Engineered Microvessel for Cell Culture in Simulated Microgravity.

As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high-throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.

Increased proliferation and adhesion properties of human dental pulp stem cells in PLGA scaffolds via simulated microgravity.

AIM: To explore the possibility of utilizing a rotary cell culture system (RCCS) to model simulated microgravity and investigate its effects on the proliferation, adhesion, migration and cytoskeletal organization of human dental pulp stem cells (hDPSCs) on poly (lactic-co-glycolic acid) (PLGA) scaffolds. METHODOLOGY: Isolated and identified hDPSCs grown on PLGA scaffolds were exposed to simulated microgravity (SMG) or normal gravity (NG) conditions for 3 days. MTT cell proliferation assays, BrdU incorporation assays, flow cytometry analysis and Western blotting were undertaken to identify the proliferation ability of hDPSCs under SMG conditions. Additionally, immunofluorescence detection, SEM observations and cell migration and adhesion assays were performed to compare adhesion, migration and cytoskeletal changes in hDPCSs subjected to SMG conditions. To further investigate the mechanisms, human pathway-focused matrix and adhesion PCR array analyses were performed. The Student's t-test was used for statistical analyses. RESULTS: SMG promoted proliferation and adhesion, decreased migration and reorganized the cytoskeletal organization of hDPSCs compared with the NG group. PCR array analyses revealed that following SMG treatment, ITGA6 (integrin alpha-6), ITGAV (integrin alpha-V), ITGB1 (integrin beta-1), LAMB1 (laminin beta-1) and TNC (tenascin-C) were significantly upregulated (P < 0.05). CONCLUSIONS: SMG may regulate the behaviour of hDPSCs grown in PLGA scaffolds in an integrin-mediated manner, which may contribute to tooth tissue engineering by increasing their expandability and scaffold adhesion.

Mineralization initiation of MC3T3-E1 preosteoblast is suppressed under simulated microgravity condition.

Microgravity decreases the differentiation of osteoblast. However, as this process is multistage and complex, the mechanism by which microgravity inhibits osteoblast differentiation is still unclear. We have previously found that 24 h acute treatment of simulated microgravity (SM) with a random positioning machine (RPM) significantly inhibited the differentiation of preosteoblasts and have explored whether osteoblasts show different response to microgravity condition at other stages, such as the mineralizing-stage. Murine MC3T3-E1 preosteoblasts induced for osteogenic differentiation for seven days were cultured either under normal gravity or SM conditions for 24 h. SM treatment significantly suppressed mineralized nodule formation. Alkaline phosphatase (ALP) activity was dramatically decreased, and the expression of ALP gene was downregulated. Expression of well-known markers and regulators for osteoblasts differentiation, including osteocalcin (OC), type I collagen α1 (Col Iα1), dentin matrix protein 1 (DMP1) and runt-related transcription factor 2 (Runx2), were downregulated. Western blot analysis showed that the phosphorylated extracellular signal-regulated kinase (p-ERK) level was lower under SM condition. Thus, the initiation of osteoblast mineralization is suppressed by SM condition, and the suppression may be through the regulation of ALP activity and the osteogenic gene expression. ERK signaling might be involved in this process. These results are relevant to the decrease of osteoblast maturation and bone formation under microgravity condition.

Advances in Microgravity Directed Tissue Engineering.

Tissue engineering aims to generate functional biological substitutes to repair, sustain, improve, or replace tissue function affected by disease. With the rapid development of space science, the application of simulated microgravity has become an active topic in the field of tissue engineering. There is a growing body of evidence demonstrating that microgravity offers excellent advantages for tissue engineering by modulating cellular morphology, metabolism, secretion, proliferation, and stem cell differentiation. To date, there have been many achievements in constructing bioartificial spheroids, organoids, or tissue analogs with or without scaffolds in vitro under simulated microgravity conditions. Herein, the current status, recent advances, challenges, and prospects of microgravity related to tissue engineering are reviewed. Current simulated-microgravity devices and cutting-edge advances of microgravity for biomaterials-dependent or biomaterials-independent tissue engineering to offer a reference for guiding further exploration of simulated microgravity strategies to produce engineered tissues are summarized and discussed.

