The effects of neurectomy and hibernation on bone properties and the endocannabinoid system in marmots (Marmota flaviventris).

Hibernators have adapted a physiological mechanism allowing them to undergo long periods of inactivity without experiencing bone loss. However, the biological mechanisms that prevent bone loss are unknown. Previous studies found meaningful changes, between active and hibernating marmots, in the endocannabinoid system of many tissues, including bone. Cannabinoid receptors (CB1 and CB2) have divergent localization in bone. CB1 is predominately found on sympathetic nerve terminals, while CB2 is more abundant on bone cells and their progenitors. This study aimed to determine the contribution of innervation on endocannabinoid regulation of bone properties in hibernating (during torpor) and non-hibernating yellow-bellied marmots. Neurectomy, a model for disuse osteoporosis, was performed unilaterally in both hibernating and active marmots. Endocannabinoid concentrations were measured in bone marrow, cortical, and trabecular regions from fourth metatarsals of both hindlimbs using microflow chromatography-tandem quadrupole mass spectrometry. Trabecular bone architectural properties of fifth metatarsals were evaluated using micro-computed tomography. There were ligand-specific increases with neurectomy in active, but not hibernating, marmots. Trabecular bone architectural properties were not affected by neurectomy during hibernation, but did show some minor negative changes in active marmots. These findings suggest protection from bone loss in hibernating rodents is peripherally rather than centrally regulated. Furthermore, findings suggest even active marmots with normal metabolism are partially protected from disuse induced bone loss compared to laboratory rodents. Understanding the mechanism hibernators use to maintain bone density may guide development for novel bone loss prevention therapies.

Granular PEG hydrogels mediate osteoporotic MSC clustering via N-cadherin influencing the pro-resorptive bias of their secretory profile.

Postmenopausal osteoporosis results from a pro-resorptive bone environment, which decreases bone mineral density causing increased fracture risk. Bone marrow derived mesenchymal stem/stromal cells (MSCs) secrete factors involved in bone homeostasis, but osteoporosis mediated changes to their secretions remain understudied. Herein, we examined the secretome of MSCs isolated from ovariectomized rats (OVX rMSCs), a model of post-menopausal osteoporosis, as a function of cell-cell interactions. Specifically, we controlled clustering of OVX and SHAM rMSCs by assembling them in granular hydrogels synthesized from poly(ethylene glycol) microgels with average diameters of ∼10, 100, and 200 µm. We directed both the sizes of rMSC clusters (single cells to ∼30 cells/cluster) and the percentages of cells within clusters (∼20-90%) by controlling the scaffold pore dimensions. Large clusters of OVX rMSCs had a pro-resorptive secretory profile, with increased concentrations of Activin A, CXCL1, CX3CL1, MCP-1, TIMP-1, and TNF-ɑ, compared to SHAM rMSCs. As this pro-resorptive bias was only observed in large cell clusters, we characterized the expression of several cadherins, mediators of cell-cell contacts. N-cadherin expression was elevated (∼4-fold) in OVX relative to SHAM rMSCs, in both cell clusters and single cells. Finally, TIMP-1 and MCP-1 secretion was only decreased in large cell clusters of OVX rMSCs when N-cadherin interactions were blocked, highlighting the dependence of OVX rMSC secretion of pro-resorptive cytokines on N-cadherin mediated cell-cell contacts. Further elucidation of the N-cadherin mediated osteoporotic MSC secretome may have implications for developing therapies for postmenopausal osteoporosis. STATEMENT OF SIGNIFICANCE: Postmenopausal osteoporosis is a prevalent bone disorder that affects tens of millions of women worldwide. This disease is characterized by severe bone loss resulting from a pro-resorptive bone marrow environment, where the rates of bone resorption outpace the rates of bone deposition. The paracrine factors secreted by bone marrow MSCs can influence cell types responsible for bone homeostasis, but the osteoporosis-mediated changes to MSC secretory properties remains understudied. In this study, we used PEG-based porous granular scaffolds to study the influence of cell clustering on the secretory properties of osteoporotic MSCs. We observed increased secretion of several pro-resorptive factors by osteoporotic MSCs in large clusters. Further, we explored the dependence of this altered secretion profile on N-cadherin mediated cell-cell contacts.

