Due to the hierarchical structure of tendon tissue (fascicles→fibers→fibrils), it is essential to investigate morphology and mechanics at the nano-scopic level to better understand whole body function. Previous studies investigating changes in tendon mechanics after exercise/disease/unloading have focused on the whole tendon and fascicle levels, but limited data exists on adaptations at the collagen fibril level. Objective: Provide a new method to study previously frozen tendon tissue by investigating collagen fibril 1) morphology, 2) nano-mechanical properties, and 3) elasticity maps using atomic force microscopy (AFM). Hypothesis: This method will provide a reliable way to assess tendon structure and function at the nano-scale. Methods: C57BL/6J mouse tendons from NASA Rodent Research-1 (-80°C) were chemically separated and mounted onto glass slides then mechanically separated into tendon subunit sheets and left to air-dry. Collagen fibril morphology was tested with a JPK Nanowizard 4a in tapping mode with a Tap300Al-G cantilever (tip radius=10nm, k=40N/m). Samples were then rehydrated for mechanical testing in Quantitative Imaging (QI) mode with an SNL-10D cantilever (tip radius=2nm, k=0.06N/m). Force data acquisition took place in liquid with a set point of 2nN and Z-speed of 40μm/s. Force-microscopy (FM) and structural data were processed using JPK Data Processing Software and Gwyddion, respectively. Data: Tendon collagen fibrils exhibited uniform D-periods across 38 fibril sections (D-period: 66nm±2.1, diameter: 63nm±23.5). Quantitative analysis of the force curves (n=30) along the highest point of the fibril revealed an average Young’s Modulus (YM) of 2.53MPa±0.31. Elasticity maps (n=3) highlighting the distribution of mechanical properties were generated using FM data. Results Summary: Our tissue preparation protocol combined with AFM reliably determines collagen fibril morphology and nano-mechanical properties of mouse tendons that were previously frozen. As evidence, we present systematic YM within and across different data sets. In addition, the YM values were found to be consistent with previous measurements in tendon tissue under similar conditions. Elasticity maps produced by analysis of FM data indicate the similarity of YM values across the tendon surface. Tendon fibrils were found to have similar D-periodicity despite the variation in fibril size, which is consistent with previous literature. Conclusions: This research provides a new method to test tendon collagen fibril structure and mechanical properties at the nano-scale, presenting a novel analysis (elasticity maps) showing contrast in YM across the fibril surface. Funding: CSUPERB New Investigator Grant to JRB This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (SRT) have experienced rapid development in recent years. The findings of spaceflight-based scRNA-seq and SRT investigations are likely to improve our understanding of life in space and our comprehension of gene expression in various cell systems and tissue dynamics. However, compared to their Earth-based counterparts, gene expression experiments conducted in spaceflight have not experienced the same pace of development. Out of the hundreds of spaceflight gene expression datasets available, only five used scRNA-seq and two used SRT. One principal factor driving this disparity are the special considerations in tissue preservation that are essential for collecting high-quality data. Advancing cell and tissue preservation protocols during spaceflight can increase the quality of data obtained and enable further scRNA-seq and SRT experiments. Additionally, the growing spectrum of computational tools for SRT can help inform which SRT method is optimal for experiment goals and can assist the selection of preservation methods to use during flight. In this paper we explore the growing importance of scRNA-seq and SRT in space biology and present investigators with the challenges and considerations relevant to robust experimental design.

Abstract In spaceflight experiments, model organisms are used to assess the effects of microgravity on specific biological systems. In many cases, only one biological system is of interest to the Principal Investigator. To maximize the scientific return of experiments, the remaining spaceflight tissue is categorized, documented, and stored in the biobank at NASA Ames Research Center, which is maintained by the Ames Life Science Data Archive (ALSDA). The purpose of this study is to evaluate the state of a sample set of tissues from the ALSDA biobank. Garnering information – such as downstream functional analysis for the generation of omics datasets – from tissues is, in part, dependent on the state of sample preservation. RNA integrity number (RIN) values have been calculated for rodent liver tissues that were part of scientific payloads returned from the International Space Station (ISS). Rat livers from Spacelab Life Sciences 1 (SLS-1) and mouse livers from Commercial Biomedical Test Module 3 (CBTM-3), Rodent Research 1 (RR1), and Rodent Research 3 (RR3) were tested. It was found that mean RIN values from CBTM-3, RR1, and RR3 were suitable for downstream functional analysis (RIN > 5) while the mean RIN value for SLS-1 was not (RIN = 2.5 ± 0.1). Information from this study lays the foundation for future efforts in determining the types of assays that are most appropriate for different tissues in the ALSDA biobank and similar preservation facilities, which would aid in shaping the design of experiments.

Spaceflight causes astronauts to experience an unbalanced level of bone remodeling favoring resorption. The combination of an antiresorptive drug and weightlessness may have negative effects on the mechanical and structural properties of bone by increasing elastic stiffness but diminishing plasticity. At 16 weeks of age, skeletally postpubescent male mice (n = 32, C57BL/6) were evenly divided and subcutaneously given either zoledronate (45 µg/kg) or vehicle (saline, same volume). Three days postinjection, half of each group were divided to undergo 3 weeks of hindlimb unloading or act as a control undergoing normal ambulation (n = 8 for each of the 4 groups). Ex vivo µCT assays showed zoledronate preserving cancellous mineral, bone, and cortical bone. Hindlimb unloading reduced cortical bone. Three-point bending of the femoral mid-shaft showed zoledronate increased stiffness, ultimate force, and total energy (+28%, +17%, +15%, respectively). Hindlimb unloading reduced stiffness but increased postyield displacement, a measurement of plasticity (-29%, +20%, respectively). The combined treatment resulted in increased stiffness, with zoledronate not modulating the hindlimb unloading main effect on postyield displacement. Though stiffness increased, contrary to the hypothesis, the combination of zoledronate and hindlimb unloading did not result in a decreased plasticity.