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  • Tissue engineering intrafusal fibers: dose- and time-dependent differentiation of nuclear bag fibers in a defined in vitro system using neuregulin 1-beta-1. 18076984

    While much is known about muscle spindle structure, innervation and function, relatively few factors have been identified that regulate intrafusal fiber differentiation and spindle development. Identification of these factors will be a crucial step in tissue engineering functional muscle systems. In this study, we investigated the role of the growth factor, neuregulin 1-beta-1 (Nrg 1-beta-1) EGF, for its ability to influence myotube fate specification in a defined culture system utilizing the non-biological substrate N-1[3-(trimethoxysilyl)propyl]-diethylenetriamine (DETA). Based on morphological and immunocytochemical criteria, Nrg 1-beta-1 treatment of developing myotubes increases the ratio of nuclear bag fibers to total myotubes from 0.019 to 0.100, approximately a five-fold increase. The myotube cultures were evaluated for expression of the intrafusal fiber-specific alpha cardiac-like myosin heavy chain and for the expression of the non-specific slow myosin heavy chain. Additionally, the expression of ErbB2 receptors on all myotubes was observed, while phosphorylated ErbB2 receptors were only observed in Nrg 1-beta-1-treated intrafusal fibers. After Nrg 1-beta-1 treatment, we were able to observe the expression of the intrafusal fiber-specific transcription factor Egr3 only in fibers exhibiting the nuclear bag phenotype. Finally, nuclear bag fibers were characterized electrophysiologically for the first time in vitro. This data shows conclusively, in a serum-free system, that Nrg 1-beta-1 is necessary to drive specification of forming myotubes to the nuclear bag phenotype.
    Document Type:
    Reference
    Product Catalog Number:
    06-562
    Product Catalog Name:
    Anti-erbB-2/HER-2 Antibody

  • Skeletal muscle tissue engineering: a maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus a ... 19625080

    The use of defined in vitro systems to study the developmental and physiological characteristics of a variety of cell types is increasing, due in large part to their ease of integration with tissue engineering, regenerative medicine, and high-throughput screening applications. In this study, myotubes derived from fetal rat hind limbs were induced to develop several aspects of mature muscle including: sarcomere assembly, development of the excitation-contraction coupling apparatus and myosin heavy chain (MHC) class switching. Utilizing immunocytochemical analysis, anisotropic and isotropic band formation (striations) within the myotubes was established, indicative of sarcomere formation. In addition, clusters of ryanodine receptors were colocalized with dihydropyridine complex proteins which signaled development of the excitation-contraction coupling apparatus and transverse tubule biogenesis. The myotubes also exhibited MHC class switching from embryonic to neonatal MHC. Lastly, the myotubes survived significantly longer in culture (70-90 days) than myotubes from our previously developed system (20-25 days). These results were achieved by modifying the culture timeline as well as the development of a new medium formulation. This defined model system for skeletal muscle maturation supports the goal of developing physiologically relevant muscle constructs for use in tissue engineering and regenerative medicine as well as for high-throughput screening applications.
    Document Type:
    Reference
    Product Catalog Number:
    AB9078
    Product Catalog Name:
    Anti-Ryanodine Receptor 1 Antibody

  • Tissue engineering a complete vaginal replacement from a small biopsy of autologous tissue. 18645481

    In women, a healthy, patent vagina is important for the maintenance of a good quality of life. Apart from congenital abnormalities, such as cloacal exstrophy, intersex disorders, and an absence of the posterior two thirds of the organ, individuals may also suffer from cancer, trauma, infection, inflammation, or iatrogenic injuries leading to tissue damage and loss -- all of which require vaginal repair or replacement. Of necessity, reconstruction is often performed with nonvaginal tissue substitutes, such as segments of large intestine or skin, which are not anatomically or functionally ideal (Hendren and Atala, J Urol 1994; 152: 752; Hendren and Atala, J Pediatr Surg 1995; 30: 91). Whenever such tissue is used additional complications often ensue, such as strictures, infection, hair growth, graft shrinkage, diverticuli, and even malignancy (Filipas et al., BJU Int 2000; 85: 715; Lai and Chang, Changgeng Yi Xue Za Zhi 1999; 22: 253; Parsons et al., J Pediatr Surg 2002; 37: 629; Seccia et al., Ann Plast Surg 2002; 49: 379; Filipas, Curr Opin Urol 2001; 11: 267).
    Document Type:
    Reference
    Product Catalog Number:
    CBL212
    Product Catalog Name:
    Anti-Neurofilament 200 kDa Antibody, clone RT97

