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Ulrich R. Goessler - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of the effect of static magnetic fields combined with human hepatocyte growth factor on human satellite cell cultures.
Molecular medicine reports, 2014Co-Authors: Richard Birk, Ulrich R. Goessler, Karl Hörmann, Anne Faber, Christoph Aderhold, Ulrich Sommer, Johannes D. Schulz, Jens Stern-straeterAbstract:Tissue engineering is a promising research field, which aims to create new Functional Muscle Tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal Muscle Tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the Muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.
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Impact of static magnetic fields on human myoblast cell cultures.
International journal of molecular medicine, 2011Co-Authors: Jens Stern-straeter, Karl Hörmann, Anne Faber, Gabriel A. Bonaterra, Stefan S. Kassner, Ralf Kinscherf, Johannes D. Schulz, Alexander Sauter, Ulrich R. GoesslerAbstract:Treatment of skeletal Muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of Functional Muscle Tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called Tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-Tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal Muscle Tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal Muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal Muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal Muscle Tissue engineering approaches.
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Characterization of human myoblast differentiation for Tissue‐engineering purposes by quantitative gene expression analysis
Journal of tissue engineering and regenerative medicine, 2011Co-Authors: Jens Stern-straeter, Karl Hörmann, Gabriel A. Bonaterra, Stefan S. Kassner, Stefanie Zügel, Ralf Kinscherf, Ulrich R. GoesslerAbstract: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. Copyright © 2011 John Wiley & Sons, Ltd.
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Characterization of human myoblast cultures for Tissue engineering.
International journal of molecular medicine, 2008Co-Authors: Jens Stern-straeter, Gregor Bran, Karl Hörmann, Frank Riedel, Alexander Sauter, Ulrich R. GoesslerAbstract:Skeletal Muscle Tissue engineering, a promising specialty, aims at the reconstruction of skeletal Muscle loss. In vitro Tissue engineering attempts to achieve this goal by creating differentiated, Functional Muscle Tissue through a process in which stem cells are extracted from the patient, e.g. by Muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal Muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation. In this study, we obtained detailed information regarding the cultivation and differentiation of human myoblast cultures in different environments. By exploring optimal culture conditions for skeletal Muscle Tissue engineering, we acquired culture data for comparison with other sources of stem cells in order to find the most applicable stem cell for focussed clinical usage.
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Advances in skeletal Muscle Tissue engineering.
in Vivo, 2007Co-Authors: Jens Stern-straeter, Gregor Bran, Karl Hörmann, Frank Riedel, Ulrich R. GoesslerAbstract:Skeletal Muscle Tissue engineering is a promising interdisciplinary specialty which aims at the reconstruction of skeletal Muscle loss caused by traumatic injury congenital defects or tumor ablations. Due to the difficulty in procuring donor Tissue, the possibilities for alternative treatment like autologous grafting (e.g. Muscle flaps) are limited. This process also presents consistent problems with donor-site morbidity. Skeletal Muscle Tissue engineering tries to overcome this problem by generating new, Functional Muscle Tissue from autologous precursor cells (stem cells). Multiple stem cells from different sources can be utilized for restoration of differentiated skeletal Muscle Tissue using Tissue engineering principles. After 15 years of intensive research in this emerging field, for the first time, solutions using different strategies (e.g. embryonic stem cells, arterio-venous (AV) loop models, etc.) are being presented to resolve problems like vascularisation of Tissue engineered constructs. This article reviews recent findings in skeletal Muscle Tissue engineering and outlines its relevance to clinical applications in reconstructive surgery. Skeletal Muscle Tissue engineering represents an inter- disciplinary approach, using cell biology and engineering principles to generate Functional Muscle Tissue by imitating neo-organogenesis from mononucleated stem cells (e.g. myoblasts) to differentiated myofibers (1). It applies specific characteristics of precursor cells, scaffolds and bioactive factors in order to form, manipulate and restore skeletal Tissue phenotype and function. Skeletal Muscle Tissue, a highly specialised Tissue, is often lost due to traumatic injury, extensive surgical tumor ablation or Muscle fiber
