Neurovascular coupling in bone regeneration

The mammalian skeletal system is densely innervated by both neural and vascular networks. Peripheral nerves in the skeleton include sensory and sympathetic nerves. The crosstalk between skeletal and neural tissues is critical for skeletal development and regeneration. The cellular processes of osteogenesis and angiogenesis are coupled in both physiological and pathophysiological contexts. The cellular and molecular regulation of osteogenesis and angiogenesis have yet to be fully defined. This review will provide a detailed characterization of the regulatory role of nerves and blood vessels during bone regeneration. Furthermore, given the importance of the spatial relationship between nerves and blood vessels in bone, we discuss neurovascular coupling during physiological and pathological bone formation. A better understanding of the interactions between nerves and blood vessels will inform future novel therapeutic neural and vascular targeting for clinical bone repair and regeneration.


INTRODUCTION
Bone tissue is vital for all mammalian species as a living organ system that allows body structure and movement. Bone remodeling maintains bone strength and mineral-calcium homeostasis, and it involves bone cells, including bone-resorbing osteoclasts, bone-forming osteoblasts, osteocytes, and bonelining cells 1,2 . The skeleton has a rich innervation of both sensory and sympathetic nerve fibers in conjunction with the bone vascular system [3][4][5] .
Although it has only recently begun to be in the spotlight, the regulatory role of the skeletal, nervous system, including sensory, sympathetic, and parasympathetic nerves, has been examined for many years [3][4][5][6][7][8][9] . Each peripheral nerve fiber subtype has distinct functions within bone [10][11][12] . In addition to performing the general roles of nerve fibers, such as providing motor control of muscle fibers, delivering sensory information from the periphery to the central nervous system (CNS), and regulating autonomic functions 13 , skeletal nerve fibers also provide paracrine stimuli, such as neuropeptide Y (NPY), leptin, calcitonin gene-related peptide (CGRP), and substance P (SP) 3,[14][15][16] . Moreover, adrenergic receptors and other receptors for neuropeptides residing on osteoblasts and osteoclasts express axon guidance molecules such as semaphorins, netrins, and neurotrophins, which further potentiate nerve ingrowth 17,18 . Neurotrophins promote the survival of neurons and target innervation by activating the receptors TrkA and p75 19 . Semaphorins serve as axon guidance molecules during nervous system development 20 . The growth of peripheral nerve fibers and the initial innervation and neurotransmission into the bone tissue regulate bone remodeling and metabolism 21 . Furthermore, nerve ingrowth is an essential upstream mediator of endochondral ossification during bone growth 22 and intramembranous bone formation during growth and repair 7,8,23 .
These accumulating study findings suggest that the stimuli provided by peripheral afferent neurons represent an essential requirement for proper skeletal growth, homeostasis, and repair 7,10,11 .
Recent studies suggest that the bone vascular system plays a significant role in bone remodeling. Bone marrow arteries and capillaries are essential components of bone multicellular units [24][25][26][27] . The bone vasculature is also important for bone marrow homeostasis 28,29 . Declines in vascular function and perfusion are predicted to cause reduced bone volume, bone density, osteoblast activity, and increased osteoclastic resorption [30][31][32] . Accumulating evidence suggests that the skeletal, nervous, and vascular systems influence one another. Recent works have documented that the majority of sympathetic and parasympathetic nerves are located in association with blood vessels 10,33 . Moreover, nerves accompanying blood vessels innervate the primary and secondary ossification center during embryonic skeletal development, whereby CGRP-expressed sensory nerves are detected near osteoblasts and the area of high osteogenic activity 34,35 . However, the extent to which nerve-vessel coupling contributes to improvements in skeletal remodeling and metabolism remains an intriguing unanswered question. As such, this review presents evidence of the roles of neurovascular coupling in bone regeneration, as well as the clinical entity of heterotopic ossification (HO), and summarizes the potential clinical implications of neurovascular coupling in bone tissue.