Osteoporosis prevention in an extraordinary hibernating bear.

Disuse osteoporosis results from physical inactivity. Reduced mechanical loading of bone stimulates bone resorption leading to bone loss, decreased mechanical properties, and increased fracture risk. Compensatory mechanisms evolved in hibernators to preserve skeletal muscle and bone during the prolonged physical inactivity that occurs during annual hibernation. This paper reports the preservation of bone properties in an exceptionally old black bear that was physically inactive for about 6 months annually for 31 years. The biological mechanisms that preserve bone during prolonged disuse during hibernation are also reviewed.

Impact of Release Kinetics on Efficacy of Locally Delivered Parathyroid Hormone for Bone Regeneration Applications.

Characterizing the release profile for materials-directed local delivery of bioactive molecules and its effect on bone regeneration is an important step to improve our understanding of, and ability to optimize, the bone healing response. This study examined the local delivery of parathyroid hormone (PTH) using a thiol-ene hydrogel embedded in a porous poly(propylene fumarate) (PPF) scaffold for bone regeneration applications. The aim of this study was to characterize the degradation-controlled in vitro release kinetics of PTH from the thiol-ene hydrogels, in vivo hydrogel degradation in a subcutaneous implant model, and bone healing in a rat critical size bone defect. Tethering PTH to the hydrogel matrix eliminated the early timepoint burst release that was observed in previous in vitro work where PTH was free to diffuse out of the matrix. Only 8% of the tethered PTH was released from the hydrogel during the first 2 weeks, but by day 21, 80% of the PTH was released, and complete release was achieved by day 28. In vivo implantation revealed that complete degradation of the hydrogel alone occurred by day 21; however, when incorporated in a three-dimensional printed osteoconductive PPF scaffold, the hydrogel persisted for >56 days. Treatment of bone defects with the composite thiol-ene hydrogel-PPF scaffold, delivering either 3 or 10 µg of tethered PTH 1-84, was found to increase bridging of critical size bone defects, whereas treatment with 30 µg of tethered PTH resulted in less bone ingrowth into the defect area. Continued development of this biomaterial delivery system for PTH could lead to improved therapies for treatment of nonunion fractures and critical size bone defects.

Thiol-ene Hydrogels for Local Delivery of PTH for Bone Regeneration in Critical Size defects.

Neither allograft nor commercially available bone graft substitutes provide the same quality of bone healing as autograft. Incorporation of bioactive molecules like parathyroid hormone (PTH) within bone graft substitute materials may provide similar, if not better treatment options to grafting. The goal of this work was to develop a biomaterial system for the local delivery of PTH to large bone defects for promoting bone regeneration. PTH was loaded in a thiol-ene hydrogel at several concentrations and polymerized in and around an osteoconductive poly(propylene fumarate) (PPF) scaffold. PTH was shown to be bioactive when released from the hydrogel for up to 21 days. Eighty percent of the PTH was released by day 3 with the remaining 20% released by day 14. Bone healing was quantified in rat critical size femoral defects that were treated with hydrogel/PPF and 0, 1, 3, 10, or 30 µg of PTH. Although complete osseous healing was not observed in all samples in any one treatment group, all samples in the 10 µg PTH group were bridged fully by bone or a combination of bone and cartilage containing hypertrophic chondrocytes and endochondral ossification. Outcome measures indicated improved defect bridging by a combination of bony and cartilaginous tissue in the 10 µg treatment group compared with empty bone defects and defects treated with only hydrogel/PPF (i.e., without PTH). Given the tailorability of the hydrogel, future studies will investigate the effects of prolonged gradual PTH release on bone healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:536-544, 2020.

Parathyroid hormone for bone regeneration.