  • Tissue engineering for full-thickness burns: a dermal substitute from bench to bedside. 17888881

    Our aim was to obtain a viable and easily available dermal substitute (DS) for the definitive coverage of full-thickness burns. A DS composed of a collagen-glycosaminoglycan-chitosan dermal matrix (DM) colonized with foreskin fibroblasts (FF) is described. FF-colonized DS were compared to the DM seeded with adult dermal fibroblasts (DF). FF-colonized DS expressed more fibrillin and tropoelastin than that with DF. Reconstructed skin obtained with both FF- and DF-colonized DS similarly expressed laminin-5 and collagen VII at the dermal-epidermal junction. Both FF- and DF-colonized DS produced cutaneous wound healing mediators in a dose-dependent manner in the presence of platelet lysate. After freeze-thawing, the FF-colonized DS were recovered in culture and retained their ability to produce vascular endothelial growth factor. Grafting of DS into nude rats achieved a complete healing of a dermal-epidermal lesion with a good epidermalization.
    Document Type:
    Reference
    Product Catalog Number:
    MAB2502
    Product Catalog Name:
    Anti-Fibrillin-1 Antibody, NT, clone 26

  • Vascular tissue engineering: bioreactor design considerations for extended culture of primary human vascular smooth muscle cells. 17885337

    The influence of mechanical stimulation on cell populations not only helps maintain the specific cellular phenotype but also plays a significant role during differentiation and maturation of plastic cells. This is particularly true of tissue-engineered vascular tissue, where in vivo shear forces at the blood interface help maintain the function of the endothelium. Considerable effort has gone into the design and implementation of functional bioreactors that mimic the chemical and mechanical forces associated with the in vivo environment. Using a decellularized ex vivo porcine carotid artery as a model scaffold, we describe a number of important design criteria used to develop a vascular perfusion bioreactor and its supporting process-flow. The results of a comparative analysis of primary human vascular smooth muscle cells cultured under traditionalstatic conditions and dynamic loading are described, where the expression of MMP-2 and 9 and cathepsin-L were assessed. Continued design improvements to perfusion bioreactors may improve cellular interactions, leading to constructs with improved biological function.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3309

  • Tissue engineering of lung: the effect of extracellular matrix on the differentiation of embryonic stem cells to pneumocytes. 20001250

    We have previously differentiated lung epithelium from human and murine embryonic stem cells (mESCs) and are now exploring the potential applications of these cells, including in the engineering of lung tissue constructs. In this study, we hypothesized that the differentiation and maintenance of lung epithelium derived from mESCs can be enhanced by extracellular matrix (ECM) proteins. Our established differentiation protocol was applied to mESCs grown on a range of ECMs: collagen I, laminin 332, fibronectin, Matrigel, and, as an experimental control, gelatin. The ECMs were coated onto tissue culture plastic (TCP) and poly-DL-lactic acid (PDLLA), a biodegradable polymer we have previously shown to support the growth of mature pneumocytes. Matrigel or Laminin-332 coating of either TCP or PDLLA film resulted in enhanced surfactant protein C gene expression in differentiating mESCs, a direct indication of the upregulation of lung epithelial differentiation. For each combination, changes in the contact angle and zeta potential of protein-coated TCP and PDLLA film confirmed protein adsorption. We conclude that the choice of the coating protein can greatly affect the differentiation of ESCs, and laminin-332-coated PDLLA provided an ECM-degradable scaffold combination that is suitable for engineering of lung tissue constructs.
    Document Type:
    Reference
    Product Catalog Number:
    AP106R
    Product Catalog Name:
    Rabbit Anti-Goat IgG Antibody, Rhodamine conjugate

