Bone is richly innervated by nerve growth factor–responsive (NGF-responsive) tropomyosin receptor kinase A–expressing (TrKa-expressing) sensory nerve fibers, which are required for osteochondral progenitor expansion during mammalian skeletal development. Aside from pain sensation, little is known regarding the role of sensory innervation in bone repair. Here, we characterized the reinnervation of tissue following experimental ulnar stress fracture and assessed the impact of loss of TrkA signaling in this process. Sequential histological data obtained in reporter mice subjected to fracture demonstrated a marked upregulation of NGF expression in periosteal stromal progenitors and fracture-associated macrophages. Sprouting and arborization of CGRP⁺TrkA⁺ sensory nerve fibers within the reactive periosteum in NGF-enriched cellular domains were evident at time points preceding periosteal vascularization, ossification, and mineralization. Temporal inhibition of TrkA catalytic activity by administration of 1NMPP1 to TrkA^F592A mice significantly reduced the numbers of sensory fibers, blunted revascularization, and delayed ossification of the fracture callus. We observed similar deficiencies in nerve regrowth and fracture healing in a mouse model of peripheral neuropathy induced by paclitaxel treatment. Together, our studies demonstrate an essential role of TrkA signaling for stress fracture repair and implicate skeletal sensory nerves as an important upstream mediator of this repair process.

Bone is a highly vascularized tissue, although this aspect of bone is often overlooked. In this article, the importance of blood flow in bone repair and regeneration will be reviewed. First, the skeletal vascular anatomy, with an emphasis on long bones, the distinct mechanisms for vascularizing bone tissue, and methods for remodeling existing vasculature are discussed. Next, techniques for quantifying bone blood flow are briefly summarized. Finally, the body of experimental work that demonstrates the role of bone blood flow in fracture healing, distraction osteogenesis, osteoporosis, disuse osteopenia, and bone grafting is examined. These results illustrate that adequate bone blood flow is an important clinical consideration, particularly during bone regeneration and in at-risk patient groups.

Developing tissues dictate the amount and type of innervation they require by secreting neurotrophins, which promote neuronal survival by activating distinct tyrosine kinase receptors. Here, we show that nerve growth factor (NGF) signaling through neurotrophic tyrosine kinase receptor type 1 (TrkA) directs innervation of the developing mouse femur to promote vascularization and osteoprogenitor lineage progression. At the start of primary ossification, TrkA-positive axons were observed at perichondrial bone surfaces, coincident with NGF expression in cells adjacent to centers of incipient ossification. Inactivation of TrkA signaling during embryogenesis in TrkA(F592A) mice impaired innervation, delayed vascular invasion of the primary and secondary ossification centers, decreased numbers of Osx-expressing osteoprogenitors, and decreased femoral length and volume. These same phenotypic abnormalities were observed in mice following tamoxifen-induced disruption of NGF in Col2-expressing perichondrial osteochondral progenitors. We conclude that NGF serves as a skeletal neurotrophin to promote sensory innervation of developing long bones, a process critical for normal primary and secondary ossification.

Sensory nerves emanating from the dorsal root extensively innervate the surfaces of mammalian bone, a privileged location for the regulation of biomechanical signaling. Here, we show that NGF-TrkA signaling in skeletal sensory nerves is an early response to mechanical loading of bone and is required to achieve maximal load-induced bone formation. First, the elimination of TrkA signaling in mice harboring mutant TrkA^F592A alleles was found to greatly attenuate load-induced bone formation induced by axial forelimb compression. Next, both in vivo mechanical loading and in vitro mechanical stretch were shown to induce the profound up-regulation of NGF in osteoblasts within 1 h of loading. Furthermore, inhibition of TrkA signaling following axial forelimb compression was observed to reduce measures of Wnt/β-catenin activity in osteocytes in the loaded bone. Finally, the administration of exogenous NGF to wild-type mice was found to significantly increase load-induced bone formation and Wnt/β-catenin activity in osteocytes. In summary, these findings demonstrate that communication between osteoblasts and sensory nerves through NGF-TrkA signaling is essential for load-induced bone formation in mice.

Biallelic mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a disease characterized by calcification in the skin, eyes, and blood vessels. The function of ATP-binding cassette C6 (ABCC6) and the pathogenesis of PXE remain unclear. We used mouse models and patient fibroblasts to demonstrate genetic interaction and shared biochemical and cellular mechanisms underlying ectopic calcification in PXE and related disorders caused by defined perturbations in extracellular adenosine 5′-triphosphate catabolism. Under osteogenic culture conditions, ABCC6 mutant cells calcified, suggesting a provoked cell-autonomous defect. Using a conditional Abcc6 knockout mouse model, we excluded the prevailing pathogenic hypothesis that singularly invokes failure of hepatic secretion of an endocrine inhibitor of calcification. Instead, deficiency of Abcc6 in both local and distant cells was necessary to achieve the early onset and penetrant ectopic calcification observed upon constitutive gene targeting. ABCC6 mutant cells additionally had increased expression and activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme that degrades pyrophosphate, a major inhibitor of calcification. A selective and orally bioavailable TNAP inhibitor prevented calcification in ABCC6 mutant cells in vitro and attenuated both the development and progression of calcification in Abcc6^−/− mice in vivo, without the deleterious effects on bone associated with other proposed treatment strategies.

The skeleton is well-innervated, but only recently have the functions of this complex network in bone started to become known. Although our knowledge of skeletal sensory and sympathetic innervation is incomplete, including the specific locations and subtypes of nerves in bone, we are now able to reconcile early studies utilizing denervation models with recent work dissecting the molecular signaling between bone and nerve. In total, sensory innervation functions in bone much as it does elsewhere in the body—to sense and respond to stimuli, including mechanical loading. Similarly, sympathetic nerves regulate autonomic functions related to bone, including homeostatic remodeling and vascular tone. However, more study is required to translate our current knowledge of bone-nerve crosstalk to novel therapeutic strategies that can be effectively utilized to combat skeletal diseases, disorders of low bone mass, and age-related decreases in bone quality.