Hydrogel mechanical properties in altered gravity.

Exposure to altered gravity influences cellular behaviour in cell cultures. Hydrogels are amongst the most common materials used to produce tissue-engineering scaffolds, and their mechanical properties play a crucial role in cell-matrix interaction. However, little is known about the influence of altered gravity on hydrogel properties. Here we study the mechanical properties of Poly (ethylene glycol) diacrylate (PEGDA) and PEGDA incorporated with graphene oxide (GO) by performing tensile tests in micro and hypergravity during a Parabolic flight campaign, and by comparing them to the same tests performed in Earth gravity. We show that gravity levels do not result in a statistically significant difference in Young's modulus.

Hydrogel Formulation for Biomimetic Fibroblast Cell Culture: Exploring Effects of External Stresses and Cellular Responses.

In the process of tissue engineering, several types of stresses can influence the outcome of tissue regeneration. This outcome can be understood by designing hydrogels that mimic this process and studying how such hydrogel scaffolds and cells behave under a set of stresses. Here, a hydrogel formulation is proposed to create biomimetic scaffolds suitable for fibroblast cell culture. Subsequently, we examine the impact of external stresses on fibroblast cells cultured on both solid and porous hydrogels. These stresses included mechanical tension and altered-gravity conditions experienced during the 83rd parabolic flight campaign conducted by the European Space Agency. This study shows distinct cellular responses characterized by cell aggregation and redistribution in regions of intensified stress concentration. This paper presents a new biomimetic hydrogel that fulfills tissue-engineering requirements in terms of biocompatibility and mechanical stability. Moreover, it contributes to our comprehension of cellular biomechanics under diverse gravitational conditions, shedding light on the dynamic cellular adaptations versus varying stress environments.

How to obtain an integrated picture of the molecular networks involved in adaptation to microgravity in different biological systems?

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

How are cell and tissue structure and function influenced by gravity and what are the gravity perception mechanisms?

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

Modeled microgravity unravels the roles of mechanical forces in renal progenitor cell physiology.

BACKGROUND: The glomerulus is a highly complex system, composed of different interdependent cell types that are subjected to various mechanical stimuli. These stimuli regulate multiple cellular functions, and changes in these functions may contribute to tissue damage and disease progression. To date, our understanding of the mechanobiology of glomerular cells is limited, with most research focused on the adaptive response of podocytes. However, it is crucial to recognize the interdependence between podocytes and parietal epithelial cells, in particular with the progenitor subset, as it plays a critical role in various manifestations of glomerular diseases. This highlights the necessity to implement the analysis of the effects of mechanical stress on renal progenitor cells. METHODS: Microgravity, modeled by Rotary Cell Culture System, has been employed as a system to investigate how renal progenitor cells respond to alterations in the mechanical cues within their microenvironment. Changes in cell phenotype, cytoskeleton organization, cell proliferation, cell adhesion and cell capacity for differentiation into podocytes were analyzed. RESULTS: In modeled microgravity conditions, renal progenitor cells showed altered cytoskeleton and focal adhesion organization associated with a reduction in cell proliferation, cell adhesion and spreading capacity. Moreover, mechanical forces appeared to be essential for renal progenitor differentiation into podocytes. Indeed, when renal progenitors were exposed to a differentiative agent in modeled microgravity conditions, it impaired the acquisition of a complex podocyte-like F-actin cytoskeleton and the expression of specific podocyte markers, such as nephrin and nestin. Importantly, the stabilization of the cytoskeleton with a calcineurin inhibitor, cyclosporine A, rescued the differentiation of renal progenitor cells into podocytes in modeled microgravity conditions. CONCLUSIONS: Alterations in the organization of the renal progenitor cytoskeleton due to unloading conditions negatively affect the regenerative capacity of these cells. These findings strengthen the concept that changes in mechanical cues can initiate a pathophysiological process in the glomerulus, not only altering podocyte actin cytoskeleton, but also extending the detrimental effect to the renal progenitor population. This underscores the significance of the cytoskeleton as a druggable target for kidney diseases.

How do gravity alterations affect animal and human systems at a cellular/tissue level?

The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: "How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?" This is one of the five major scientific issues of the ESA roadmap "Biology in Space and Analogue Environments". Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.

Optimization of an Ex-Vivo Human Skin/Vein Model for Long-Term Wound Healing Studies: Ground Preparatory Activities for the 'Suture in Space' Experiment Onboard the International Space Station.

