Among the physiological consequences of extended spaceflight are loss of skeletal muscle and bone mass. One signaling pathway that plays an important role in maintaining muscle and bone homeostasis is that regulated by the secreted signaling proteins, myostatin (MSTN) and activin A. Here, we used both genetic and pharmacological approaches to investigate the effect of targeting MSTN/activin A signaling in mice that were sent to the International Space Station. Wild type mice lost significant muscle and bone mass during the 33 d spent in microgravity. Muscle weights of Mstn^−/− mice, which are about twice those of wild type mice, were largely maintained during spaceflight. Systemic inhibition of MSTN/activin A signaling using a soluble form of the activin type IIB receptor (ACVR2B), which can bind each of these ligands, led to dramatic increases in both muscle and bone mass, with effects being comparable in ground and flight mice. Exposure to microgravity and treatment with the soluble receptor each led to alterations in numerous signaling pathways, which were reflected in changes in levels of key signaling components in the blood as well as their RNA expression levels in muscle and bone. These findings have implications for therapeutic strategies to combat the concomitant muscle and bone loss occurring in people afflicted with disuse atrophy on Earth as well as in astronauts in space, especially during prolonged missions. Significance Among the major health challenges for astronauts during prolonged space travel are loss of muscle mass and loss of bone mass. Here, we investigated the effects of targeting the signaling pathway mediated by the secreted signaling molecules, myostatin and activin A, in mice sent to the International Space Station. We show that targeting this signaling pathway has significant beneficial effects in protecting against both muscle and bone loss in microgravity, suggesting that this strategy may be effective in preventing or treating muscle and bone loss not only in astronauts on prolonged missions but also in people with disuse atrophy on Earth, such as in older adults or in individuals who are bedridden or wheelchair-bound from illness.

Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.

There is significant clinical demand for functional tendon grafts in human and veterinary medicine. Tissue engineering techniques combining cells, scaffolds, and environmental stimuli may circumvent the shortcomings of traditional transplantation processes. In this study, the influence of cyclic mechanical stimulation on graft maturation and cellular phenotype was assessed in an equine model. Decellularized tendon scaffolds from four equine sources were seeded with syngeneic bone marrow‐derived mesenchymal stem cells and subjected to 0%, 3%, or 5% strain at 0.33 Hz for up to 1 h daily for 11 days. Cells cultured at 3% strain integrated deep into their scaffolds, altered extracellular matrix composition, adopted tendon‐like gene expression profiles, and increased construct elastic modulus and ultimate tensile strength to native levels. This bioreactor protocol is therefore suitable for cultivating replacement tendon material or as an in vitro model for studying differentiation of stem cells toward tendon. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:911–918, 2015.

Loss-of-function mutations in the Notch ligand, Jagged1 (Jag1), result in multi-system developmental pathologies associated with Alagille syndrome (ALGS). ALGS patients present with skeletal manifestations including hemi-vertebrae, reduced bone mass, increased fracture incidence and poor bone healing. However, it is not known whether the increased fracture risk is due to altered bone homeostasis (primary) or nutritional malabsorption due to chronic liver disease (secondary). To determine the significance of Jag1 loss in bone, we characterized the skeletal phenotype of two Jag1-floxed conditional knockout mouse models: Prx1-Cre;Jag1(f/f) to target osteoprogenitor cells and their progeny, and Col2.3-Cre;Jag1(f/f) to target mid-stage osteoblasts and their progeny. Knockout phenotypes were compared to wild-type (WT) controls using quantitative micro-computed tomography, gene expression profiling and mechanical testing. Expression of Jag1 and the Notch target genes Hes1 and Hey1 was downregulated in all Jag1 knockout mice. Osteoblast differentiation genes were downregulated in whole bone of both groups, but unchanged in Prx1-Cre;Jag1(f/f) cortical bone. Both knockout lines exhibited changes in femoral trabecular morphology including decreased bone volume fraction and increased trabecular spacing, with males presenting a more severe trabecular osteopenic phenotype. Prx1-Cre;Jag1(f/f) mice showed an increase in marrow mesenchymal progenitor cell number and, counterintuitively, developed increased cortical thickness resulting from periosteal expansion, translating to greater mechanical stiffness and strength. Similar alterations in femoral morphology were observed in mice with canonical Notch signaling disrupted using Prx1-Cre-regulatable dominant-negative mastermind like-protein (dnMAML). Taken together, we report that 1) Jag1 negatively regulates the marrow osteochondral progenitor pool, 2) Jag1 is required for normal trabecular bone formation and 3) Notch signaling through homotypic Jag1 signaling in osteochondral progenitors, but not mature osteoblasts, inhibits periosteal expansion. Therefore, Jag1 signaling within the osteoblast lineage regulates bone metabolism in a compartment-dependent manner. Moreover, loss of Jag1 function in osteoblast lineage cells may contribute to the skeletal phenotype associated with ALGS.

Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

Each year, 33% of US citizens suffer from a musculoskeletal condition that requires medical intervention, with direct medical costs approaching $1 trillion USD per year. Despite the ubiquity of skeletal dysfunction, there are currently limited safe and efficacious bone growth factors in clinical use. Notch is a cell–cell communication pathway that regulates self-renewal and differentiation within the mesenchymal/osteoblast lineage. The principal Notch ligand in bone, Jagged-1, is a potent osteoinductive protein that positively regulates post-traumatic bone healing in animals. This report describes the temporal regulation of Notch during intramembranous bone formation using marrow ablation as a model system and demonstrates decreased bone formation following disruption of Jagged-1 in mesenchymal progenitor cells. Notch gain-of-function using recombinant Jagged-1 protein on collagen scaffolds promotes healing of craniofacial (calvarial) and appendicular (femoral) surgical defects in both mice and rats. Localized delivery of Jagged-1 promotes bone apposition and defect healing, while avoiding the diffuse bone hypertrophy characteristic of the clinically problematic bone morphogenetic proteins. It is concluded that Jagged-1 is a bone-anabolic agent with therapeutic potential for regenerating traumatic or congenital bone defects.