Delayed healing and/or non-union occur in approximately 5-10% of the fractures that occur annually in the United States. Segmental bone loss increases the probability of non-union. Though grafting can be an effective treatment for segmental bone loss, autografting is limited for large defects since a limited amount of bone is available for harvest. Parathyroid hormone (PTH) is a key regulator of calcium homeostasis in the body and plays an important role in bone metabolism. Presently PTH is FDA approved for use as an anabolic treatment for osteoporosis. The anabolic effect PTH has on bone has led to research on its use for bone regeneration applications. Numerous studies in animal models have indicated enhanced fracture healing as a result of once daily injections of PTH. Similarly, in a human case study, non-union persisted despite treatment attempts with internal fixation, external fixation, and autograft in combination with BMP-7, until off label use of PTH1-84 was utilized. Use of a biomaterial scaffold to locally deliver PTH to a defect site has also been shown to improve bone formation and healing around dental implants in dogs and drill defects in sheep. Thus, PTH may be used to promote bone regeneration and provide an alternative to autograft and BMP for the treatment of large segmental defects and non-unions. This review briefly summarizes the unmet clinical need for improved bone regeneration techniques and how PTH may help fill that void by both systemically and locally delivered PTH for bone regeneration applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2586-2594, 2018.

The challenges of promoting osteogenesis in segmental bone defects and osteoporosis.

Conventional clinical management of complex bone healing scenarios continues to result in 5-10% of fractures forming non-unions. Additionally, the aging population and prevalence of osteoporosis-related fractures necessitate the further exploration of novel ways to augment osteogenesis in this special population. This review focuses on the current clinical modalities available, and the ongoing clinical and pre-clinical research to promote osteogenesis in segmental bone defects, delayed unions, and osteoporosis. In summary, animal models of fracture repair are often small animals as historically significant large animal models, like the dog, continue to gain favor as companion animals. Small rodents have well-documented limitations in comparing to fracture repair in humans, and few similarities exist. Study design, number of studies, and availability of funding continue to limit large animal studies. Osteoinduction with rhBMP-2 results in robust bone formation, although long-term quality is scrutinized due to poor bone mineral quality. PTH 1-34 is the only FDA approved osteo-anabolic treatment to prevent osteoporotic fractures. Limited to 2 years of clinical use, PTH 1-34 has further been plagued by dose-related ambiguities and inconsistent results when applied to pathologic fractures in systematic human clinical studies. There is limited animal data of PTH 1-34 applied locally to bone defects. Gene therapy continues to gain popularity among researchers to augment bone healing. Non-integrating viral vectors and targeted apoptosis of genetically modified therapeutic cells is an ongoing area of research. Finally, progenitor cell therapies and the content variation of patient-side treatments (e.g., PRP and BMAC) are being studied. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1559-1572, 2018.

Seasonal Changes in Endocannabinoid Concentrations between Active and Hibernating Marmots (Marmota flaviventris).

Hibernation is a naturally occurring model for studying diseases such as obesity and osteoporosis. Hibernators, marmots (Marmota flaviventris) among them, are able to nearly double their body mass by increasing fat stores prior to hibernation without the negative consequences of obesity. They are also physically inactive for extended periods of time without experiencing negative effects on the skeleton. The endocannabinoid system is involved in modulating neural signaling, circannual rhythms, behavior, appetite, thermogenesis, and bone and energy metabolism. These systems are also altered to maintain homeostasis during hibernation. This study aims to better understand the involvement of the endocannabinoid system in the regulation of physiological processes during hibernation by quantifying the seasonal variation of endocannabinoids and endocannabinoid-like ligands in both active and hibernating marmots. We hypothesized that there would be significant changes in endocannabinoid concentrations at the tissue level in marmots between active and hibernating states. Concentrations were measured in brain, serum, brown adipose tissue, white adipose tissue, bone marrow, cortical bone, and trabecular bone using microflow chromatography coupled with tandem quadrupole mass spectrometry. Significant changes were found, such as a 30-fold decrease in 2-arachidonoyl glycerol (2-AG) in cortical bone during hibernation. Many endocannabinoid and endocannabinoid-like ligands decreased in brown adipose tissue, white adipose tissue, and cortical bone, while several ligands increased in bone marrow. This result supports our hypothesis and suggests the possibility of a peripherally controlled shift in energy metabolism, reduction in bone metabolism, and suppression of the immune system during hibernation.