NERVE AND VASCULAR DISTRIBUTION WITHIN THE BONE
The skeleton is densely innervated by sensory and sympathetic nerves. Peptidergic sensory nerve fibers are prevalent in the skeletal system of vertebrate species and express the neuropeptides CGRP and SP 3,36 . The general role of sensory nerves is to transmit internal and external information from the periphery to the CNS for processing of sensory information. The autonomic nervous system (ANS), including sympathetic and parasympathetic nerves, is a part of the efferent peripheral nervous system (PNS) that innervates and regulates all organs and involuntary functions in the body. Sympathetic nerves are abundant in bone and express tyrosine hydroxylase (TH) 10,36,37 . Both sensory and autonomic nerve fibers in bone are closely linked to bone metabolism and bone remodeling cellular activities.
Although sensory and autonomic nerve fibers have been shown to have mixed effects, both types of nerve fibers are strongly associated with blood vessels during skeletal development and bone repair. During development, blood vessels run into the medullary cavity with their accompanying nerves along the shaft within the bone 38,39 , and the collection of sinusoidal vessels returns to the central nutrient vein along with nerves 38,40 . In cortical bone, sensory and sympathetic nerve fibers are largely confined to vascularized Haversian canals 41 . Moreover, CGRP + and NF200 + sensory nerve fibers are associated with blood vessels located in the Haversian canals, and TH + sympathetic nerve fibers are observed in the bone marrow and wrapping around blood vessels 42 . In our prior observations, Thy1-YFP pan-neuronal reporter activity closely paralleled CD31 + blood vessels during calvarial defect healing 8 . A similar nerve-vessel coupling was observed by Chartier et al., in which CGRP + sensory nerves and TH + sympathetic nerves were colocalized with CD31 + blood vessels in the periosteum 41 . Moreover, neuropeptide Y (NPY) fibers were identified around cerebral blood vessels 43 and in the medullary cavity accompanying blood vessels in the long bones 11 .
In summary, the skeleton is covered by both neural and vascular networks. Their intimate spatial relationship suggests that they modulate each other. However, the interaction between the nerves and vessels remains largely unknown, which has been a trending topic of research in recent years.

NEUROVASCULAR COUPLING DURING BONE REPAIR AND REGENERATION
Nerve signals during fracture repair and regeneration processes Primary sensory and sympathetic axons covering the periosteal bone are required for bone tissue repair and fracture healing by rearranging and reinnervating the nerve fibers into injury sites 3,44 . The nerve growth factor (NGF) is the most important neurotrophin and neuropeptide involved in the regulation of the growth, maintenance, proliferation, and survival of sensory neurons 45 . Tropomyosin receptor kinase A (TrkA) is the high-affinity neurotrophin receptor for NGF, and TrkA + sensory neurons are extensively innervated in the periosteum 42,46 . A previous study in mouse long bone stress fracture repair showed that NGF expression within a nascent callus was considerably upregulated at 3 days and up to 7 days postfracture with a concomitant increase in axonal ingrowth and sprouting, followed by vascularization and ossification 7 . Consistent with this study, our recent work demonstrated that during calvarial bone defect healing, neurotrophic NGF and TrkA signaling play a crucial role in normal calvarial bone regeneration 8 . Moreover, Tomlinson et al. reported that communications between osteoblasts and sensory nerves via NGF-TrkA signaling are required for mechanical loading-induced bone formation 47 . In contrast, studies in murine injury models have revealed that surgical and chemical denervation of sensory nerves resulted in impaired fracture healing and decreased bone formation [48][49][50][51] . In sensory nerves, the disruption of TrkA catalytic activity by the small molecule 1NMPP1 impairs the sequential events of long bone fracture and flat bone defect healing. This disruption also leads to reduced osteoblast activity and abrogated blood vessels in the defect area 7,8 . In addition to NGF-TrkA signaling, common sensory nerves that express CGRP and SP have been shown to play a role in rat tibial fracture healing, whereby sensory nerve innervation was present on the periosteal layers and influenced bone regeneration by stimulating newly produced bony calli 52,53 . The regulatory role of sensory nerves in bone formation was also observed in both mammalian models and human patients with nerve dysfunction. Moreover, reduced peripheral innervation was associated with delayed fracture healing 54 , and the loss of the sensory nerves led to a reduction in new bone quality in rabbits 55 .