Propagation of Dental and Respiratory Cells and Organs in Microgravity.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

Establishment of three-dimensional tissue-engineered bone constructs under microgravity-simulated conditions.

Bone constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In our work, the relationships between the composition and mechanical properties of engineered bone constructs were studied by culturing bone marrow mesenchymal stem cells (BMSCs) on ceramic bovine bone scaffolds in different environments: static flasks and dynamic culture system in rotating vessels-which was a National Aeronautics and Space Administration-recommended, ground-based, microgravity-simulating system. After 15 days of cultivation, osteogenicity was determined according to DNA and alkaline phosphatase (ALP) analysis. DNA content and ALP were higher for cells grown on dynamic culture. Subsequently, the two kinds of engineered bone constructs were selected for transplantation into Sprague-Dawley rat cranial bone defects. After 24 weeks of in vivo implantation, the engineered bone constructs under dynamic culture were found to repair the defects better, with the engineered constructs showing histologically better bone connection. Thus, this dynamic system provides a useful in vitro model to construct the functional role and effects of osteogenesis in the proliferation, differentiation, and maturation of BMSCs. These findings suggest that the hydrodynamic microgravity conditions in tissue-culture bioreactors can modulate the composition, morphology, and function of the engineered bone.

Ultrasound evaluation of sinus fluid levels in swine during microgravity conditions.

BACKGROUND: Acute rhinosinusitis is a common problem that could occur in space secondary to absence of gravity-dependent drainage or odontogenic or external sources of infection. The purpose of this study was to determine the efficacy of ultrasound to determine sinus fluid distribution levels in swine and to assess the accuracy of ultrasound in the animal during normal and microgravity conditions. METHODS: Anesthetized swine had a catheter placed through a frontal bone window to allow aliquots of a viscous solution to be injected at 1 G (N = 4) or during brief microgravity parabolic flights (N = 4). Ultrasound examinations were performed with a high frequency probe during baseline and fluid-induced conditions. RESULTS: There was a consistent air-fluid level interface seen on ultrasound examination with the injection of 1 ml of fluid during 1-G conditions. Microgravity conditions caused the rapid (< 10 s) dissolution of the air-fluid level associated with dispersion of the fluid to the walIs of the sinus cavity in a uniform fashion. The air-fluid interface was recreated with return to 1 G. CONCLUSIONS: Ultrasound is a reliable diagnostic test for assessing fluid levels; these experiments demonstrate the technique can be used during microgravity conditions with attention to altered fluid behavior in the absence of gravity.

[Probability of gravitational polarization of blood cells].

To study lucigenin-dependent chemiluminescence (ChL), samples of whole heparinized blood and isolated neutrophils of human blood were put in special cells between an indifferent wet dialysis membrane and a plate of solid material (polymer, metal or other) stimulating activation of polymorphonuclear leukocytes and monocytes. Production of ChL-registered O2 active forms depended on whether the stimulant was above or below cells. Search for possible artifacts made it clear that the trigger of the phenomenon is located inside cell or proximately. Some aspects of stimulation were amiss, i.e. independence from adhesive properties of the plate, role of erythrocytes, reversibility of the gravity-driven component in the sequence of processes leading to active oxygen production and others. However, the question as to whether it is cell or surrounding cell media that gets polarized by gravity is still an open question. This phenomenon may have implications for cell functioning in microgravity; under the 1 g conditions, it can facilitate understanding of cell reconstruction brought on by reorientation relative to the gravity vector.

Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations.

Weightlessness is known to effect cellular functions by as yet undetermined processes. Many experiments indicate a role of the cytoskeleton and microtubules. Under appropriate conditions in vitro microtubule preparations behave as a complex system that self-organises by a combination of reaction and diffusion. This process also results in the collective transport and organisation of any colloidal particles present. In large centimetre-sized samples, self-organisation does not occur when samples are exposed to a brief early period of weightlessness. Here, we report both space-flight and ground-based (clinorotation) experiments on the effect of weightlessness on the transport and segregation of colloidal particles and chromosomes. In centimetre-sized containers, both methods show that a brief initial period of weightlessness strongly inhibits particle transport. In miniature cell-sized containers under normal gravity conditions, the particle transport that self-organisation causes results in their accumulation into segregated regions of high and low particle density. The gravity dependence of this behaviour is strongly shape dependent. In square wells, neither self-organisation nor particle transport and segregation occur under conditions of weightlessness. On the contrary, in rectangular canals, both phenomena are largely unaffected by weightlessness. These observations suggest, depending on factors such as cell and embryo shape, that major biological functions associated with microtubule driven particle transport and organisation might be strongly perturbed by weightlessness.