Mimicking the effects of spaceflight on bone: Combined effects of disuse and chronic low-dose rate radiation exposure on bone mass in mice.

During spaceflight, crewmembers are subjected to biomechanical and biological challenges including microgravity and radiation. In the skeleton, spaceflight leads to bone loss, increasing the risk of fracture. Studies utilizing hindlimb suspension (HLS) as a ground-based model of spaceflight often neglect the concomitant effects of radiation exposure, and even when radiation is accounted for, it is often delivered at a high-dose rate over a very short period of time, which does not faithfully mimic spaceflight conditions. This study was designed to investigate the skeletal effects of low-dose rate gamma irradiation (8.5 cGy gamma radiation per day for 20 days, amounting to a total dose of 1.7 Gy) when administered simultaneously to disuse from HLS. The goal was to determine whether continuous, low-dose rate radiation administered during disuse would exacerbate bone loss in a murine HLS model. Four groups of 16 week old female C57BL/6 mice were studied: weight bearing + no radiation (WB+NR), HLS + NR, WB + radiation exposure (WB+RAD), and HLS+RAD. Surprisingly, although HLS led to cortical and trabecular bone loss, concurrent radiation exposure did not exacerbate these effects. Our results raise the possibility that mechanical unloading has larger effects on the bone loss that occurs during spaceflight than low-dose rate radiation.

Arctic Ground Squirrels Limit Bone Loss during the Prolonged Physical Inactivity Associated with Hibernation.

Prolonged disuse (e.g., physical inactivity) typically results in increased bone porosity, decreased mineral density, and decreased bone strength, leading to increased fracture risk in many mammals. However, bears, marmots, and two species of ground squirrels have been shown to preserve macrostructural bone properties and bone strength during long seasons of hibernation while they remain mostly inactive. Some small hibernators (e.g., 13-lined ground squirrels) show microstructural bone loss (i.e., osteocytic osteolysis) during hibernation, which is not seen in larger hibernators (e.g., bears and marmots). Arctic ground squirrels (Urocitellus parryii) are intermediate in size between 13-lined ground squirrels and marmots and are perhaps the most extreme rodent hibernator, hibernating for up to 8 mo annually with body temperatures below freezing. The goal of this study was to quantify the effects of hibernation and inactivity on cortical and trabecular bone properties in arctic ground squirrels. Cortical bone geometrical properties (i.e., thickness, cross-sectional area, and moment of inertia) at the midshaft of the femur were not different in animals sampled over the hibernation and active seasons. Femoral ultimate stress tended to be lower in hibernators than in summer animals, but toughness was not affected by hibernation. The area of osteocyte lacunae was not different between active and hibernating animals. There was an increase in osteocytic lacunar porosity in the hibernation group due to increased lacunar density. Trabecular bone volume fraction in the proximal tibia was unexpectedly greater in the hibernation group than in the active group. This study shows that, similar to other hibernators, arctic ground squirrels are able to preserve many bone properties during hibernation despite being physically inactive for up to 8 mo.

The effects of hibernation and forced disuse (neurectomy) on bone properties in arctic ground squirrels.