This study is preliminary to an experiment to be performed onboard the International Space Station (ISS) and on Earth to investigate how low gravity influences the healing of sutured human skin and vein wounds. Its objective was to ascertain whether these tissue explants could be maintained to be viable ex vivo for long periods of time, mimicking the experimental conditions onboard the ISS. We developed an automated tissue culture chamber, reproducing and monitoring the physiological tensile forces over time, and a culture medium enriched with serelaxin (60 ng/mL) and (Zn(PipNONO)Cl) (28 ng/mL), known to extend viability of explanted organs for transplantation. The results show that the human skin and vein specimens remained viable for more than 4 weeks, with no substantial signs of damage in their tissues and cells. As a further clue about cell viability, some typical events associated with wound repair were observed in the tissue areas close to the wound, namely remodeling of collagen fibers in the papillary dermis and of elastic fibers in the vein wall, proliferation of keratinocyte stem cells, and expression of the endothelial functional markers eNOS and FGF-2. These findings validate the suitability of this new ex vivo organ culture system for wound healing studies, not only for the scheduled space experiment but also for applications on Earth, such as drug discovery purposes.

Role of fibroblasts in wound healing and tissue remodeling on Earth and in space.

Wound healing (WH) and the role fibroblasts play in the process, as well as healing impairment and fibroblast dysfunction, have been thoroughly reviewed by other authors. We treat these topics briefly, with the only aim of contextualizing the true focus of this review, namely, the microgravity-induced changes in fibroblast functions involved in WH. Microgravity is a condition typical of spaceflight. Studying its possible effects on fibroblasts and WH is useful not only for the safety of astronauts who will face future interplanetary space missions, but also to help improve the management of WH impairment on Earth. The interesting similarity between microgravity-induced alterations of fibroblast behavior and fibroblast dysfunction in WH impairment on Earth is highlighted. The possibility of using microgravity-exposed fibroblasts and WH in space as models of healing impairment on Earth is suggested. The gaps in knowledge on fibroblast functions in WH are analyzed. The contribution that studies on fibroblast behavior in weightlessness can make to fill these gaps and, consequently, improve therapeutic strategies is considered.

Hypoxia/Ischemia-Induced Rod Microglia Phenotype in CA1 Hippocampal Slices.

The complexity of microglia phenotypes and their related functions compels the continuous study of microglia in diseases animal models. We demonstrated that oxygen-glucose deprivation (OGD) induced rapid, time- and space-dependent phenotypic microglia modifications in CA1 stratum pyramidalis (SP) and stratum radiatum (SR) of rat organotypic hippocampal slices as well as the degeneration of pyramidal neurons, especially in the outer layer of SP. Twenty-four h following OGD, many rod microglia formed trains of elongated cells spanning from the SR throughout the CA1, reaching the SP outer layer where they acquired a round-shaped amoeboid phagocytic head and phagocytosed most of the pyknotic, damaged neurons. NIR-laser treatment, known to preserve neuronal viability after OGD, prevented rod microglia formation. In CA3 SP, pyramidal neurons were less damaged, no rod microglia were found. Thirty-six h after OGD, neuronal damage was more pronounced in SP outer and inner layers of CA1, rod microglia cells were no longer detectable, and most microglia were amoeboid/phagocytic. Damaged neurons, more numerous 36 h after OGD, were phagocytosed by amoeboid microglia in both inner and outer layers of CA1. In response to OGD, microglia can acquire different morphofunctional phenotypes which depend on the time after the insult and on the subregion where microglia are located.

Robot-assisted surgery in space: pros and cons. A review from the surgeon's point of view.

The target of human flight in space has changed from permanence on the International Space Station to missions beyond low earth orbit and the Lunar Gateway for deep space exploration and Missions to Mars. Several conditions affecting space missions had to be considered: for example the effect of weightlessness and radiations on the human body, behavioral health decrements or communication latency, and consumable resupply. Telemedicine and telerobotic applications, robot-assisted surgery with some hints on experimental surgical procedures carried out in previous missions, had to be considered as well. The need for greater crew autonomy in health issues is related to the increasing severity of medical and surgical interventions that could occur in these missions, and the presence of a highly trained surgeon on board would be recommended. A surgical robot could be a valuable aid but only inasfar as it is provided with multiple functions, including the capability to perform certain procedures autonomously. Space missions in deep space or on other planets present new challenges for crew health. Providing a multi-function surgical robot is the new frontier. Research in this field shall be paving the way for the development of new structured plans for human health in space, as well as providing new suggestions for clinical applications on Earth.

Effect of space flight on the behavior of human retinal pigment epithelial ARPE-19 cells and evaluation of coenzyme Q10 treatment.