Three-dimensional simulated microgravity culture improves the proliferation and odontogenic differentiation of dental pulp stem cell in PLGA scaffolds implanted in mice.

Tooth regeneration through stem cell-based therapy is a promising treatment for tooth decay and loss. Human dental pulp stem cells (hDPSCs) have been widely identified as the stem cells with the most potential for tooth tissue regeneration. However, the culture of hDPSCs in vitro for tissue engineering is challenging, as cells may proliferate slowly or/and differentiate poorly in vivo. Dynamic three­dimensional (3D) simulated microgravity (SMG) created using the rotary cell culture system is considered to an effective tool, which contributes to several cell functions. Thus, the present study aimed to investigate the effect of dynamic 3D SMG culture on the proliferation and odontogenic differentiation abilities of hDPSCs in poly (lactic­co­glycolic acid) (PLGA) scaffolds in nude mice. The hDPSCs on PLGA scaffolds were maintained separately in the 3D SMG culture system and static 3D cultures with osteogenic medium for 7 days in vitro. Subsequently, the cell­PLGA complexes were implanted subcutaneously on the backs of nude mice for 4 weeks. The results of histological and immunohistochemical examinations of Ki­67, type I collagen, dentin sialoprotein and DMP­1 indicated that the proliferation and odontogenic differentiation abilities of the hDPSCs prepared in the 3D SMG culture system were higher, compared with those prepared in the static culture system. These findings suggested that dynamic 3D SMG culture likely contributes to tissue engineering by improving the proliferation and odontogenic differentiation abilities of hDPSCs in vivo.

Aerobic anti-gravity exercise in patients with Charcot-Marie-Tooth disease types 1A and X: A pilot study.

Background: Charcot-Marie-Tooth (CMT) disease is a hereditary neuropathy associated with impaired walking capacity. Some patients are too weak in the lower extremity muscles to walk at gravity with sufficient intensity or duration to gain benefit. Aim: The aim was to investigate the effect of aerobic anti-gravity exercise in weak patients with CMT 1A and X. Methods: Five adult patients performed moderate-intensity aerobic anti-gravity exercise 3/week for 10 weeks. Results: There was a significant positive difference in Berg balance scale and postural stability test between test occasions, and walking distance in the 6-min walk test trended to increase. Conclusions: The study indicates that the anti-gravity treadmill training of patients with CMT should be pursued in larger CMT cohorts.

[Fractionation of hydrogen stable isotopes in the human body].

Fractionation of hydrogen stable isotopes was studied in 9 human subjects in a chamber with normal air pressure imitating a space cabin. Mass-spectrometry of isotopes in blood, urine, saliva, and potable water evidenced increases in the contents of heavy H isotope (deuterium) in the body liquids as compared with water. These results support one of the theories according to which the human organism eliminates heavy stable isotopes of biogenous chemical elements.

Craniomandibular System and Postural Balance after 3-Day Dry Immersion.

The objective of the study was to determine the influence of simulated microgravity by exposure to dry immersion on the craniomandibular system. Twelve healthy male volunteers participated in a 3-day dry immersion study. Before and immediately after exposure we measured maximal bite force using piezoresistive sensors. The mechanical properties of the jaw and cervical muscles were evaluated before, during, and after dry immersion using MyotonPRO. Because recent studies reported the effects of jaw motor activity on the postural stability of humans, stabilometric measurements of center of pressure were performed before and after dry immersion in two mandibular positions: rest position without jaw clenching, and intercuspidal position during voluntary teeth clenching. Results revealed no significant changes of maximal bite force after dry immersion. All postural parameters were significantly altered by dry immersion. There were however no significant differences in stabilometric data according to mandibular position. Moreover the masseter tonicity increased immediately after the end of dry immersion period. Dry immersion could be used as a valid model for studying the effects of microgravity on human subjects. However, 3 days appear insufficient in duration to evaluate the effects of weightlessness on maximal bite force. Our research suggests a link between postural disturbance after dry immersion and masseter tonicity.

