Sexual dimorphism during integrative endocrine and immune responses to ionizing radiation in mice.

Exposure to cosmic ionizing radiation is an innate risk of the spaceflight environment that can cause DNA damage and altered cellular function. In astronauts, longitudinal monitoring of physiological systems and interactions between these systems are important to consider for mitigation strategies. In addition, assessments of sex-specific biological responses in the unique environment of spaceflight are vital to support future exploration missions that include both females and males. Here we assessed sex-specific, multi-system immune and endocrine responses to simulated cosmic radiation. For this, 24-week-old, male and female C57Bl/6J mice were exposed to simplified five-ion, space-relevant galactic cosmic ray (GCRsim) radiation at 15 and 50 cGy, to simulate predicted radiation exposures that would be experienced during lunar and Martian missions, respectively. Blood and adrenal tissues were collected at 3- and 14-days post-irradiation for analysis of immune and endocrine biosignatures and pathways. Sexually dimorphic adrenal gland weights and morphology, differential total RNA expression with corresponding gene ontology, and unique immune phenotypes were altered by GCRsim. In brief, this study offers new insights into sexually dimorphic immune and endocrine kinetics following simulated cosmic radiation exposure and highlights the necessity for personalized translational approaches for astronauts during exploration missions.

The Impact of Microgravity on Immunological States.

As we explore other planetary bodies, astronauts will face unique environmental and physiological challenges. The human immune system has evolved under Earth's gravitational force. Consequently, in the microgravity environment of space, immune function is altered. This can pose problematic consequences for astronauts on deep space missions where medical intervention will be limited. Studying the unique environment of microgravity has its challenges, yet current research has uncovered immunological states that are probable during exploration missions. As microgravity-induced immune states are uncovered, novel countermeasure developments and personalized mitigation programs can be designed to improve astronaut health. This can also benefit immune-related monitoring programs for disorders on Earth. This is a comprehensive review, including gaps in knowledge, of simulated and spaceflight microgravity studies in human and rodent models.

Astrocytes regulate vascular endothelial responses to simulated deep space radiation in a human organ-on-a-chip model.

Central nervous system (CNS) damage by galactic cosmic ray radiation is a major health risk for human deep space exploration. Simulated galactic cosmic rays or their components, especially high Z-high energy particles such as 56Fe ions, cause neurodegeneration and neuroinflammation in rodent models. CNS damage can be partially mediated by the blood-brain barrier, which regulates systemic interactions between CNS and the rest of the body. Astrocytes are major cellular regulators of blood-brain barrier permeability that also modulate neuroinflammation and neuronal health. However, astrocyte roles in regulating CNS and blood-brain barrier responses to space radiation remain little understood, especially in human tissue analogs. In this work, we used a novel high-throughput human organ-on-a-chip system to evaluate blood-brain barrier impairments and astrocyte functions 1-7 days after exposure to 600 MeV/n 56Fe particles and simplified simulated galactic cosmic rays. We show that simulated deep space radiation causes vascular permeability, oxidative stress, inflammation and delayed astrocyte activation in a pattern resembling CNS responses to brain injury. Furthermore, our results indicate that astrocytes have a dual role in regulating radiation responses: they exacerbate blood-brain barrier permeability acutely after irradiation, followed by switching to a more protective phenotype by reducing oxidative stress and pro-inflammatory cytokine and chemokine secretion during the subacute stage.

Differential Single Cell Responses of Embryonic Stem Cells Versus Embryoid Bodies to Gravity Mechanostimulation.

The forces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. Conversely, disuse on Earth or during spaceflight, reduces normal mechanical loading of organisms, resulting in altered cell and tissue function. Although gravity mechanical loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how distinct cell types respond to gravity mechanostimulation during early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-min pulse of 50 g hypergravity (HG)/5 kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the embryoid body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the HG pulse. Single cell t-distributed stochastic neighbor mapping shows limited transcriptome shifts in response to the HG pulse in either ESCs or EBs; this pulse however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program. More specifically, HG resulted in upregulation of self-renewal and angiogenesis genes in ESCs, while in EBs, HG loading was associated with upregulation of Gene Ontology-pathways for multicellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs show HG promotes maintenance of transitory cell phenotypes in early development; including EB cluster co-expression of markers for progenitor, post-implant epiblast, and primitive endoderm phenotypes with HG pulse but expression exclusivity in the non-pulsed clusters. Pseudotime analysis identified three branching cell types susceptible to HG induction of cell fate decisions. In totality, this study provides novel evidence that ESC maintenance and EB development can be regulated by gravity mechanostimulation and that stem cells committed to a differentiation program are more sensitive to gravity-induced changes to their transcriptome.