In regard to the ANS contribution to the bone healing process, in contrast to the sensory nerves, NPY-positive autonomic nerves were found to be decreased at the fracture or defect regions at early time points, while the presence of NPY-expressing nerve ingrowth and sprouting through Y1 receptor-associated signaling was observed. This accelerated the late phase of bone formation by increasing the osteogenic differentiation of mesenchymal stem cells (MSCs) [56][57][58] . A recent finding has demonstrated that the disruption of sympathetic nerve stimulation via sympathectomy hinders motor function and locomotion and delays fracture callus formation in the late phase of the bone healing process 59 . Although the relationship between the nervous system and the cells that drive bone formation is not fully understood, it is known that neurotransmitters can affect osteoblast formation 60,61 . In addition, osteoblasts are regulated by peripheral nerve-derived norepinephrine 62,63 , and bone contributes to the completeness of the microenvironment by accommodating nerves in the bones to provide bone stability 13 . Recently, our group used spatial transcriptomics of the developing cranium to reveal the importance of nerve signaling in the regulation of bone patterning and the differentiation of bone precursors, highlighting the important role of neural signaling in tissue regulation 23 .
Given our data and previously reported outcomes demonstrating the essential role of NGF-TrkA signaling, CGRP-and SPassociated sensory nerves, and autonomic nerve fibers in bone repair and remodeling, both sensory and autonomic innervation are employed for stimulating vascular ingrowth and sprouting, increasing osteogenic differentiation, and facilitating the delivery of osteogenic factors to the fracture site.

Vascular signals during bone repair and regeneration processes
Crucial roles of the vascular system for bone and bone marrow function, such as bone homeostasis and hematopoietic activity, have also been suggested. Bone blood vessels deliver O 2 , nutrients, and systemic hormones to bone and bone marrow, and they remove waste products. Bone blood vessels also transport immune and precursor cells to and from bone and bone marrow and are fundamental components of the bone multicellular unit 24,25,64 . In addition, blood vessels enriching bone plays a significant role in hematopoietic stem cell (HSC) niches, which are essential for bone homeostasis 29 . It is presumed that the dysfunction of bone and bone marrow is highly associated with dysfunction in the skeletal vascular system 31,32,65 . For example, declines in bone mass were found to be associated with diminished bone blood flow and impaired vasodilator function of bone blood vessels [66][67][68] in rats and humans.
Normally, skeletal blood vessel sprouting and vascular functionrelated substances, such as nitric oxide, are augmented quickly following fracture incidence due to rapid increases in blood flow to the fracture region 69 . Blood supply is a challenge for bone repair and regeneration. To achieve proper bone repair, the local microcirculation must be intact. Many factors stimulate angiogenesis, some of which include fibroblast growth factors, transforming growth factors, platelet-derived growth factors, and vascular endothelial growth factors (VEGF) 70,71 . VEGF has been shown to increase angiogenesis, which increases vascular permeability and the recruitment of MSCs and osteoprogenitor cells. VEGF has the ability to attract MSCs and promote differentiation into osteogenic cell types. Studies have shown that when VEGF is inhibited, bone formation and invasion of the vasculature are decreased 71,72 . When the VEGF receptor is antagonized, this inhibits bone regeneration by bone morphogenetic protein (BMP) 4 and 2, as osteogenesis is induced by these osteogenic cytokines. Kaigler et al. showed that when VEGF is inhibited, angiogenesis and osteogenesis are inhibited 73 . However, new vascularization and bone regeneration occurred with the controlled release of VEGF. In addition, BMPs act synergistically with VEGF to enhance cell survival, cartilage formation, resorption, bone formation, and mineralization 71,72 . BMPs and VEGF synergistically enhance angiogenesis, osteogenic genes, and MSC responses to BMP6, while the ratio of VEGF/BMP is an important mediator. A high ratio of VEGF/BMP2 was found to produce well-formed mineralized bone but a decreased amount of bone compared to a low ratio of VEGF/BMP2 22 .

