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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1995

Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

NASA Discipline Radiation Health↗
NASA NTRS2002

Mechanical stimulation improves tissue-engineered human skeletal muscle

Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

Non-NASA Center↗
NASA NTRS2011

Robotic Scaffolds for Tissue Engineering and Organ Growth

The aim of tissue engineering (TE) is to restore tissue and organ functions with minimal host rejection. TE is seen as a future solution to solve the crisis of donor organs for transplant, which faces a shortage expected only to increase in the future. In this innovation, a flexible and configurable scaffold has been conceived that mechanically stresses cells that are seeded on it, stimulating them to increased growth. The influence of mechanical stress/ loading on cell growth has been observed on all forms of cells. For example, for cartilages, studies in animals, tissue explants, and engineered tissue scaffolds have all shown that cartilage cells (chondrocytes) modify their extracellular matrix in response to loading. The chondrocyte EMC production response to dynamics of the physical environment (in vivo cartilage development) illustrates a clear benefit (better growth) when stressed. It has been shown that static and dynamic compression regulates PRG4 biosynthesis by cartilage explants. Mechanical tissue stimulation is beneficial and (flexible) scaffolds with movable components, which are able to induce mechanical stimulation, offer advantages over the fixed, rigid scaffold design. In addition to improved cell growth from physical/mechanical stimulation, additional benefits include the ability to increase in size while preserving shape, or changing shape. By making scaffolds flexible, allowing relative movement between their components, adding sensing (e.g., for detecting response of cells to drug release and to mechanical actions), building controls for drug release and movement, and building even simple algorithms for mapping sensing to action, these structures can actually be made into biocompatible and biodegradable robots. Treating them as robots is a perspective shift that may offer advantages in the design and exploitation of these structures of the future.

Stoica, Adrian↗
NASA NTRS1999

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

Non-NASA Center↗
NASA NTRS1996

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myoribers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Vandenburgh, Herman↗
NASA NTRS1996

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid Implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postimtotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Vandenburgh, Herman↗
NASA NTRS1997

Biomaterials for tissue engineering: summary

This article summarizes presentations and discussion at the workshop "Enabling Biomaterial Technology for Tissue Engineering," which was held during the Fifth World Biomaterials Congress in May 1996. Presentations covered the areas of material substrate architecture, barrier effects, and cellular response, including analysis of biomaterials challenges involved in producing specific tissue-engineered products.

Non-NASA Center↗
NASA NTRS1996

The importance of new processing techniques in tissue engineering

The use of polymer scaffolds in tissue engineering is reviewed and processing techniques are examined. The discussion of polymer-scaffold processing explains fiber bonding, solvent casting and particulate leaching, membrane lamination, melt molding, polymer/ceramic fiber composite-foam processing, phase separation, and high-pressure processing.

Non-NASA Center↗
NASA NTRS1996

A Simplified Method for Tissue Engineering Skeletal Muscle Organoids in Vitro

Tissue-engineered three dimensional skeletal muscle organ-like structures have been formed in vitro from primary myoblasts by several different techniques. This report describes a simplified method for generating large numbers of muscle organoids from either primary embryonic avian or neonatal rodent myoblasts, which avoids the requirements for stretching and other mechanical stimulation.

Shansky, Janet↗
NASA NTRS2002

Tissue engineering skeletal muscle for orthopaedic applications

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

Review↗
NASA NTRS1999

Tissue Engineered Skeletal Myofibers can Directly "Sense" Gravitational Force Changes

Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures. Embryonic avian muscle cells were isolated and BAMs tissue engineered as described previously. The myoblasts proliferate and fuse into aligned postmitotic myofibers after ten to fourteen days in vitro. A cylindrical muscle-like structure containing several thousand myofibers is formed which is approximately 30 mm in length, 2-3 mm in diameter, and attached at each end. For the Space Shuttle experiments, the BAMs were transferred to 55 mL bioreactor cartridges (6 BAMs/cartridge). At Kennedy Space Center, the cartridges were mounted in two Space Tissue Loss (STL) Modules (three to four cartridges per Module) and either maintained as ground controls or loaded in a Mid-Deck locker of the Space Shuttle. The BAM cartridges were continuously perfused during the experiment at 1.5 mL/ min with tissue culture medium. Eighteen BAMs were flown for nine days on Mission STS66 while eighteen BAMs served as ground controls. The complete experiment was repeated on Mission STS77 with twenty four BAMs in each group. BAMs could be maintained in a healthy state for at least 30 days in the perfusion bioreactor cartridges. The BAM muscle fibers directly detected both the loss of gravity and the reloading effects of 1 x g. While total cellular metabolism and total protein degradation rates were not altered during 9 to 10 days in Space, protein synthesis rates were significantly reduced and resulted in significant myofiber atrophy compared to ground controls. One g reloading of the flight muscle cells post-flight significantly increased protein synthesis rates and the synthesis rates of myosin heavy chain, fibronectin, and collagen. Tissue cultured muscle cells can directly "sense" changes in gravity and provide a valid model to begin the study of countermeasures. Based on our ground based experiments, and the experiments of others, growth hormone and/or insulin-like growth factors are attractive protein therapeutics which may assisting attenuating skeletal muscle wasting in space. Our laboratory is developing a new cell-based delivery system for this and other potential therapeutic factors for attenuating muscle and bone wasting.

Vandenburgh, Herman H.↗
NASA NTRS2005

Tissue Engineering Using Transfected Growth-Factor Genes

A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

Madry, Henning↗
NASA NTRS2003

Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

Martin, Ivan↗
NASA NTRS1998

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

NASA Discipline Musculoskeletal↗
NASA NTRS1999

Tissue Engineering Organs for Space Biology Research

Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures.

Vandenburgh, H. H.↗
NASA NTRS1998

Attenuation of skeletal muscle wasting with recombinant human growth hormone secreted from a tissue-engineered bioartificial muscle

Skeletal muscle wasting is a significant problem in elderly and debilitated patients. Growth hormone (GH) is an anabolic growth factor for skeletal muscle but is difficult to deliver in a therapeutic manner by injection owing to its in vivo instability. A novel method is presented for the sustained secretion of recombinant human GH (rhGH) from genetically modified skeletal muscle implants, which reduces host muscle wasting. Proliferating murine C2C12 skeletal myoblasts stably transduced with the rhGH gene were tissue engineered in vitro into bioartificial muscles (C2-BAMs) containing organized postmitotic myofibers secreting 3-5 microg of rhGH/day in vitro. When implanted subcutaneously into syngeneic mice, C2-BAMs delivered a sustained physiologic dose of 2.5 to 11.3 ng of rhGH per milliliter of serum. rhGH synthesized and secreted by the myofibers was in the 22-kDa monomeric form and was biologically active, based on downregulation of a GH-sensitive protein synthesized in the liver. Skeletal muscle disuse atrophy was induced in mice by hindlimb unloading, causing the fast plantaris and slow soleus muscles to atrophy by 21 to 35% ( < 0.02). This atrophy was significantly attenuated 41 to 55% (p < 0.02) in animals that received C2-BAM implants, but not in animals receiving daily injections of purified rhGH (1 mg/kg/day). These data support the concept that delivery of rhGH from BAMs may be efficacious in treating muscle-wasting disorders.

NASA Discipline Musculoskeletal↗