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De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

Military literature has demonstrated the utility and safety of tourniquets in preventing mortality for some time, paving the way for increased use of tourniquets in civilian settings, including perioperatively to provide a bloodless surgical field. However, tourniquet use is not without risk and the subsequent effects of tissue ischemia can impede downstream rehabilitative efforts to regenerate and salvage nerve, muscle, tissue and bone in the limb. Limb ischemia studies in both the mouse and pig models have indicated not only that there is residual flow past the tourniquet by means of microcirculation, but also that recovery from tissue ischemia is dependent upon this microcirculation. Here we expand upon these previous studies using portable Near-Infrared Imaging to quantify residual plasma flow distal to the tourniquet in mice, pigs, and humans and leverage this flow to show that plasma can be supersaturated with oxygen to reduce intracellular hypoxia and promote tissue salvage following tourniquet placement. Our findings provide a mechanism of delivery for the application of oxygen, tissue preservation solutions, and anti-microbial agents prior to tourniquet release to improve postoperative recovery. In the current environment of increased tourniquet use, techniques which promote distal tissue preservation and limb salvage rates are crucial.

Cell metabolism coordinates the biochemical reactions that produce carbon and ATP in order for the cell to proliferate, differentiate, and respond to environmental changes. Cell type determines metabolic demand, so proliferating skeletal progenitors and differentiated osteoblasts exhibit different levels of cell metabolism. Limb regeneration is an energetically demanding process that involves multiple types of tissues and cell functions over time. Dysregulation of cell metabolism in aged mice results in impaired regeneration, a defect that can be rescued in part by the administration of oxaloacetate (OAA). A better understanding of how cell metabolism regulates regeneration in general, and how these changes can be modulated to benefit potential regenerative strategies in the future is needed. Here we sought to better understand the effects of OAA on young mice and determine whether the same mechanism could be tapped to improve regeneration without an aged-defect. We also asked which dosing time periods were most impactful for promoting regenerative outcomes, and whether these effects were sustained after dosing was stopped. Consistent with our findings in aged mice we found that OAA enhanced regeneration by accelerating bone growth, even beyond control measures, by increasing trabecular thickness, decreasing trabecular spacing, and improving the patterning by decreasing the taper, making the regenerated bone more like an unamputated digit. Our data suggests that the decrease in spacing, an improvement over aged mice, may be due to a decrease in hypoxia-driven vasculature. Our findings suggest that OAA, and similar metabolites, may be a strong tool to promote regenerative strategies and investigate the mechanisms that link cell metabolism and regeneration.

Osteogenesis imperfecta (OI) type V is the second most common form of OI, distinguished by hyperplastic callus formation and calcification of the interosseous membranes, in addition to the bone fragility. It is caused by a recurrent, dominant pathogenic variant (c.-14C>T) in interferon-induced transmembrane protein 5 ( IFITM5 ). Here, we generated a conditional Rosa26- knockin mouse model to study the mechanistic consequences of the recurrent mutation. Expression of the mutant Ifitm5 in osteo-chondroprogenitor or chondrogenic cells resulted in low bone mass and growth retardation. Mutant limbs showed impaired endochondral ossification, cartilage overgrowth, and abnormal growth plate architecture. The cartilage phenotype correlates with the pathology reported in patients with OI type V. Surprisingly, expression of mutant Ifitm5 in mature osteoblasts caused no obvious skeletal abnormalities. In contrast, earlier expression in osteo-chondroprogenitors was associated with an increase in the skeletal progenitor cell population within the periosteum. Lineage tracing showed that chondrogenic cells expressing the mutant Ifitm5 had decreased differentiation into osteoblastic cells in diaphyseal bone. Moreover, mutant IFITM5 disrupted early skeletal homeostasis in part by activating ERK signaling and downstream SOX9 protein, and inhibition of these pathways partially rescued the phenotype in mutant animals. These data identify the contribution of a signaling defect altering osteo-chondroprogenitor differentiation as a driver in the pathogenesis of OI type V.