G. B. Stark - One of the best experts on this subject based on the ideXlab platform.
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Impact of electrical stimulation on three‐dimensional myoblast cultures ‐ a real‐time RT‐PCR study
Journal of cellular and molecular medicine, 2005Co-Authors: J. Stern-straeter, Alexander D. Bach, Lars Stangenberg, V.t. Foerster, Raymund E. Horch, G. B. Stark, Justus P. BeierAbstract:Several focal skeletal Muscle diseases, including tumours and trauma lead to a limited loss of Functional Muscle Tissue. There is still no suitable clinical approach for treating such defects. A promising approach could be the Tissue engineering of skeletal Muscle. However, a clinically reliable differentiation stimulus for three-dimensional (3-D) cultures is necessary for this process, and this condition has not yet been established. In order to quantify and analyze the differentiation potential of electrical cell stimulation, primary myoblasts were stimulated within a 3-D fibrin- matrix. Gene expression of MyoD, myogenin and AChR-epsilon were measured by real-time RT-PCR over a time period of eight days, showing immediate down-regulation of all marker genes. For Tissue engineering approaches, cell multiplication is crucial for acquisition of sufficient Tissue volumes for reconstruction. Therefore, all experiments were performed with high and low passaged myoblasts, demonstrating higher transcript rates of marker genes in lowpassage cells. Our findings strongly suggest a reconsideration of electrical stimulation in Muscle Tissue engineering.
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Expression of Trisk 51, agrin and nicotinic-acetycholine receptor ε-subunit during Muscle development in a novel three-dimensional Muscle-neuronal co-culture system
Cell and Tissue Research, 2003Co-Authors: A. D. Bach, J. P. Beier, G. B. StarkAbstract:The purpose of our study was to create Functional Muscle Tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional Muscle-neuronal cultures were established by co-cultivating primary rat skeletal Muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of Muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) ε-subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR γ-subunit to the ε-subunit without neural agrin activity.
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Expression of Trisk 51, agrin and nicotinic-acetycholine receptor ε-subunit during Muscle development in a novel three-dimensional Muscle-neuronal co-culture system
Cell and tissue research, 2003Co-Authors: Alexander D. Bach, J. P. Beier, G. B. StarkAbstract:The purpose of our study was to create Functional Muscle Tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional Muscle-neuronal cultures were established by co-cultivating primary rat skeletal Muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of Muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) e-subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR γ-subunit to the e-subunit without neural agrin activity.
Anthony Atala - One of the best experts on this subject based on the ideXlab platform.
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the influence of electrospun aligned poly epsilon caprolactone collagen nanofiber meshes on the formation of self aligned skeletal Muscle myotubes
Biomaterials, 2008Co-Authors: Jin San Choi, George J. Christ, Anthony Atala, Sang Jin Lee, James J. YooAbstract:Current treatment options for restoring large skeletal Muscle Tissue defects due to trauma or tumor ablation are limited by the host Muscle Tissue availability and donor site morbidity of Muscle flap implantation. Creation of implantable Functional Muscle Tissue that could restore Muscle defects may bea possible solution. To engineer Functional Muscle Tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(epsilon-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered Muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal Muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal Muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced Muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal Muscle cells may provide implantable Functional Muscle Tissues for patients with large Muscle defects.
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the influence of electrospun aligned poly epsilon caprolactone collagen nanofiber meshes on the formation of self aligned skeletal Muscle myotubes
Biomaterials, 2008Co-Authors: Jin San Choi, George J. Christ, Anthony AtalaAbstract:Abstract 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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Angiogenic gene modification of skeletal Muscle cells to compensate for ageing-induced decline in bioengineered Functional Muscle Tissue.
BJU international, 2008Co-Authors: Dawn M. Delo, Anthony Atala, Daniel Eberli, J. Koudy Williams, Karl-erik Andersson, Shay SokerAbstract:OBJECTIVE To explore the effects of ageing on the viability of bioengineered striated Muscle Tissue in vivo, and if this viability can be enhanced by concurrent neovascularization, as its utility for the treatment of stress urinary incontinence (SUI) might be reduced if Muscle cells are derived from old patients. MATERIALS AND METHODS Myoblasts were obtained and expanded in culture from young (2 weeks), mature (3 months) and old (24 months) mice, and were engineered to express vascular endothelial growth factor (VEGF) to stimulate neovascularization. Myoblasts were injected subcutaneously into male nude mice and after 2 and 4 weeks, the engineered Muscle Tissues were harvested. RESULTS Bioengineered Muscle Tissues were formed in all groups, but the engineered Muscles formed by myoblasts from old mice were smaller and less contractile. However, the bioengineered Muscles expressing VEGF had a greater mass and better contractility in all age groups. CONCLUSION This pilot study showed that there was an age-related decline in the size and function of bioengineered Muscle; however, there was an improvement in volume and function when the Muscle cells were expressing VEGF.