Bone loss is a well-known medical consequence of disuse such as in long-term space flight. Immobilization in many animals mimics the effects of space flight on bone mineral density. Decreases in metabolism are also thought to contribute to a loss of skeletal mass. Hibernating mammals provide a natural model of disuse and metabolic suppression. Hibernating ground squirrels have been shown to maintain bone strength despite long periods of disuse and decreased metabolism during torpor. This study examined if the lack of bone loss during torpor was a result of the decrease in metabolic rate during torpor or an evolutionary change in these animals affording protection against disuse. We delineated changes in bone density during natural disuse (torpor) and forced disuse (sciatic neurectomy) in the hind limbs of the arctic ground squirrel (AGS) over an entire year. We hypothesized that the animals would be resistant to bone loss due to immobilization and disuse during the winter hibernation season when metabolism is depressed but not the summer active season. This hypothesis was not supported. The animals maintained bone density (dual-energy X-ray absorptiometry) and most bone structural and mechanical properties in both seasons. This was observed in both natural and forced disuse, regardless of the known metabolic rate increase during the summer. However, trabecular bone volume fraction (microcomputed tomography) in the distal femur was lower in neurectomized AGS at the study endpoint. These results demonstrate a need to better understand the relationship between skeletal load (use) and bone density that may lead to therapeutics or strategies to maintain bone density in disuse conditions.

Exploring the Bone Proteome to Help Explain Altered Bone Remodeling and Preservation of Bone Architecture and Strength in Hibernating Marmots.

Periods of physical inactivity increase bone resorption and cause bone loss and increased fracture risk. However, hibernating bears, marmots, and woodchucks maintain bone structure and strength, despite being physically inactive for prolonged periods annually. We tested the hypothesis that bone turnover rates would decrease and bone structural and mechanical properties would be preserved in hibernating marmots (Marmota flaviventris). Femurs and tibias were collected from marmots during hibernation and in the summer following hibernation. Bone remodeling was significantly altered in cortical and trabecular bone during hibernation with suppressed formation and no change in resorption, unlike the increased bone resorption that occurs during disuse in humans and other animals. Trabecular bone architecture and cortical bone geometrical and mechanical properties were not different between hibernating and active marmots, but bone marrow adiposity was significantly greater in hibernators. Of the 506 proteins identified in marmot bone, 40 were significantly different in abundance between active and hibernating marmots. Monoaglycerol lipase, which plays an important role in fatty acid metabolism and the endocannabinoid system, was 98-fold higher in hibernating marmots compared with summer marmots and may play a role in regulating the changes in bone and fat metabolism that occur during hibernation.

Black bears with longer disuse (hibernation) periods have lower femoral osteon population density and greater mineralization and intracortical porosity.

Intracortical bone remodeling is persistent throughout life, leading to age related increases in osteon population density (OPD). Intracortical porosity also increases with age in many mammals including humans, contributing to bone fragility and fracture risk. Unbalanced bone resorption and formation during disuse (e.g., physical inactivity) also increases intracortical porosity. In contrast, hibernating bears are a naturally occurring model for the prevention of both age-related and disuse osteoporoses. Intracortical bone remodeling is decreased during hibernation, but resorption and formation remain balanced. Black bears spend 0.25-7 months in hibernation annually depending on climate and food availability. We found longer hibernating bears demonstrate lower OPD and higher cortical bone mineralization than bears with shorter hibernation durations, but we surprisingly found longer hibernating bears had higher intracortical porosity. However, bears from three different latitudes showed age-related decreases in intracortical porosity, indicating that regardless of hibernation duration, black bears do not show the disuse- or age-related increases in intracortical porosity which is typical of other animals. This ability to prevent increases in intracortical porosity likely contributes to their ability to maintain bone strength during prolonged periods of physical inactivity and throughout life. Improving our understanding of the unique bone metabolism in hibernating bears will potentially increase our ability to develop treatments for age- and disuse-related osteoporoses in humans.

Yellow-bellied marmots (Marmota flaviventris) preserve bone strength and microstructure during hibernation.