Headache under simulated microgravity is related to endocrine, fluid distribution, and tight junction changes.

Head-down-tilted bed rest (HDTBR) induces headaches similar to headaches during space flights. The objective of this investigation was to study hematological, endocrinological, fluid changes and tight junctions in HDTBR-induced headaches as a proxy for space headache. The randomized crossover HDTBR design by the European Space Agency included 12 healthy, nonheadache male subjects. Before, during, and after confined HDTBR periods, epinephrine (urine), cortisol (saliva), hematological, endothelium markers, and fluid distribution parameters were measured. Headaches were assessed with a validated headache questionnaire. Compared with baseline, HDTBR in all subjects was associated with higher hematocrit, hemoglobin, and epinephrine levels, higher erythrocyte counts, and lower relative plasma volumes (all P < 0.05). In total, 26 headache episodes occurred. In subjects with headaches during HDTBR, epinephrine levels were exaggerated (vs headache-free subjects; HDTBR day 3; 5.1 ± 1.7 vs 3.4 ± 2.4; P = 0.023), cortisol levels were decreased (vs headache-free subjects; HDTBR day 1; 0.37 ± 0.16 vs 0.50 ± 0.20; P < 0.001) and the tight junction marker zonulin was elevated (vs headache-free subjects in HDTBR days 1, 3, 5; P < 0.05). HDTBR induces hemoconcentration and fluid redistribution in all subjects. During headache episodes, endocrinological changes, fluid distribution, and tight junctions were more pronounced, suggesting an additional role in headache pathophysiology.

[Effects of simulated weightlessness on rats mandible, lumbar vertebra and femur].

OBJECTIVE: To observe the effect of weightlessness simulation on rats mandible, lumbar vertebra and femur. METHOD: Twenty five Wistar rats were randomly arranged into control group (n=10) and tail-suspension group (n=15 rats). The experiment lasted for 28 d. Then the mandible, lumbar vertebra and femur were excised and the histological structure of condylar process of mandible, molar and premolar area of mandible body, first lumbar, head, middle segment and condyle of femur were examined. RESULT: After tail-suspension there was no distinct change in bone density and bone mass of condylar process. But the degree of interlacement of trabecular bone increased. The bone substance of mandible was very dense in both control and experimental groups. There was no stimulated difference of bone structure and bone mass between the two groups. There was no marked change in the thickness of periodontal membrane. The bone lamella in premolar area of experimental group arranged regularly. And its maturity was higher than that in molar area. The line of bone hyperplasia in molar area of experimental group arranged disorderly and irregularly, which means that much bone remodeling occurred. The component of trabecular bone decreased in femur and lumbar vertebra and the thickness was uneven. The interlacement and connection among trabecular bones was poor. CONCLUSION: Four weeks of stimulated weightlessness can lead to osteoporosis in acantha and femur [correction of feurar] but has no distinct effect on mandible.

Effect of magnetic field on the crystalline structure of magnetostrictive TbFe2 alloy solidified unidirectionally in microgravity.

We performed unidirectional solidification experiments on TbFe(2) alloy in a static magnetic field in microgravity of 10(-4) g for 10 sec obtained by a 490 m free fall of the Japan microgravity center (JAMIC). When the magnetic field strength was increased from zero to 4.5 x 10(-2) T during unidirectional solidification in microgravity, a [1 1 1] crystallographic alignment dominated, and the maximum magnetostriction constant increased from 1,000 ppm to 4,000 ppm. For unidirectional solidification in normal gravity, the maximum magnetostriction constant remained at 2,000 ppm with increasing magnetic field. The columnar structure grows and orients along the magnetic field. TbFe(2) crystals grow in microgravity predominantly in the same direction as the magnetic field.

Chest wall mechanics in sustained microgravity.