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cyclic mechanical preconditioning improves engineered Muscle contraction
Tissue Engineering Part A, 2008Co-Authors: Du Geon Moon, George J. Christ, Joel D. Stitzel, Anthony Atala, James J. YooAbstract:The inability to engineer clinically relevant Functional Muscle Tissue remains a major hurdle to successful skeletal Muscle reconstructive procedures. This article describes an in vitro preconditioning protocol that improves the contractility of engineered skeletal Muscle after implantation in vivo. Primary human Muscle precursor cells (MPCs) were seeded onto collagen-based acellular Tissue scaffolds and subjected to cyclic strain in a computer-controlled bioreactor system. Control constructs (static culture conditions) were run in parallel. Bioreactor preconditioning produced viable Muscle Tissue constructs with unidirectional orientation within 5 days, and in vitro-engineered constructs were capable of generating contractile responses after 3 weeks of bioreactor preconditioning. MPC-seeded constructs preconditioned in the bioreactor for 1 week were also implanted onto the latissimus dorsi Muscle of athymic mice. Analysis of Tissue constructs retrieved 1 to 4 weeks postimplantation showed that bioreactor-preconditioned constructs, but not statically cultured control Tissues, generated tetanic and twitch contractile responses with a specific force of 1% and 10%, respectively, of that observed on native latissimus dorsi. To our knowledge, this is the largest force generated for Tissue-engineered skeletal Muscle on an acellular scaffold. This finding has important implications to the application of Tissue engineering and regenerative medicine to skeletal Muscle replacement and reconstruction.
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Tissue bioreactor system for the creation and maturation of organized Functional Tissues for surgical reconstruction
Journal of the American College of Surgeons, 2007Co-Authors: Du Geon Moon, Bryan W. Tillman, George J. Christ, Joel D. Stitzel, James J. Yoo, Anthony AtalaAbstract:INTRODUCTION: One of the major challenges in engineering clinically applicable Functional Tissues for reconstructive procedures is the lack of a bioreactor system that would accelerate cellular organization and Tissue formation. We developed a computerized bioreactor system that could allow for enhanced bladder Tissue formation. In this study we investigated whether organized Functional Muscle Tissue could be engineered using the 3-D bioreactor system.
Jens Stern-straeter - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of the effect of static magnetic fields combined with human hepatocyte growth factor on human satellite cell cultures.
Molecular medicine reports, 2014Co-Authors: Richard Birk, Ulrich R. Goessler, Karl Hörmann, Anne Faber, Christoph Aderhold, Ulrich Sommer, Johannes D. Schulz, Jens Stern-straeterAbstract:Tissue engineering is a promising research field, which aims to create new Functional Muscle Tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal Muscle Tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the Muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.
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Influence of Static Magnetic Fields Combined with Human Insulin-like Growth Factor 1 on Human Satellite Cell Cultures
In vivo (Athens Greece), 2014Co-Authors: Richard Birk, J. U. Sommer, D. Haas, Anne Faber, Christoph Aderhold, Johannes David Schultz, Karl Hoermann, Jens Stern-straeterAbstract:Tissue engineering represents a promising research field, targeting the creation of new Functional Muscle Tissue in vitro. The aim of the present study was to show the influence of static magnetic fields (SMF) and insulin-like growth factor-1 (IGF1), as enhancing stimuli on human satellite cell cultures, which are preferred sources of stem cells in engineering skeletal Muscle Tissue. To detect effects on myogenic maturation and proliferation, AlamarBlue® proliferation, assay and semi-quantitative reverse transcription-polymerase chain reaction of following markers was performed: desmin (DES), myogenic factor-5 (MYF5), myogenic differentiation antigen-1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1). As a distinct marker of differentiation, immunohistochemical staining and fusion index determination was performed on satellite cell cultures stimulated with IGF1 and IGF1-plus-SMF with an intensity of 80 mT. Proliferation was increased by additional SMF application to IGF1-stimulated cell cultures on the first day of myogenesis. Relative gene expression of measured markers was increased by IGF1 application in the first days of myogenesis except for ACTA1. Additional SMF application enhanced this effect. Nevertheless we were unable to demonstrate the formation of contractile Muscle Tissue. Immunhistochemical staining verified Muscle origin and all markers were displayed.
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Impact of static magnetic fields on human myoblast cell cultures.
International journal of molecular medicine, 2011Co-Authors: Jens Stern-straeter, Karl Hörmann, Anne Faber, Gabriel A. Bonaterra, Stefan S. Kassner, Ralf Kinscherf, Johannes D. Schulz, Alexander Sauter, Ulrich R. GoesslerAbstract:Treatment of skeletal Muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of Functional Muscle Tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called Tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-Tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal Muscle Tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal Muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal Muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal Muscle Tissue engineering approaches.