Reduced skeletal loading typically results in decreased bone strength and increased fracture risk for humans and many other animals. Previous studies have shown bears are able to prevent bone loss during the disuse that occurs during hibernation. Studies with smaller hibernators, which arouse intermittently during hibernation, show that they may lose bone at the microstructural level. These small hibernators, like bats and squirrels, do not utilize intracortical remodeling. However, slightly larger mammals like marmots do. In this study we examined the effects of hibernation on bone structural, mineral, and mechanical properties in yellow-bellied marmots (Marmota flaviventris). This was done by comparing cortical bone properties in femurs and trabecular bone properties in tibias from marmots killed before hibernation (fall) and after hibernation (spring). Age data were not available for this study; however, based on femur length the post-hibernation marmots were larger than the pre-hibernation marmots. Thus, cross-sectional properties were normalized by allometric functions of bone length for comparisons between pre- and post-hibernation. Cortical thickness and normalized cortical area were higher in post-hibernation samples; no other normalized cross-sectional properties were different. No cortical bone microstructural loss was evident in osteocyte lacunar measurements, intracortical porosity, or intracortical remodeling cavity density. Osteocyte lacunar area, porosity, and density were surprisingly lower in post-hibernation samples. Trabecular bone volume fraction was not different between pre- and post-hibernation. Measures of both trabecular and cortical bone mineral content were higher in post-hibernation samples. Three-point bending failure load, failure energy, elastic energy, ultimate stress, and yield stress were all higher in post-hibernation samples. These results support the idea that, like bears, marmots are able to prevent disuse osteoporosis during hibernation, thus preventing increased fracture risk and promoting survival of the extreme environmental conditions that occur in hibernation.

Six months of disuse during hibernation does not increase intracortical porosity or decrease cortical bone geometry, strength, or mineralization in black bear (Ursus americanus) femurs.

Disuse typically uncouples bone formation from resorption, leading to bone loss which compromises bone mechanical properties and increases the risk of bone fracture. Previous studies suggest that bears can prevent bone loss during long periods of disuse (hibernation), but small sample sizes have limited the conclusions that can be drawn regarding the effects of hibernation on bone structure and strength in bears. Here we quantified the effects of hibernation on structural, mineral, and mechanical properties of black bear (Ursus americanus) cortical bone by studying femurs from large groups of male and female bears (with wide age ranges) killed during pre-hibernation (fall) and post-hibernation (spring) periods. Bone properties that are affected by body mass (e.g. bone geometrical properties) tended to be larger in male compared to female bears. There were no differences (p>0.226) in bone structure, mineral content, or mechanical properties between fall and spring bears. Bone geometrical properties differed by less than 5% and bone mechanical properties differed by less than 10% between fall and spring bears. Porosity (fall: 5.5+/-2.2%; spring: 4.8+/-1.6%) and ash fraction (fall: 0.694+/-0.011; spring: 0.696+/-0.010) also showed no change (p>0.304) between seasons. Statistical power was high (>72%) for these analyses. Furthermore, bone geometrical properties and ash fraction (a measure of mineral content) increased with age and porosity decreased with age. These results support the idea that bears possess a biological mechanism to prevent disuse and age-related osteoporoses.

Grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) prevent trabecular bone loss during disuse (hibernation).

Disuse typically causes an imbalance in bone formation and bone resorption, leading to losses of cortical and trabecular bone. In contrast, bears maintain balanced intracortical remodeling and prevent cortical bone loss during disuse (hibernation). Trabecular bone, however, is more detrimentally affected than cortical bone in other animal models of disuse. Here we investigated the effects of hibernation on bone remodeling, architectural properties, and mineral density of grizzly bear (Ursus arctos horribilis) and black bear (Ursus americanus) trabecular bone in several skeletal locations. There were no differences in bone volume fraction or tissue mineral density between hibernating and active bears or between pre- and post-hibernation bears in the ilium, distal femur, or calcaneus. Though indices of cellular activity level (mineral apposition rate, osteoid thickness) decreased, trabecular bone resorption and formation indices remained balanced in hibernating grizzly bears. These data suggest that bears prevent bone loss during disuse by maintaining a balance between bone formation and bone resorption, which consequently preserves bone structure and strength. Further investigation of bone metabolism in hibernating bears may lead to the translation of mechanisms preventing disuse-induced bone loss in bears into novel treatments for osteoporosis.