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NASA NTRS2001

Monoclonal L-citrulline immunostaining reveals nitric oxide-producing vestibular neurons

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

NASA Discipline Neuroscience↗

NASA NTRS2012

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Benardini, James N.↗

NASA NTRS1994

Post-embedding tem signal-to-noise ratio of S-100

We assessed the reactivity of purified S-100 antiserum in immuno-electron microscopy by counting the number of gold particles per microns 2 over inner ear tissues embedded in different media. Sections containing predominantly Schwann's cell cytoplasm and nucleus, afferent fiber axoplasm and myelin sheath of chick cochleae were reacted with anti-S-100 IgG, an antibody to a calcium binding protein of neuronal tissues, then labeled with anti-IgG-gold conjugate. This investigation was conducted because previously published procedures, unmodified, did not yield acceptable results. Preparation of all specimens was identical. Only the medium (PolyBed 812, Araldite or Spurr epoxies; and LR White, LR Gold or Lowicryl plastics) was changed. The medium was made the changing variable because antigens available in post-embedding immuno-electron microscopy are decreased by heat, either used and/or released during polymerization of the embedding medium. The results indicate that: (a) none of the embedding media above provided optimal signal-to-noise ratio for all parts of the nerve stained in the same section; (b) aggregation of gold particles over cells was highest in embedding media with high background labeling over areas devoid of tissue (noise); (c) aggregation occurred randomly throughout both cellular and acellular regions; and (d) particles aggregated less and were distributed more evenly in tissues from media yielding good ultrastructural integrity.

Non-NASA Center↗

NASA NTRS1999

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Review, Tutorial↗

NASA NTRS1999

Model system for plant cell biology: GFP imaging in living onion epidermal cells

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

NASA Discipline Plant Biology↗

NASA NTRS1996

Rapid Bacterial Testing for Spacecraft Water

Evaluations of the fluorogenic stains and probes will continue. E. coli 0157:H7 will be used as the reference strain for optimizing protocols. We anticipate the continued use of the fluorescent antibodies (TRITC and FITC labeled) in conjunction with CTC, Rhl23, DiBAC4(3), DAPI and acridine orange. Chemunex, the manufacturer of the ChemScan analyzer system, also makes a fluorogenic probe, Chemchrome B, which will be incorporated into the suite of probes to evaluate once their system is on site. Regardless of the combination of stains and probes all will be evaluated on membrane filters. Development of a FISH protocol that will be applicable to our conditions will be continued. Complimentary 16s rRNA probes to Ps. aeruginosa and currently in our laboratory will be evaluated first. Once this protocol has been adequately optimized other probes will be ordered for u a select number of other species. Currently, protocols to evaluate the effects of disinfection and the resulting lethality, injury on stain and/or probe specificity and reliability are being developed. E. coli 0157:H7 is the reference strain and chlorine the disinfectant the reference protocol is being developed around. Upon completion of this work, the resulting protocol will be extended to other species and disinfectants (e.g., iodine). Similar disinfectant experiments will then be conducted on the same species after starvation to evaluate the effects of starvation on disinfection resistance and the applicability of the stains and probes. Development of the immunomagnetic separation system will continue. Combined with the rapid methods described above, with enumeration by the ChemScan, we anticipate that this will provide a highly sensitive technique for the detection of specific, active bacteria.

Lisle, John T.↗

NASA NTRS1993

Activity-induced regulation of myosin isoform distribution - Comparison of two contractile activity programs

This study examined the role of specific types of contractile activity in regulating myosin heavy chain (MHC) isoform expression in rodent soleus. A combination of hindlimb suspension (SN) and two programmed contractile training activity paradigms, either isometric contractile activity (ST-IM) or high-load slowly shortening isovelocity activity, were utilized. Both training paradigms increased muscle mass compared with SN alone. However, only ST-IM resulted in a partial prevention of the suspension-induced decrease in type I MHC. With the use of a fluorescently labeled antibody to type IIa MHC, the distribution of MHCs among fibers was examined immunohistochemically. In SN, the percentage of cells staining positive for type IIa MHC was increased but the staining intensity of the positively staining cells was unchanged compared with control cells. In the ST-IM soleus, the percentage of positively staining fibers was unchanged but the intensity of the positively staining cells was decreased compared with SN values. These results suggest that 1) isometric contractile activity is more effective than isovelocity activity in preventing suspension-induced shifts in soleus MHC distribution and 2) changes associated with both suspension and training occur in only a small number of fibers, with the majority of fibers apparently unresponsive to these interventions.

Diffee, Gary M.↗

NASA NTRS2010

Microfluidics microFACS for Life Detection

A prototype micro-scale Fluorescent Activated Cell Sorter (microFACS) for life detection has been built and is undergoing testing. A functional miniature microfluidics instrument with the ability to remotely distinguish live or dead bacterial cells from abiotic particulates in ice or permafrost of icy bodies of the solar system would be of fundamental value to NASA. The use of molecular probes to obtain the bio-signature of living or dead cells could answer the most fundamental question of Astrobiology: Does life exist beyond Earth? The live-dead fluorescent stains to be used in the microFACS instrument function only with biological cell walls. The detection of the cell membranes of living or dead bacteria (unlike PAH's and many other Biomarkers) would provide convincing evidence of present or past life. This miniature device rapidly examine large numbers of particulates from a polar ice or permafrost sample and distinguish living from dead bacteria cells and biological cells from mineral grains and abiotic particulates and sort the cells and particulates based on a staining system. Any sample found to exhibit fluorescence consistent with living cells could then be used in conjunction with a chiral labeled release experiment or video microscopy system to seek addition evidence for cellular metabolism or motility. Results of preliminary testing and calibration of the microFACS prototype instrument system with pure cultures and enrichment assemblages of microbial extremophiles will be reported.

Platt, Donald W.↗

NASA NTRS1997

Immunolocalization of integrin-like proteins in Arabidopsis and Chara

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

Non-NASA Center↗

NASA NTRS1998

Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at approximately 40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle alpha-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

NASA Discipline Cardiopulmonary↗

NASA NTRS1995

The statolith compartment in Chara rhizoids contains carbohydrate and protein

In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

NASA Discipline Plant Biology↗

NASA NTRS1988

In vitro differentiation of quail neural crest cells into sensory-like neuroblasts

Data are presented that demonstrate the ability of quail neural-crest embrionic cells grown as primary culture to differentiate in vitro into sensorylike neuroblasts. After 7-14 days of growth as primary culture, many of the putative sensory neuroblasts displayed substance P (SP)-like immunoreactivity and some exhibited histochemical carbonic anhydrase activity. Double staining experiments showed that the SP-like immunoreactive neuroblasts did not contain detectable levels of tyrosine hydroxylase or dopamine-beta-hydroxylase. The neuronal nature of the cultured sensorylike neuroblasts was further documented by double labeling for antibodies against the 68 kDa neurofilament polypeptide and substance P.

Sieber-Blum, Maya↗

NASA NTRS2002

L-citrulline immunostaining identifies nitric oxide production sites within neurons

The cellular and subcellular localization of L-citrulline was analyzed in the adult rat brain and compared with that of traditional markers for the presence of nitric oxide synthase. Light, transmission electron, and confocal laser scanning microscopy were used to study tissue sections processed for immunocytochemistry employing a monoclonal antibody against L-citrulline or polyclonal anti-neuronal nitric oxide synthase sera, and double immunofluorescence to detect neuronal nitric oxide synthase and L-citrulline co-localization. The results demonstrate that the same CNS regions and cell types are labeled by neuronal nitric oxide synthase polyclonal antisera and L-citrulline monoclonal antibodies, using both immunocytochemistry and immunofluorescence. Short-term pretreatment with a nitric oxide synthase inhibitor reduces L-citrulline immunostaining, but does not affect neuronal nitric oxide synthase immunoreactivity. In the vestibular brainstem, double immunofluorescence studies show that many, but not all, neuronal nitric oxide synthase-positive cells co-express L-citrulline, and that local intracellular patches of intense L-citrulline accumulation are present in some neurons. Conversely, all L-citrulline-labeled neurons co-express neuronal nitric oxide synthase. Cells expressing neuronal nitric oxide synthase alone are interpreted as neurons with the potential to produce nitric oxide under other stimulus conditions, and the subcellular foci of enhanced L-citrulline staining are viewed as intracellular sites of nitric oxide production. This interpretation is supported by ultrastructural observations of subcellular foci with enhanced L-citrulline and/or neuronal nitric oxide synthase staining that are located primarily at postsynaptic densities and portions of the endoplasmic reticulum. We conclude that nitric oxide is produced and released at focal sites within neurons that are identifiable using L-citrulline as a marker. Copyright 2002 IBRO.

NASA Discipline Neuroscience↗

NASA NTRS2000

Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure

BACKGROUND: To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice. METHODS AND RESULTS: Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7+/-0.7 versus 3.1+/-0.2 and 5. 7+/-0.4 versus 2.7+/-0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0+/-0.8 and 25.2+/-1.8 versus 14. 1+/-0.5 microm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for beta(1)-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides. CONCLUSIONS: Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

NASA Discipline Cardiopulmonary↗

NASA NTRS1995

Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior

The spatial distribution, soma size and oxidative enzyme activity of gamma and alpha motoneurons innervating muscle fibres in the deep (away from the surface of the muscle) and superficial (close to the surface of the muscle) portions of the tibialis anterior in normal rats were determined. The deep portion had a higher percentage of high oxidative fibres than the superficial portion of the muscle. Motoneurons were labelled by retrograde neuronal transport of fluorescent tracers: Fast Blue and Nuclear Yellow were injected into the deep portion and Nuclear Yellow into the superficial portion of the muscle. Therefore, motoneurons innervating the deep portion were identified by both a blue fluorescent cytoplasm and a golden-yellow fluorescent nucleus, while motoneurons innervating the superficial portion were identified by only a golden-yellow fluorescent nucleus. After staining for succinate dehydrogenase activity on the same section used for the identification of the motoneurons, soma size and succinate dehydrogenase activity of the motoneurons were measured. The gamma and alpha motoneurons innervating both the deep and superficial portions were located primarily at L4 and were intermingled within the same region of the dorsolateral portion of the ventral horn in the spinal cord. Mean soma size was similar for either gamma or alpha motoneurons in the two portions of the muscle. The alpha motoneurons innervating the superficial portion had a lower mean succinate dehydrogenase activity than those innervating the deep portion of the muscle. An inverse relationship between soma size and succinate dehydrogenase activity of alpha, but not gamma, motoneurons innervating both the deep and superficial portions was observed. Based on three-dimensional reconstructions within the spinal cord, there were no apparent differences in the spatial distribution of the motoneurons, either gamma or alpha, associated with the deep and superficial compartments of the muscle. The data provide evidence for an interdependence in the oxidative capacity between a motoneuron and its target muscle fibres in two subpopulations of motoneurons from the same motor pool, i.e. the same muscle.

NASA Discipline Musculoskeletal↗

NASA NTRS2000

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

Evaluation Studies↗

NASA NTRS1995

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

NASA Discipline Musculoskeletal↗

NASA NTRS1993

Embryonic mouse pre-metatarsal development in organ culture

Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.

Non-NASA Center↗