for human bladder cells

Collagen is highly conserved across species and has been used extensively for tissue regeneration; however, its mechanical properties are limited. A recent advance using plastic compression of collagen gels to achieve much higher concentrations significantly increases its mechanical properties at the neo‐tissue level. This controlled, cell‐independent process allows the engineering of biomimetic scaffolds. We have evaluated plastic compressed collagen scaffolds seeded with human bladder smooth muscle cells inside and urothelial cells on the gel surface for potential urological applications. Bladder smooth muscle and urothelial cells were visualized using scanning electron microscopy, conventional histology and immunohistochemistry; cell viability and proliferation were also quantified for 14 days in vitro. Both cell types tested proliferated on the construct surface, forming dense cell layers after 2 weeks. However, smooth muscle cells seeded within the construct, assessed with the Alamar blue assay, showed lower proliferation. Cellular distribution within the construct was also evaluated, using confocal microscopy. After 14 days of in vitro culture, 30% of the smooth muscle cells were found on the construct surface compared to 0% at day 1. Our results provide some evidence that cell‐seeded plastic compressed collagen has significant potential for bladder tissue regeneration, as these materials allow efficient cell seeding inside the construct as well as cell proliferation. Copyright © 2009 John Wiley & Sons, Ltd.     

A novel engineered dermis for in vitro photodamage research

The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three‐dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non‐human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast‐populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo‐aged dermis could be detected in the fibroblast‐populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright © 2016 John Wiley & Sons, Ltd.     

Dermal cells distribution on laser‐structured ormosils

Several dermal substitutes for skin grafting are now commercially available, although their performance still needs improvement. Most artificial dermises have a lower take rate than autologous grafts and require more time for sufficient vascular ingrowth to overlay the skin graft. Herein we characterize new two‐dimensional scaffolds for tissue‐engineering applications, which were fabricated by two‐photon polymerization (2PP) of ormosils hybrid materials. For the 2PP experiments, a Ti:sapphire laser was used to induce the photopolymerization. In this study we showed that the polymeric structures with controlled architectures produced via 2PP could be used as scaffolds for the in vitro culture and proliferation of human dermal fibroblasts. Fluorescence microscopy revealed that the fibroblasts' orientation was guided by the scaffold geometry, consisting of ormosils lines or grids. This 'dermal equivalent' was investigated for its ability to accommodate epidermal cells. To evaluate this interaction, two experimental approaches were hence used: (a) fibroblast–melanocyte co‐cultures; and (b) fibroblast–keratinocyte organotypic cultures. During their growth on ormosil scaffolds, productive interaction of fibroblasts with both epidermal cell types was found. Moreover, this pseudo‐dermis was shown to support the growth of keratinocytes for up to 8 days after their seeding. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications.     

P(NIPAAM–co‐HEMA) thermoresponsive hydrogels: an alternative approach for muscle cell sheet engineering

Loss of skeletal muscle tissue caused by traumatic injury or damage due to myopathies produces a deficit of muscle function for which there is still no clinical treatment. Transplantation of myogenic cells, themselves or combined with materials, has been proposed to increase the regenerative capacity of skeletal muscle but it is hampered by many limitations, such as low cell survival and engraftment or immunological reaction and low biocompatibility of the exogenous materials. Recently, myoblast sheet engineering, obtained with thermoresponsive culture dishes, has attracted attention as a new technique for muscle damage treatment. For this purpose, a series of thermoresponsive hydrogels, constituted by poly(N‐isopropylacrylamide‐co‐2‐hydroxyethylmethacrylate) [p(NIPAAM‐co‐HEMA)] were synthesized by a simple and inexpensive free‐radical polymerization of the two co‐monomers with a redox initiator. Different ratios of N‐isopropylacrylamide (NIPAAm) and 2‐hydroxyethylmethacrylate (HEMA) have been examined to evaluate the effects on physicochemical, mechanical and optical hydrogel properties. The murine muscle cell line C₂C₁₂ has been exploited to test the cytotoxicity of the thermoresponsive hydrogels, depending on different synthesis conditions. In this study, we have identified a thermoresponsive hydrogel that allows cell adhesion and viability, together with the detachment of viable sheet of muscle cells, giving the chance to develop further applications for muscle damage and disease. Copyright © 2014 John Wiley & Sons, Ltd.     