Astronauts on board the International Space Station (ISS) are exposed to the damaging effects of microgravity and cosmic radiation. One of the most critical and sensitive districts of an organism is the eye, particularly the retina, and > 50% of astronauts develop a complex of alterations designated as spaceflight-associated neuro-ocular syndrome. However, the pathogenesis of this condition is not clearly understood. In the current study, we aimed to explore the cellular and molecular effects induced in the human retinal pigment ARPE-19 cell line by their transfer to and 3-day stay on board the ISS in the context of an experiment funded by the Agenzia Spaziale Italiana. Treatment of cells on board the ISS with the well-known bioenergetic, antioxidant, and antiapoptotic coenzyme Q10 was also evaluated. In the ground control experiment, the cells were exposed to the same conditions as on the ISS, with the exception of microgravity and radiation. The transfer of ARPE-19 retinal cells to the ISS and their living on board for 3 days did not affect cell viability or apoptosis but induced cytoskeleton remodeling consisting of vimentin redistribution from the cellular boundaries to the perinuclear area, underlining the collapse of the network of intermediate vimentin filaments under unloading conditions. The morphological changes endured by ARPE-19 cells grown on board the ISS were associated with changes in the transcriptomic profile related to the cellular response to the space environment and were consistent with cell dysfunction adaptations. In addition, the results obtained from ARPE-19 cells treated with coenzyme Q10 indicated its potential to increase cell resistance to damage.

Effect of Microgravity on Endothelial Cell Function, Angiogenesis, and Vessel Remodeling During Wound Healing.

Wound healing is a complex phenomenon that involves different cell types with various functions, i.e., keratinocytes, fibroblasts, and endothelial cells, all influenced by the action of soluble mediators and rearrangement of the extracellular matrix (ECM). Physiological angiogenesis occurs in the granulation tissue during wound healing to allow oxygen and nutrient supply and waste product removal. Angiogenesis output comes from a balance between pro- and antiangiogenic factors, which is finely regulated in a spatial and time-dependent manner, in order to avoid insufficient or excessive nonreparative neovascularization. The understanding of the factors and mechanisms that control angiogenesis and their change following unloading conditions (in a real or simulated space environment) will allow to optimize the tissue response in case of traumatic injury or medical intervention. The potential countermeasures under development to optimize the reparative angiogenesis that contributes to tissue healing on Earth will be discussed in relation to their exploitability in space.

NIR Laser Photobiomodulation Induces Neuroprotection in an In Vitro Model of Cerebral Hypoxia/Ischemia.

Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm2; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

Effect of NIR Laser Therapy by MLS-MiS Source on Fibroblast Activation by Inflammatory Cytokines in Relation to Wound Healing.

The fine control of inflammation following injury avoids fibrotic scars or impaired wounds. Due to side effects by anti-inflammatory drugs, the research is continuously active to define alternative therapies. Among them, physical countermeasures such as photobiomodulation therapy (PBMT) are considered effective and safe. To study the cellular and molecular events associated with the anti-inflammatory activity of PBMT by a dual-wavelength NIR laser source, human dermal fibroblasts were exposed to a mix of inflammatory cytokines (IL-1ß and TNF-α) followed by laser treatment once a day for three days. Inducible inflammatory key enzymatic pathways, as iNOS and COX-2/mPGES-1/PGE2, were upregulated by the cytokine mix while PBMT reverted their levels and activities. The same behavior was observed with the proangiogenic factor vascular endothelial growth factor (VEGF), involved in neovascularization of granulation tissue. From a molecular point of view, PBMT retained NF-kB cytoplasmatic localization. According to a change in cell morphology, differences in expression and distribution of fundamental cytoskeletal proteins were observed following treatments. Tubulin, F-actin, and α-SMA changed their organization upon cytokine stimulation, while PBMT reestablished the basal localization. Cytoskeletal rearrangements occurring after inflammatory stimuli were correlated with reorganization of membrane α5ß1 and fibronectin network as well as with their upregulation, while PBMT induced significant downregulation. Similar changes were observed for collagen I and the gelatinolytic enzyme MMP-1. In conclusion, the present study demonstrates that the proposed NIR laser therapy is effective in controlling fibroblast activation induced by IL-1ß and TNF-α, likely responsible for a deleterious effect of persistent inflammation.

Effect of Unloading Condition on the Healing Process and Effectiveness of Platelet Rich Plasma as a Countermeasure: Study on In Vivo and In Vitro Wound Healing Models.

Wound healing is a very complex process that allows organisms to survive injuries. It is strictly regulated by a number of biochemical and physical factors, mechanical forces included. Studying wound healing in space is interesting for two main reasons: (i) defining tools, procedures, and protocols to manage serious wounds and burns eventually occurring in future long-lasting space exploration missions, without the possibility of timely medical evacuation to Earth; (ii) understanding the role of gravity and mechanical factors in the healing process and scarring, thus contributing to unravelling the mechanisms underlying the switching between perfect regeneration and imperfect repair with scarring. In the study presented here, a new in vivo sutured wound healing model in the leech (Hirudo medicinalis) has been used to evaluate the effect of unloading conditions on the healing process and the effectiveness of platelet rich plasma (PRP) as a countermeasure. The results reveal that microgravity caused a healing delay and structural alterations in the repair tissue, which were prevented by PRP treatment. Moreover, investigating the effects of microgravity and PRP on an in vitro wound healing model, it was found that PRP is able to counteract the microgravity-induced impairment in fibroblast migration to the wound site. This could be one of the mechanisms underlying the effectiveness of PRP in preventing healing impairment in unloading conditions.