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Characterization of human myoblast differentiation for Tissue‐engineering purposes by quantitative gene expression analysis
Journal of tissue engineering and regenerative medicine, 2011Co-Authors: Jens Stern-straeter, Karl Hörmann, Gabriel A. Bonaterra, Stefan S. Kassner, Stefanie Zügel, Ralf Kinscherf, Ulrich R. GoesslerAbstract: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. Copyright © 2011 John Wiley & Sons, Ltd.
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Characterization of human myoblast cultures for Tissue engineering.
International journal of molecular medicine, 2008Co-Authors: Jens Stern-straeter, Gregor Bran, Karl Hörmann, Frank Riedel, Alexander Sauter, Ulrich R. GoesslerAbstract:Skeletal Muscle Tissue engineering, a promising specialty, aims at the reconstruction of skeletal Muscle loss. In vitro Tissue engineering attempts to achieve this goal by creating differentiated, Functional Muscle Tissue through a process in which stem cells are extracted from the patient, e.g. by Muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal Muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation. In this study, we obtained detailed information regarding the cultivation and differentiation of human myoblast cultures in different environments. By exploring optimal culture conditions for skeletal Muscle Tissue engineering, we acquired culture data for comparison with other sources of stem cells in order to find the most applicable stem cell for focussed clinical usage.
James J. Yoo - One of the best experts on this subject based on the ideXlab platform.
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the influence of electrospun aligned poly epsilon caprolactone collagen nanofiber meshes on the formation of self aligned skeletal Muscle myotubes
Biomaterials, 2008Co-Authors: Jin San Choi, George J. Christ, Anthony Atala, Sang Jin Lee, James J. YooAbstract:Current treatment options for restoring large skeletal Muscle Tissue defects due to trauma or tumor ablation are limited by the host Muscle Tissue availability and donor site morbidity of Muscle flap implantation. Creation of implantable Functional Muscle Tissue that could restore Muscle defects may bea possible solution. To engineer Functional Muscle Tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(epsilon-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered Muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal Muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal Muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced Muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal Muscle cells may provide implantable Functional Muscle Tissues for patients with large Muscle defects.
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cyclic mechanical preconditioning improves engineered Muscle contraction
Tissue Engineering Part A, 2008Co-Authors: Du Geon Moon, George J. Christ, Joel D. Stitzel, Anthony Atala, James J. YooAbstract:The inability to engineer clinically relevant Functional Muscle Tissue remains a major hurdle to successful skeletal Muscle reconstructive procedures. This article describes an in vitro preconditioning protocol that improves the contractility of engineered skeletal Muscle after implantation in vivo. Primary human Muscle precursor cells (MPCs) were seeded onto collagen-based acellular Tissue scaffolds and subjected to cyclic strain in a computer-controlled bioreactor system. Control constructs (static culture conditions) were run in parallel. Bioreactor preconditioning produced viable Muscle Tissue constructs with unidirectional orientation within 5 days, and in vitro-engineered constructs were capable of generating contractile responses after 3 weeks of bioreactor preconditioning. MPC-seeded constructs preconditioned in the bioreactor for 1 week were also implanted onto the latissimus dorsi Muscle of athymic mice. Analysis of Tissue constructs retrieved 1 to 4 weeks postimplantation showed that bioreactor-preconditioned constructs, but not statically cultured control Tissues, generated tetanic and twitch contractile responses with a specific force of 1% and 10%, respectively, of that observed on native latissimus dorsi. To our knowledge, this is the largest force generated for Tissue-engineered skeletal Muscle on an acellular scaffold. This finding has important implications to the application of Tissue engineering and regenerative medicine to skeletal Muscle replacement and reconstruction.
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Tissue bioreactor system for the creation and maturation of organized Functional Tissues for surgical reconstruction
Journal of the American College of Surgeons, 2007Co-Authors: Du Geon Moon, Bryan W. Tillman, George J. Christ, Joel D. Stitzel, James J. Yoo, Anthony AtalaAbstract:INTRODUCTION: One of the major challenges in engineering clinically applicable Functional Tissues for reconstructive procedures is the lack of a bioreactor system that would accelerate cellular organization and Tissue formation. We developed a computerized bioreactor system that could allow for enhanced bladder Tissue formation. In this study we investigated whether organized Functional Muscle Tissue could be engineered using the 3-D bioreactor system.