Interaction between electrical stimulation,protein coating and matrix elasticity: a complex effect on muscle fibre maturation

In skeletal muscle tissue engineering, it remains a challenge to produce mature, functional muscle tissue. Mimicking the in vivo niche in in vitro culture might overcome this problem. Niche components include, for example, extracellular matrix proteins, neighbouring cells, growth factors and physical factors such as the elasticity of the matrix. Previously, we showed the effects of matrix stiffness and protein coating on proliferation and differentiation of muscle progenitor cells in a two‐dimensional (2D) situation. In the present study we have investigated the additional effect of electrical stimulation. More precisely, we investigated the effect of electrical stimulation on primary myoblast maturation when cultured on top of Matrigel^?‐ or laminin‐coated substrates with varying elasticities. The effect of electrical stimulation on differentiation and maturation was found to be dependent on coating and stiffness. Although electrical stimulation enhanced myoblast maturation, the effect was mild. We therefore conclude that, with the current regimen, electrical stimulation is not essential to create functional, mature muscle tissue. Copyright © 2010 John Wiley & Sons, Ltd.     

Current opportunities and challenges in skeletal muscle tissue engineering

The purpose of this article is to give a concise review of the current state of the art in tissue engineering (TE) of skeletal muscle and the opportunities and challenges for future clinical applicability. The endogenous progenitor cells of skeletal muscle, i.e. satellite cells, show a high proneness to muscular differentiation, in particular exhibiting the same characteristics and function as its donor muscle. This suggests that it is important to use an appropriate progenitor cell, especially in TE facial muscles, which have a exceptional anatomical and fibre composition compared to other skeletal muscle. Muscle TE requires an instructive scaffold for structural support and to regulate the proliferation and differentiation of muscle progenitor cells. Current literature suggests that optimal scaffolding could comprise of a fibrin gel and cultured monolayers of muscle satellite cells obtained through the cell sheet technique. Tissue‐engineered muscle constructs require an adequate connection to the vascular system for efficient transport of oxygen, carbon dioxide, nutrients and waste products. Finally, functional and clinically applicable muscle constructs depend on adequate neuromuscular junctions with neural cells. To reach this, it seems important to apply optimal electrical, chemotropic and mechanical stimulation during engineering and discover other factors that influence its formation. Thus, in addition to approaches for myogenesis, we discuss the current status of strategies for angiogenesis and neurogenesis of TE muscle constructs and the significance for future clinical use. Copyright © 2009 John Wiley & Sons, Ltd.     

The 1999 Moyer award. Burn injury induces skeletal muscle apoptosis and the activation of caspase pathways in rats

Burn injury induces many metabolic disorders, including altered protein kinetics with muscle weakness. The skeletal muscle weakness that occurs as a result of the loss of muscle mass causes hypoventilation and dependence on respirators, a condition that increases morbidity and mortality. The presence or absence of apoptosis in muscle, which can be a cause of the loss of muscle mass, was studied in rats after they had received scald burns to 40% of their body surface areas. The potential pro-apoptotic pathways that were activated were also examined. The burn injury produced did not directly destroy the muscle beneath; muscles just beneath the burned surface showed dramatic apoptotic changes according to assessments with the cell death enzyme-linked immunosorbent assay and in situ TdT-mediated dUTP-X nick-end labeling staining. The extent of apoptosis reached a peak on postburn days 3 and 7. Of note is that apoptosis was also confirmed in muscles at sites distant from the burn injury (eg, tibialis anterior) on both postburn days 3 and 7, a condition that is suggestive of the systemic effects of pro-apoptotic factors. To show that heat itself causes the initiation of the pro-apoptotic signaling, muscle-derived C2C12 cells were subjected to heat treatment at 55 degrees C. Ceramide, a key apoptotic second messenger, was observed to increase in the caveolae fraction but not in non-caveolae fraction of these muscle cells. In muscle tissue from burned rats, stress-activated protein kinase (a downstream-signaling kinase of ceramide) was activated soon after burn injury; this finding is consistent with the hypothesis that ceramide plays a role in burn-induced apoptosis. Caspase-1, -3, and -9, important final apoptotic enzymes involved with the downstream signaling of stress-activated protein kinase, were also activated after burn injury in muscle tissue from burned rats. These findings confirm the hypothesis that apoptosis occurs in skeletal muscle and that major apoptotic pathways are activated after a burn injury. Further characterization of these apoptotic signaling cascades may provide new therapeutic targets for the prevention of burn-induced muscle wasting.     

