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NASA NTRS1996

Effects of Microgravity on Quail Eye Development

During embryonic development, the most exposed tissue of the eye, the cornea, becomes differentially bulged outward because of constant intraocular pressure (IOP). The component cells of the cornea secrete a unique, paracrystalline extracellular matrix (the stroma) composed of orthogonal plies of collagen fibrils and proteoglycans. The cornea remains avascular, becomes transparent, and becomes more densely innervated than any other region on the surface of the body. Corneas from chicken embryos that flew on STS-47 contain many more cellular processes in the outermost region of the stroma (Bowman's Layer) than any corresponding region of control corneas. These processes appear to be cross-sections of cytoplasmic extensions of cells and are found in that region of Bowman's Layer immediately beneath the basal lamina of the corneal epithelium. Here, we propose to compare corneas of quail that flew in space on Mir-1 with those of ground controls to determine if the same unusual cellular processes are seen as in the space-flown chicken corneas. In the central regions of such space-flown corneas, the processes appear to be either portions of basal epithelial cells whose pseudopodial extensions have migrated down through their own basal lamina into the stroma, or corneal nerves that have innervated the corneal stroma in an unusual manner. Eyeballs of embryos fixed on Mir-1, control embryos fixed at KSC and clinostated embryos fixed at KSU, will provide corneas for this study. Electron microscopy will be used to assess the distribution of the cellular processes in Bowman's Layer in the central region of each cornea. Attempts also will be made to determine the relative glycosaminoglycan distributions in the corneal stromas by indirect immunofluorscence and to record whole-mount staining patterns of the corneal nerves.

Conrad, Gary W.↗

NASA NTRS2003

Decorin modulates matrix mineralization in vitro

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.

NASA Discipline Musculoskeletal↗

NASA NTRS2001

Differential expression of decorin and biglycan genes during mouse tooth development

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.

Non-NASA Center↗

NASA NTRS2000

Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

Non-NASA Center↗

NASA NTRS1999

If walls could talk

The plant cell wall is very complex, both in structure and function. The wall components and the mechanical properties of the wall have been implicated in conveying information that is important for morphogenesis. Proteoglycans, fragments of polysaccharides and the structural integrity of the wall may relay signals that influence cellular differentiation and growth control. Furthering our knowledge of cell wall structure and function is likely to have a profound impact on our understanding of how plant cells communicate with the extracellular environment.

Review↗

NASA NTRS1999

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Non-NASA Center↗

NASA NTRS1994

A fiber matrix model for fluid flow and streaming potentials in the canaliculi of an osteon

A theoretical model is developed to predict the fluid shear stress and streaming potential at the surface of osteocytic processes in the lacunar-canalicular porosity of an osteon when the osteon is subject to mechanical loads that are parallel or perpendicular to its axis. The theory developed in Weinbaum et al. (31) for the flow through a proteoglycan matrix in a canaliculus is employed in a poroelastic model for the osteon. Our formulation is a generalization of that of Petrov et al. (17). Our model predicts that, in order to satisfy the measured frequency dependence of the phase and magnitude of the SGP in macroscopic bone samples, the fiber spacing in the fluid annulus must lie in the narrow range 6-7 nm typical of the spacing of GAG sidechains along a protein monomer. The model predictions for the local SGP profiles in the osteon agree with the experimental observations of Starkebaum et al. (24). The theory predicts that the pore pressure relaxation time, tau d, for a 150-300 microns diameter osteon with the foregoing matrix structure is approximately 0.03-0.13 sec, and that the amplitude of the mean fluid shear stress on the membrane of the osteocytic process at the mean areal radius of the osteon has a maximum at 28 Hz if tau d = 0.06 sec. This maximum, which is independent of the magnitude of the loading, could be important in vivo since the recent experiments of Turner et al. (28) and McLeod et al. (15) have a peak in the strain frequency spectrum between 20 and 30 Hz that also appears to be independent of the type (magnitude) of loading. Numerical predictions for the amplitude of the average fluid shear stress on the osteocytic membrane at the mean areal radius of the osteon show that the fluid shear stress associated with the low amplitude 20-30 Hz spectral strain component is at least as large as the average fluid shear stress associated with the high amplitude 1 Hz stride component, although the latter loading is an order of magnitude larger, and has a magnitude that lies within the middle of the range, 6-30 dynes/cm2, where fluid shear stresses in tissue culture studies with osteoblast monolayers have elicited an intracellular Ca++ response (31). The implications of these results for intracellular electrical communication are discussed.

Non-NASA Center↗

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↗