Mouse digit amputation provides a useful model of bone growth after injury, in that the injury promotes intramembranous bone formation in an adult animal. The digit tip is composed of skin, nerves, blood vessels, bones, and tendons, all of which regenerate after digit tip amputation, making it a powerful model for multi-tissue regeneration. Bone integrity relies upon a balanced remodeling between bone resorption and formation, which, when disrupted, results in changes to bone architecture and biomechanics, particularly during aging. In this study, we used recently developed techniques to evaluate bone patterning differences between young and aged regenerated bone. This analysis suggests that aged mice have altered trabecular spacing and patterning and increased mineral density of the regenerated bone. To further characterize the biomechanics of regenerated bone, we measured elasticity using a micro-computed tomography image-processing method combined with nanoindentation. This analysis suggests that the regenerated bone demonstrates decreased elasticity compared with the uninjured bone, but there is no significant difference in elasticity between aged and young regenerated bone. These data highlight distinct architectural and biomechanical differences in regenerated bone in both young and aged mice and provide a new analysis tool for the digit amputation model to aid in evaluating the outcomes for potential therapeutic treatments to promote regeneration.

The mouse digit tip amputation model is an excellent model of bone regeneration, but its size and shape present an obstacle for biomechanical testing. As a result, assessing the structural quality of the regenerated bone in this model has focused on mineral density and bone architecture analysis. Here we describe an image-processing based method for assessment of mechanical properties in the regenerated digit by using micro-computed tomography mineral density data to calculate spatially discrete Young’s modulus values throughout the entire distal third phalange. Further, we validate this method through comparison to nanoindentation-measured values for Young’s modulus. Application to a set of regenerated and unamputated digits shows that regenerated bone has a lower Young’s modulus compared to the uninjured digit, with a similar trend for experimental hardness values. Importantly, this method heightens the utility of the digit regeneration model, allows for more impactful treatment evaluation using the model, and introduces an analysis platform that can be used for other bones that do not conform to a standard long-bone model.

The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to metabolic flexibility–promoting mitochondrial energy production and maintaining homeostasis. In bone, metabolic profiles are tightly regulated and the loss of SIRT3 has deleterious effects on bone volume in vivo and on osteoblast differentiation in vitro . Despite the prominent role of this protein in bone stem cell proliferation, metabolic activity, and differentiation, the importance of SIRT3 for regeneration after bone injury has never been reported. We show here, using the mouse digit amputation model, that SIRT3 deficiency has no impact on the regenerative capacity and architecture of bone and soft tissue. Regeneration occurs in SIRT3 deficient mice in spite of the reduced oxidative metabolic profile of the periosteal cells. These data suggest that bone regeneration, in contrast to homeostatic bone turnover, is not reliant upon active SIRT3, and our results highlight the need to examine known roles of SIRT3 in the context of injury.

Purpose of Review The purpose of this review is to outline the principles of clinical genetic testing and to provide practical guidance to clinicians in navigating genetic testing for patients with suspected monogenic forms of osteoporosis. Recent Findings Heritability assessments and genome-wide association studies have clearly shown the significant contributions of genetic variations to the pathogenesis of osteoporosis. Currently, over 50 monogenic disorders that present primarily with low bone mass and increased risk of fractures have been described. The widespread availability of clinical genetic testing offers a valuable opportunity to correctly diagnose individuals with monogenic forms of osteoporosis, thus instituting appropriate surveillance and treatment. Summary Clinical genetic testing may identify the appropriate diagnosis in a subset of patients with low bone mass, multiple or unusual fractures, and severe or early-onset osteoporosis, and thus clinicians should be aware of how to incorporate such testing into their clinical practices.

Bone is an essential, healing structure in vertebrates that ensures daily function. However, the regenerative capacity of bone declines with age, compromising quality of life in the elderly and increasing cost of care. Here, for the first time, the elasticity of regenerated bone in a mouse digit amputation model is evaluated in order to better investigate biomechanics of skeletal regeneration. Amputation of the distal one third of the digit (third phalangeal element P3) results in de novo regeneration of the digit, where analyzing the structural quality of this regenerated bone is a challenging task due to its small scale and triangular shape. To date, the evaluation of structural quality of the P3 bone has primarily focused on mineral density and bone architecture. This work describes an image-processing based method for assessment of elasticity in the whole P3 bone by using microcomputed tomography-generated mineral density data to calculate spatially discrete elastic modulus values across the entire P3 bone volume. Further, we validate this method through comparison to nanoindentation-measured values for elastic modulus. Application to a set of regenerated and unamputated digits shows that regenerated bone has a lower elastic modulus compared to the uninjured digit, with a similar trend for experimental hardness values. This method will be impactful in predicting and evaluating the regenerative outcomes of potential treatments and heightens the utility of the P3 regenerative model. Competing Interest Statement The authors have declared no competing interest.