Human endothelial colony‐forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold‐based tissue engineering

One of the major challenges in tissue engineering is to supply larger three‐dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)‐derived endothelial colony‐forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue‐culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo‐expanded ECFCs are a novel, very potent cell source for scaffold‐based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Transplanted fibroblast cell sheets promote migration of hepatic progenitor cells in the incised host liver in allogeneic rat model

Cell sheet engineering has been noted as a new and valuable approach in the tissue‐engineering field. The objective of this study was to explore a procedure to induce hepatic progenitor cells and biliary duct structures in the liver. Sprague–Dawley rat dermal fibroblast (DF) sheets were transplanted into the incised surface of the liver of F344 nude rats. In the control group, an incision was made without transplantation of the DF sheets. Bile duct (BD)‐like structures and immature hepatocyte‐like cells were observed in the DF sheet transplant sites. These BD‐like structures were cytokeratin‐8‐positive, while the hepatocyte‐like cells were both OV‐6‐positive and α‐fetoprotein‐positive as well. The proliferation and differentiation of liver progenitor cells were not influenced by hepatectomy. We also transplanted DF sheets transfected with a plasmid encoding the enhanced yellow fluorescent protein target to mitochondria (pEYFP–Mito) by electroporation, and found that the new structures were pEYFP–Mito‐negative. We observed new BD‐like structures and immature hepatocytes after transplantation of DF sheets onto incised liver surfaces, and clarified that the origin of these BD‐like structures and hepatocyte‐like cells was the recipient liver. The present study described an aspect of the hepatic differentiation process induced at the site of liver injury. Copyright © 2013 John Wiley & Sons, Ltd.     

Human progenitor‐derived endothelial cells vs. venous endothelial cells for vascular tissue engineering: an in vitro study

The isolation of endothelial progenitor cells from human peripheral blood generates a great hope in vascular tissue engineering because of particular benefit when compared with mature endothelial cells. We explored the capability of progenitor‐derived endothelial cells (PDECs) to line fibrin and collagen scaffolds in comparison with human saphenous and umbilical cord vein endothelial cells (HSVECs and HUVECs): (a) in a static situation, allowing definition of the optimal cell culture conditions with different media and cell‐seeding densities to check cell behaviour; (b) under shear stress conditions (flow chambers or tubular vascular constructs), allowing investigation of cell response and mRNA expression on both substrates by oligonucleotide microarray analysis and quantitative real‐time PCR. Well characterized PDECs: (a) could not be expanded adequately with the usual mature ECs culture media; (b) were able to colonize and grow on fibrin glue; (c) exhibited higher resistance to oxidative stress than HSVECs and HUVECs; (d) withstood physiological shear stress when lining both substrates in flow chambers, and their gene expression was regulated; (e) colonized a collagen‐impregnated vascular prosthesis and were able to sense mechanical forces. Our results provide an improved qualification of PDECs for vascular tissue engineering. Copyright © 2010 John Wiley & Sons, Ltd.     

TGF‐β1 enhances contractility in engineered skeletal muscle

Scaffoldless engineered 3D skeletal muscle tissue created from satellite cells offers the potential to replace muscle tissue that is lost due to severe trauma or disease. Transforming growth factor‐beta 1 (TGF‐β1) plays a vital role in mediating migration and differentiation of satellite cells during the early stages of muscle development. Additionally, TGF‐β1 promotes collagen type I synthesis in the extracellular matrix (ECM) of skeletal muscle, which provides a passive elastic substrate to support myofibres and facilitate the transmission of force. To determine the role of TGF‐β1 in skeletal muscle construct formation and contractile function in vitro, we created tissue‐engineered 3D skeletal muscle constructs with varying levels of recombinant TGF‐β1 added to the cell culture medium. Prior to the addition of TGF‐β1, the primary cell population was composed of 75% Pax7‐positive cells. The peak force for twitch, tetanus and spontaneous force were significantly increased in the presence of 2.0 ng/ml TGF‐β1 when compared to 0, 0.5 and 1.0 ng/ml TGF‐β1. Visualization of the cellular structure with H&E and with immunofluorescence staining for sarcomeric myosin heavy chains and collagen type I showed denser regions of better organized myofibres in the presence of 2.0 ng/ml TGF‐β1 versus 0, 0.5 and 1.0 ng/ml. The addition of 2.0 ng/ml TGF‐β1 to the culture medium of engineered 3D skeletal muscle constructs enhanced contractility and extracellular matrix organization. Copyright © 2012 John Wiley & Sons, Ltd.