Space habitats for bioengineering and surgical repair: addressing the requirement for reconstructive and research tissues during deep-space missions.

Numerous technical scenarios have been developed to facilitate a human return to the Moon, and as a testbed for a subsequent mission to Mars. Crews appointed with constructing and establishing planetary bases will require a superior level of physical ability to cope with the operational demands. However, the challenging environments of nearby planets (e.g. geological, atmospheric, gravitational conditions) as well as the lengthy journeys through microgravity, will lead to progressive tissue degradation and an increased susceptibility to injury. The isolation, distance and inability to evacuate in an emergency will require autonomous medical support, as well as a range of facilities and specialised equipment to repair tissue damage on-site. Here, we discuss the design requirements of such a facility, in the form of a habitat that would concomitantly allow tissue substitute production, maintenance and surgical implantation, with an emphasis on connective tissues. The requirements for the individual modules and their operation are identified. Several concepts are assessed, including the presence of adjacent wet lab and medical modules supporting the gradual implementation of regenerative biomaterials and acellular tissue substitutes, leading to eventual tissue grafts and, in subsequent decades, potential tissues/organ-like structures. The latter, currently in early phases of development, are assessed particularly for researching the effects of extreme conditions on representative analogues for astronaut health support. Technical solutions are discussed for bioengineering in an isolated planetary environment with hypogravity, from fluid-gel bath suspended manufacture to cryostorage, cell sourcing and on-site resource utilisation for laboratory infrastructure. Surgical considerations are also discussed.

Trabecular bone organoids: a micron-scale 'humanised' prototype designed to study the effects of microgravity and degeneration.

Bone is a highly responsive organ, which continuously adapts to the environment it is subjected to in order to withstand metabolic demands. These events are difficult to study in this particular tissue in vivo, due to its rigid, mineralised structure and inaccessibility of the cellular component located within. This manuscript presents the development of a micron-scale bone organoid prototype, a concept that can allow the study of bone processes at the cell-tissue interface. The model is constructed with a combination of primary female osteoblastic and osteoclastic cells, seeded onto femoral head micro-trabeculae, where they recapitulate relevant phenotypes and functions. Subsequently, constructs are inserted into a simulated microgravity bioreactor (NASA-Synthecon) to model a pathological state of reduced mechanical stimulation. In these constructs, we detected osteoclastic bone resorption sites, which were different in morphology in the simulated microgravity group compared to static controls. Once encapsulated in human fibrin and exposed to analogue microgravity for 5 days, masses of bone can be observed being lost from the initial structure, allowing to simulate the bone loss process further. Constructs can function as multicellular, organotypic units. Large osteocytic projections and tubular structures develop from the initial construct into the matrix at the millimetre scale. Micron-level fragments from the initial bone structure are detected travelling along these tubules and carried to sites distant from the native structure, where new matrix formation is initiated. We believe this model allows the study of fine-level physiological processes, which can shed light into pathological bone loss and imbalances in bone remodelling.

Organotypic Culture of Bone-Like Structures Using Composite Ceramic-Fibrin Scaffolds.

We have developed an organotypic culture system that allows the production of bone tissue features on a centimeter scale. A composite, calcium phosphate-strained fibrin gel system is able to organize itself in the presence of osteoblastic cells, creating basic hierarchical units as seen in vivo, and can be modified to produce a range of other tissues that require such directional structuring. Constructs evolve over time into multi-compositional structures containing a high mineral content and terminally differentiated, osteocyte-like cells. These tissues can be cultured over extended durations (exceeding 1 year) and are responsive to a variety of chemical and biological agents. The platform can reduce the number of animals used in experimentation by acting as an intermediate stage in which more personalized research conditions can be generated. We provide a thorough description of the protocol used to successfully culture and modify this system, as well as guidance on compositional characterization. © 2019 by John Wiley & Sons, Inc.

A novel method for the collection of nanoscopic vesicles from an organotypic culture model.

Nanovesicles, exosomes and other membrane bound particles excreted by cells are currently gaining research attention since they have been shown to play a significant role in many biologically related processes. Vesicles are now thought to mediate cellular communication, transmission of some diseases and pathologically mediated calcification. Matrix vesicles have long been proposed to be central to the controlled mineralisation of bone. They remain relatively poorly studied, however, since they are challenging to extract from biological media. One difficulty is the presence of a mineral content in comparison to pure lipid vesicles, meaning that standard separation process such as ultracentrifugation are unable to precisely separate on the basis of size or weight. In this paper we report the separation of matrix vesicles from an organotypic bone culture system using a process of immunoprecipitation. Matrix vesicles were extracted using polymeric beads that were modified with an antibody for tissue non-specific alkaline phosphatase (TNALP), a surface marker abundant in bone-derived vesicles. The vesicles isolated were positive for adenosine triphosphate (ATP), the substrate for TNALP and were demonstrated to have a high-binding affinity to type I collagen, the principal collagen type found in bone. This protocol enables more detailed study of the process and regulation of mineralisation.