Cdkn1a deletion or suppression by cyclic stretch enhance the osteogenic potential of bone marrow mesenchymal stem cell-derived cultures.

CDKN1A/P21 is a potent inhibitor of cell cycle progression and its overexpression is thought to be associated with inhibition of normal bone regenerative osteogenesis during spaceflight. To test whether CDKN1A/P21 regulates osteogenesis in response to mechanical loading we studied cyclic stretch versus static culture of Cdkn1a-/- (null) or wildtype primary mouse bone marrow osteoprogenitors during 21-day ex-vivo mineralization assays. Cyclically stretched Cdkn1a-/- cells are 3.95-fold more proliferative than wildtype, while static Cdkn1a-/- cells show a 2.50-fold increase. Furthermore, stage-specific single cell RNAseq analyses show expression of Cdkn1a is strongly suppressed by cyclic stretch in early and late osteoblasts, and minimally in the progenitor population. Lastly, both stretch and/or Cdkn1a deletion cause population shift from osteoprogenitors to osteoblasts, also indicating increased differentiation. Collectively, our results support the hypothesis that Cdkn1a constitutively plays a mechano-reversible anti-proliferative role during osteogenesis and suggests a new molecular target to counter regenerative deficits caused by disuse.

Mechanobiological Assessment of TMJ Disc Surfaces: Nanoindentation and Transmission Electron Microscopy.

OBJECTIVES: Temporomandibular disc is a mechanically robust fibrocartilage tissue exhibiting highly elastic compressive, shear, and tensile moduli with structurally dense extracellular matrix that supports functional loading of the joint. The aim of this study was to illustrate structural complexities of the superior and inferior disc surfaces, to demonstrate the robust mechanical ability of the disc as a whole may be due to depth-dependent regional/layered variation, and also to provide characterization data imperative for future tissue engineering efforts focused on restoring function to the joint. MATERIAL AND METHODS: Nanoindentation was used to assess tissue zones in conjunction with detailed Transmission Electron Microscopy to define structural attributes that influence the temporomandibular disc function. RESULTS: The disc architecture adjacent to the superior surface was shown to have three distinct regional segments within the interface layer: 1-a surface peripheral layer; 2-subsurface region; and 3-a layer of helical matrix bundles. The inferior surface displayed an interface layer (20 µm) that showed limited cell populations with little depth-dependent structural variation, a stiffer elastic modulus and reduced energy dissipation compared to the superior surface. These data indicate that the primary function of the inferior surface is resistance to compression rather than load distribution during joint motion. CONCLUSIONS: These are the first works that demonstrate that the superior central surface of the he temporomandibular disc is structured in depth-dependent isometric layers, each of which provides different mechanical function supporting the bulk tissue's properties. From a clinical perspective these data have potential to define regions susceptible to fatigue that may translate to diagnostic criteria to better define the stages of dysfunction.

Engineered microporosity: enhancing the early regenerative potential of decellularized temporomandibular joint discs.

The temporomandibular joint (TMJ) disc is susceptible to numerous pathologies that may lead to structural degradation and jaw dysfunction. The limited treatment options and debilitating nature of severe temporomandibular disorders has been the primary driving force for the introduction and development of TMJ disc tissue engineering as an approach to alleviate this important clinical issue. This study aimed to evaluate the efficacy of laser micropatterning (LMP) ex vivo-derived TMJ disc scaffolds to enhance cellular integration, a major limitation to the development of whole tissue implant technology. LMP was incorporated into the decellularized extracellular matrix scaffold structure using a 40 W CO2 laser ablation system to drill an 8×16 pattern with a bore diameter of 120 µm through the scaffold thickness. Disc scaffolds were seeded with human neonatal-derived umbilical cord mesenchymal stem cells differentiated into chondrocytes at a density of 900 cells per mm(2) and then assessed on days 1, 7, 14, and 21 of culture. Results derived from histology, PicoGreen DNA quantification, and cellular metabolism assays indicate that the LMP scaffolds improve cellular remodeling compared to the unworked scaffold over the 21-day culture period. Mechanical analysis further supports the use of the LMP showing the compressive properties of the LMP constructs closely represent native disc mechanics. The addition of an artificial path of infiltration by LMP culminated in improved chondrocyte adhesion, dispersion, and migration after extended culture aiding in recapitulating the native TMJ disc characteristics.