Effect of NIR laser therapy by MLS-MiS source against neuropathic pain in rats: in vivo and ex vivo analysis.

Neuropathic pain is characterized by an uncertain etiology and by a poor response to common therapies. The ineffectiveness and the frequent side effects of the drugs used to counteract neuropathic pain call for the discovery of new therapeutic strategies. Laser therapy proved to be effective for reducing pain sensitivity thus improving the quality of life. However, its application parameters and efficacy in chronic pain must be further analyzed. We investigated the pain relieving and protective effect of Photobiomodulation Therapy in a rat model of compressive mononeuropathy induced by Chronic Constriction Injury of the sciatic nerve (CCI). Laser (MLS-MiS) applications started 7 days after surgery and were performed ten times over a three week period showing a reduction in mechanical hypersensitivity and spontaneous pain that started from the first laser treatment until the end of the experiment. The ex vivo analysis highlighted the protective role of laser through the myelin sheath recovery in the sciatic nerve, inhibition of iNOS expression and enhancement of EAAT-2 levels in the spinal cord. In conclusion, this study supports laser treatment as a future therapeutic strategy in patients suffering from neuropathic pain induced by trauma.

Glyoxylate cycle activity in Pinus pinea seeds during germination in altered gravity conditions.

This work inserts in the research field regarding the effects of altered gravity conditions on biological plant processes. Pinus pinea seeds germination was studied in simulated microgravity (2x10-3g) and hypergravity (20g) conditions. The effects of simulated gravity were evaluated monitoring the levels of the key enzymes, involved in the main metabolic pathway during germination process of lipid-rich seeds (oilseeds): isocitrate lyase and malate synthase for glyoxylate cycle, 3-hydroxyacyl-CoA dehydrogenase for beta-oxidation, isocitrate dehydrogenase for Krebs cycle, pyruvate kinase for glycolysis and glucose 6 phosphate dehydrogenase for pentose phosphate shunt. The simulated micro and hypergravity conditions were obtained by a Random Position Machine and a Hyperfuge, respectively. Results show that the levels of some tested enzymes, at different lag times of the germination process, have the same trend of controls (g = 1), but with significant differences from quantitative point of view. They are higher in microgravity conditions and lower in hypergravity ones, suggesting that, from a biochemical point of view, the germination process results accelerated in microgravity conditions and delayed in hypergravity ones. These biochemical results show a good correlation with morphological ones, obtained with the measurement of the length of the seeds sprouting radicle. These results give promising indications regarding the possibility to grow plant with lipid-rich seeds in spatial environment, to obtain food sources for astronauts during long term space missions and to reconstitute new atmosphere.

Photobiomodulation therapy by NIR laser in persistent pain: an analytical study in the rat.

Over the past three decades, physicians have used laser sources for the management of different pain conditions obtaining controversial results that call for further investigations. In order to evaluate the pain relieving possibilities of photobiomodulation therapy (PBMT), we tested two near infrared (NIR) laser systems, with different power, against various kinds of persistent hyperalgesia animal models. In rats, articular pain was reproduced by the intra-articular injection of sodium monoiodoacetate (MIA) and complete Freund's adjuvant (CFA), while compressive neuropathy was modelled by the chronic constriction injury of the sciatic nerve (CCI). In MIA and CFA models, (NIR) laser (MLS-Mphi, ASA S.r.l., Vicenza, Italy) application was started 14 days after injury and was performed once a day for a total of 13 applications. In MIA-treated animals, the anti-hyperalgesic effect of laser began 5 min after treatment and vanished after 60 min. The subsequent applications evoked similar effects. In CFA-treated rats, laser efficacy started 5 min after treatment and disappeared after 180 min. In rats that underwent CCI, two treatment protocols with similar fluence but different power output were tested using a new experimental device called Multiwave Locked System laser (MLS-HPP). Treatments began 7 days after injury and were performed during 3 weeks for a total of 10 applications. Both protocols reduced mechanical hyperalgesia and hindlimb weight bearing alterations until 60 min after treatment with a higher efficacy recorded for the animals treated using the higher power output. In conclusion, this study supports laser therapy as a potential treatment for immediate relief of chronic articular or neuropathic pain.

Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts.

Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.