We assessed the effects of sustained weightlessness on chest wall mechanics in five astronauts who were studied before, during, and after the 10-day Spacelab D-2 mission (n = 3) and the 180-day Euromir-95 mission (n = 2). We measured flow and pressure at the mouth and rib cage and abdominal volumes during resting breathing and during a relaxation maneuver from midinspiratory capacity to functional residual capacity. Microgravity produced marked and consistent changes (Delta) in the contribution of the abdomen to tidal volume [DeltaVab/(DeltaVab + DeltaVrc), where Vab is abdominal volume and Vrc is rib cage volume], which increased from 30.7 +/- 3. 5 (SE)% at 1 G head-to-foot acceleration to 58.3 +/- 5.7% at 0 G head-to-foot acceleration (P < 0.005). Values of DeltaVab/(DeltaVab + DeltaVrc) did not change significantly during the 180 days of the Euromir mission, but in the two subjects DeltaVab/(DeltaVab + DeltaVrc) was greater on postflight day 1 than on subsequent postflight days or preflight. In the two subjects who produced satisfactory relaxation maneuvers, the slope of the Konno-Mead plot decreased in microgravity; this decrease was entirely accounted for by an increase in abdominal compliance because rib cage compliance did not change. These alterations are similar to those previously reported during short periods of weightlessness inside aircrafts flying parabolic trajectories. They are also qualitatively similar to those observed on going from upright to supine posture; however, in contrast to microgravity, such postural change reduces rib cage compliance.

Deposition and dispersion of 1-micrometer aerosol boluses in the human lung: effect of micro- and hypergravity.

We performed bolus inhalations of 1-micrometer particles in four subjects on the ground (1 G) and during parabolic flights both in microgravity (microG) and in approximately 1.6 G. Boluses of approximately 70 ml were inhaled at different points in an inspiration from residual volume to 1 liter above functional residual capacity. The volume of air inhaled after the bolus [the penetration volume (Vp)] ranged from 200 to 1,500 ml. Aerosol concentration and flow rate were continuously measured at the mouth. The deposition, dispersion, and position of the bolus in the expired gas were calculated from these data. For Vp >/=400 ml, both deposition and dispersion increased with Vp and were strongly gravity dependent, with the greatest deposition and dispersion occurring for the largest G level. At Vp = 800 ml, deposition and dispersion increased from 33.9% and 319 ml in microG to 56.9% and 573 ml at approximately 1.6 G, respectively (P < 0.05). At each G level, the bolus was expired at a smaller volume than Vp, and this volume became smaller with increasing Vp. Although dispersion was lower in microG than in 1 G and approximately 1.6 G, it still increased steadily with increasing Vp, showing that nongravitational ventilatory inhomogeneity is partly responsible for dispersion in the human lung.

Alterations in skeletal perfusion with simulated microgravity: a possible mechanism for bone remodeling.

Bone loss occurs as a consequence of exposure to microgravity. Using the hindlimb-unloaded rat to model spaceflight, this study had as its purpose to determine whether skeletal unloading and cephalic fluid shifts alter bone blood flow. We hypothesized that perfusion would be diminished in the hindlimb bones and increased in skeletal structures of the forelimbs and head. Using radiolabeled microspheres, we measured skeletal perfusion during control standing and after 10 min, 7 days, and 28 days of hindlimb unloading (HU). Femoral and tibial perfusion were reduced with 10 min of HU, and blood flow to the femoral shaft and marrow were further diminished with 28 days of HU. Correspondingly, the mass of femora (-11%, P < 0. 05) and tibiae (-6%, P < 0.1) was lowered with 28 days of HU. In contrast, blood flow to the skull, mandible, clavicle, and humerus was increased with 10 min HU but returned to control levels with 7 days HU. Mandibular (+10%, P < 0.05), clavicular (+18%, P < 0.05), and humeral (+8%, P < 0.1) mass was increased with chronic HU. The data demonstrate that simulated microgravity alters bone perfusion and that such alterations correspond to unloading-induced changes in bone mass. These results support the hypothesis that alterations in bone blood flow provide a stimulus for bone remodeling during periods of microgravity.

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.