  • Tissue engineering the monosynaptic circuit of the stretch reflex arc with co-culture of embryonic motoneurons and proprioceptive sensory neurons. 22594977

    The sensory circuit of the stretch reflex arc is composed of intrafusal muscle fibers and their innervating proprioceptive neurons that convert mechanical information regarding muscle length and tension into action potentials that synapse onto the homonymous motoneurons in the ventral spinal cord which innervate the extrafusal fibers of the same muscle. To date, the in vitro synaptic connection between proprioceptive sensory neurons and spinal motoneurons has not been demonstrated. A functional in vitro system demonstrating this connection would enable the understanding of feedback by the integration of sensory input into the spinal reflex arc. Here we report a co-culture of rat embryonic motoneurons and proprioceptive sensory neurons from dorsal root ganglia (DRG) in a defined serum-free medium on a synthetic silane substrate (DETA). Furthermore, we have demonstrated functional synapse formation in the co-culture by immunocytochemistry and electrophysiological analysis. This work will be valuable for enabling in vitro model systems for the study of spinal motor control and related pathologies such as spinal cord injury, muscular dystrophy and spasticity by improving our understanding of the integration of the mechanosensitive feedback mechanism.
    Document Type:
    Reference
    Product Catalog Number:
    MAB365
    Product Catalog Name:
    Anti-Nerve Growth Factor Receptor Antibody, extracellular, clone 192-IgG

  • A paradigm for functional tissue engineering of articular cartilage via applied physiologic deformational loading. 14964720

    Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3391
    Product Catalog Name:
    Anti-Collagen Type I Antibody, clone 5D8-G9

  • Autologous minced muscle grafts: a tissue engineering therapy for the volumetric loss of skeletal muscle. 23885064

    Volumetric muscle loss (VML) results in a large void deficient in the requisite materials for regeneration for which there is no definitive clinical standard of care. Autologous minced muscle grafts (MG), which contain the essential components for muscle regeneration, may embody an ideal tissue engineering therapy for VML. The purpose of this study was to determine if orthotopic transplantation of MG acutely after VML in the tibialis anterior muscle of male Lewis rats promotes functional tissue regeneration. Herein we report that over the first 16 wk postinjury, MG transplantation 1) promotes remarkable regeneration of innervated muscle fibers within the defect area (i.e., de novo muscle fiber regeneration); 2) reduced evidence of chronic injury in the remaining muscle mass compared with nonrepaired muscles following VML (i.e., transplantation attenuated chronically upregulated transforming growth factor-β1 gene expression and the presence of centrally located nuclei in 30% of fibers observed in nonrepaired muscles); and 3) significantly improves net torque production (i.e., ∼55% of the functional deficit in nonrepaired muscles was restored). Additionally, voluntary wheel running was shown to reduce the heightened accumulation of extracellular matrix deposition observed within the regenerated tissue of MG-repaired sedentary rats 8 wk postinjury (collagen 1% area: sedentary vs. runner, ∼41 vs. 30%), which may have been the result of an augmented inflammatory response [i.e., M1 (CCR7) and M2 (CD163) macrophage expression was significantly greater in runner than sedentary MG-repaired muscles 2 wk postinjury]. These findings support further exploration of autologous minced MGs for the treatment of VML.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells. 22421311

    Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and its demonstrated ability to support bone formation both in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASCs), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol (HFIP)), pore sizes (250-500 μm vs. 500-1000 μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes, with decellularized trabecular bone (DCB) included as a "gold standard". The scaffolds were seeded with hASCs and cultured for 7 weeks in osteogenic medium. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400-600 μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes, as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2 and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple