Endochondral bone formation in embryonic mouse pre-metatarsals

Long term exposure to a reduced gravitational environment has a deleterious effect on bone. The developmental events which occur prior to initial bone deposition will provide insight into the regulation of mature bone physiology. We have characterized a system in which the events preceding bone formation take place in an isolated in vitro organ culture environment. We show that cultured pre-metatarsal tissue parallels development of pre-metatarsal tissue in the embryo. Both undergo mesenchyme differentiation and morphogenesis to form a cartilage rod, which resembles the future bone, followed by terminal chondrocyte differentiation in a definite morphogenetic pattern. These sequential steps occur prior to osteoblast maturation and bone matrix deposition in the developing organism. Alkaline phosphatase (ALP) activity is a distinctive enzymatic marker for mineralizing tissues. We have measured this activity throughout pre-metatarsal development and show (a) where in the tissue it is predominantly found, and (b) that this is indeed the mineralizing isoform of the enzyme.     

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the nasa rotating wall vessel bioreactor

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.     

Simulated microgravity culture system for a 3-D carcinoma tissue model

An in vitro organotypic culture model is needed to understand the complexities of carcinoma tissue consisting of carcinoma cells, stromal cells, and extracellular matrices. We developed a new in vitro model of carcinoma tissue using a rotary cell culture system with four disposable vessels (RCCS-4D) that provides a simulated microgravity condition. Solid collagen gels containing human pancreatic carcinoma NOR-P1 cells and fibroblasts or minced human pancreatic carcinoma tissue were cultured under a simulated microgravity condition or a static Ig condition for seven days. NOR-P1 cultures subjected to the simulated microgravity condition showed greater numbers of mitotic, cycling (Ki-67-positive), nuclear factor-kappa B-activating cells, and a lower number of apoptotic cells than were shown by cultures subjected to the static Ig condition. In addition, human pancreatic carcinoma specimens cultured under the simulated microgravity condition maintained the heterogeneous composition and cellular activity (determined by the cycling cell ratio and mitotic index) of the original carcinoma tissue better than static culture conditions. This new 3-D rotary cell culture system with four disposal vessels may be useful for in vitro studies of complex pancreatic carcinoma tissue.     

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research. © 1993 Wiley-Liss, Inc.     

Prospects for use of microgravity-based bioreactors to study three-dimensional host—tumor interactions in human neoplasia

Microgravity offers unique advantages for the cultivation of mammalian tissues because the lack of gravity-induced sedimentation supports three-dimensional growth in batch culture in aqueous medium. Bioreactors that simulate microgravity but operate in unit gravity provide conditions that permit human epithelial cells to grow to densities approaching 10⁷ cells/ml on microcarriers in suspension, in masses up to 1 cm in diameter, and under conditions of low shear stress. While useful for many different applications in tissue culture, this culture system is especially useful for the analysis of the microenvironment in which host matrix and cells interact with infiltrating tumor cells. Growth in the microgravity-based bioreactor has supported morphological differentiation of human colon carcinoma cells when cultured with normal human stromal cells. Furthermore, these co-cultures produced factors that stimulated goblet cell production in normal colon cells in an in vivo bioassay. Early experiments also suggest that the microgravity environment will not alter the ability of epithelial cells to recognize and associate with each other and with constituents of basement membrane and extracellular matrix. These findings suggest that cells grown in bioreactors that simulate aspects of microgravity or under actual microgravity conditions will produce tissues and substances in sufficient quantity and at high enough concentration to promote characterization of molecules that control differentiation and neoplastic transformation. © 1993 Wiley-Liss, Inc.     

Effects of chronic exposure to simulated microgravity on skeletal muscle cell proliferation and differentiation

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.     

A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli

We sought to develop a practical and representative model to study the interactions of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) with human intestinal tissue. For this purpose, human intestinal epithelial HCT-8 cells were cultured under low-shear microgravity conditions in a rotating cell culture system. After 10 days, layered cell aggregates, or 'organoids', developed. Three lines of evidence indicated that these organoids exhibited traits characteristic of normal tissue. First, the organoids expressed normal intestinal tissue markers in patterns that suggested greater cellular differentiation in the organoids than conventionally grown monolayers. Second, the organoids produced higher levels of intestinally expressed disaccharidases and alkaline phosphatase on a cell basis than did conventionally cultured monolayers. Third, HCT-8 organoid tissue developed microvilli and desmosomes characteristic of normal tissue, as revealed by electron microscopy. Because the low-shear microgravity condition is proposed by modelling studies to more closely approximate conditions in the intestinal microvilli, we also tested the impact of microgravity of bacterial growth and virulence gene expression. No influence on growth rates was observed but intimin expression by EHEC was elevated during culture in microgravity as compared with normal gravity. That the responses of HCT-8 organoids to infection with wild-type EPEC or EHEC under microgravitational conditions approximated infection of normal tissue was demonstrated by the classical appearance of the resultant attaching and effacing lesions. We concluded that the low shear microgravity environment promoted growth of intestinal cell organoids with greater differentiation than was seen in HCT-8 cells maintained in conventional tissue culture and provided a reduced gravity environment for study of bacterial-host cell interactions.     

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.     

Impact of microgravity on radiobiological processes and efficiency of DNA repair

To study the influence of microgravity on radiobiological processes in space, space experiments have been performed, using an on-board 1×g reference centrifuge as in-flight control. The trajectory of individual heavy ions was localized in relation to the biological systems by use of the Biostack concept, or an additional high dose of radiation was applied either before the mission or during the mission from an on-board radiation source. In embryonic systems, such as early developmental stages of Drosophila melanogaster and Carausius morosus, the occurrence of chromosomal translocations and larval malformations was dramatically increased in response to microgravity and radiation. It has been hypothesized that these synergistic effects might be caused by an interference of microgravity with DNA repair processes. However, recent studies on bacteria, yeast cells and human fibroblasts suggest that a disturbance of cellular repair processes in the microgravity environment might not be a complete explanation for the reported synergism of radiation and microgravity. As an alternative explanation, an impact of microgravity on signal transduction, on the metabolic/physiological state or on the chromatin structure at the cellular level, or modification of self-assembly, intercellular communication, cell migration, pattern formation or differentiation at the tissue and organ level should be considered.     

微重力条件下细胞培养和组织工程研究进展

随着空间生命科学研究的发展,人们将细胞、组织培养技术与微重力环境相结合产生了组织工程研究的一个新领域——微重力组织工程。模拟微重力条件下细胞培养和组织构建研究表明,微重力环境有利于细胞的三维生长,形成具有功能的组织样结构,培养后的三维组织无论从形态上还是基因表达上都更接近于正常的机体组织。这种微重力对细胞的作用效应,将可能为未来组织工程和再生医学研究提供一条新途径。该文概述了近十年来国内外微重力组织工程相关研究的最新进展。     

Advanced bioreactor with controlled application of multi-dimensional strain for tissue engineering

Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues. To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control. We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function. Well-controlled mechanical strains (resolution of < 0.1 micron for translational and < 0.1 degree for rotational strain) and dissolved oxygen tension (between 0%-95% +/- 1%) could be applied to the developing tissue, while maintaining temperature at 37 +/- 0.2 degrees C about developing tissue over prolonged periods of operation. A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90 degrees rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port. Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature (37 +/- 0.2 degrees C), pH (7.4 +/- 0.02), and pO2 (20 +/- 0.5%) over 14 days in culture. The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).     

Effects of clinostat-microgravity on bone and calcium metabolism in rats

Decreases in bone minerals and tissue volume after space flight have been observed in humans and animals, with a variety of results. Such data obtained from space flight experiments have given unsatisfactory results due to short periods of space flight and differences in age, body weights, and strain of animals used. Therefore, ground-based animal models have been developed in order to elucidate changes in bone affected by space flight. For example, a tail-suspended rat model has been established to study the effects of microgravity on bones by producing hind limb unloading. However, problems with this model due to the remaining forelimb loading and the unusual changes in blood current require the development of a new model simulating the physiological conditions of space flight. So we developed a three-dimension clinostat as an apparatus to produce a simulated microgravity similar to space flight by rotating rats equally in all directions. The purpose of the present study is to examine the effects of clinostat-microgravity on bone metabolism in rats.     

3D Bone tissue engineered with bioactive microspheres in simulated microgravity

Three-dimensional (3D) osteoblast cell cultures were obtained in rotating-wall vessels (RWV), simulating microgravity. Three types of bioactive microcarriers, specifically modified bioactive glass particles, bioceramic hollow microspheres, and biodegradable bioactive glass-polymer composite microspheres, were developed and used with osteoblasts. The surfaces of composite microspheres fully transformed into bone apatite after 2-wk immersion in simulated physiological fluid, which demonstrated their bone-bonding ability. The motion of microcarriers in RWVs was photographically recorded and numerically analyzed. The trajectories of hollow microspheres showed that they migrated and eventually stayed around at the central region of the RWV. At their surfaces, shear stresses were low. In contrast, solid glass or polymer particles moved toward and finally bounced off the outer wall of the RWVs. Cell culture studies in the RWV using bone marrow stromal cells showed that the cells attached to and formed 3D aggregates with the hollow microspheres. Extracellular matrix and mineralization were observed in the aggregates. Cell culture studies also confirmed the ability of the composite microspheres to support 3D bone-like tissue formation. These data suggest that the new hollow bioceramic microspheres and degradable composite microspheres can be used as microcarriers for 3D bone tissue engineering in microgravity. They also have potential applications as drug delivery systems.     

Mechanisms of gravity-dependent changes in the bone tissue

The most typical changes for the bone under the space flight conditions and a long-term hypokinesia are the following: the decreasing in bone mass, the demineralization and a reducing of a mechanical strength. It can lead to osteopenia and osteoporosis development. Also it increases the risk of fractures of supporting bones. Osteopenies, caused by the microgravity, are partially connected with the increasing of a reduction of trabecular bones. [Cytological mechanisms of gravity-dependent reactions in a bone tissue remain in many respects not clear. The study purpose was the analysis of some ultrastructural changes in bone tissue cells of the monkeys (Macaca mulatta), staying during 2 weeks onboard the biosatellite "Bion-11".     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

The Effect of the Microgravity Rotating Culture System on the Chondrogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

We investigated the influence of the microgravity rotating culture system on the chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs). During chondrogenic induction, MSCs combined with polyglycolic acid (PGA) were cultured by static culture or microgravity rotating culture and chondrocyte formation was confirmed by toluidine blue staining. Furthermore, the mRNA and protein expressions of a specific cartilage extracellular matrix protein (collagen type II and Aggrecan) were evaluated by real-time RT-PCR and western blot, respectively. Toluidine blue staining indicated the OD values of proteoglycans semi-determination were higher in the microgravity rotating culture group than the static culture group. Following chondrogenic induction, mRNA and proteins of collagen type II and Aggrecan were more significantly expressed in cells of the microgravity rotating culture group compared with the controls. Compared with routine three-dimensional static culture, the microgravity rotating culture system was more effective for the construction of tissue-engineered cartilage in vitro.     

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.