Neurovascular crosstalk and potential sources of bone repair and regeneration
Neurovascular coupling is a combination of vascular smooth muscle cells, neurons, and astrocyte glial cells. Both neurons and astrocytes respond to increases in vasoactive metabolites that adjust blood flow. Glutamate released from presynaptic neurons in the presence of oxygen stimulates the activation of nitric oxide synthase (NOS), which produces NO to act directly as a dilator on parenchymal arterioles 74 . Astrocytes respond to glutamate in the presence of oxygen to activate a cascading pathway to produce arachidonic acid, which leads to the production of epoxyeicosatrienoic acid and prostaglandins 75 . By having dual stimuli, there is an even greater effect on the increase in blood flow 76 . The neurovascular theory for nerves in bone emerged in the 1980s following the observation that blood flow was increased in the joints of patients with diabetic neuropathy 77 . In addition, the crucial functions of both blood vessels and nerve stimuli in hematopoietic niches have been described. Nociceptive nerves are required to induce HSC mobilization, while sympathetic and sensory nerves collaboratively maintain HSC homeostasis in the bone marrow 78,79 .
After a bone is fractured, growth factors and cytokines recruit osteoprogenitor cells to the affected site 80,81 . As the bone remodels, angiogenesis allows for an invasion of vascular networks into the bone to restore normal circulation to remove/replace the necrotic bone tissue 82 , and nerve fibers are closely associated with blood vessels 7,22 . Our results showed that nerve ingrowth occurred before revascularization and contributed to vascular regrowth 8 . Moreover, impaired nerve reinnervation is accompanied by decreased vascularity and reduced osteogenic capacity, which delayed bone defect healing 8 . Single-cell RNA sequencing data from a traumainduced HO model revealed that NGF was highly expressed in perivascular stem cells and that NGF-TrkA signaling increased angiogenesis and vascular regeneration 9,83-85 . The inhibition of TrkA signaling resulted in delayed vascular invasion in primary and secondary ossification centers, which further reduced the length and volume of the femur in a rodent model 22 . We also demonstrated that chemical genetic inhibition of TrkA signaling led to blunted sensory nerve reinnervation and diminished revascularization, thereby impairing fracture healing 7 .
VEGF is one of the primary mediators promoting angiogenesis while binding to VEGF receptors 1 and 2 promotes bone repair and has direct effects on the nervous systems in terms of axon branching and survival 86 . Conversely, NGF exhibits angiogenic properties 87 . The injection of NGF directly into the site of the defect enhances vascularization and osteogenesis 84 . SP could promote angiogenesis and osteogenic differentiation by upregulating VEGF expression and enhancing BMP2 signaling 61,88,89 . In addition, cholinergic neurons release the neuropeptide vasoactive intestinal peptide, which improves cranial defect healing and bone formation by increasing VEGF expression in MSCs and stimulating angiogenesis 90 .
Clinically, vascularization and neurotization are important in the care of bone trauma, tumors, and infection. A study by Fan et al. showed how tissue-engineered bone (TEB) is affected by implanted vessels and nerves by using a mouse model where the femur was exposed, and osteotomy was performed 91 . Bone tissue gradually increased in TEB along with the femoral blood vessels and saphenous nerve implantation at the defect site 91 . However, new bone formation was highest following saphenous nerve implantation, whereas vascular implantation led to more and better-quality blood vessels 91 . Interestingly, vascular implantation led to increased CGRP and NPY expression, which was presumably associated with the nerves in the walls of the blood vessels, which in turn stimulated neurotization and osteoblast activity and inhibited osteoclasts 91 . Moreover, the levels of CGRP and NPY were found at the highest levels with saphenous nerve implantation, which led to better neurotization of the TEB and more blood vessels 91 . Taken together, these findings demonstrate that the increase in vasculature was due to the capillary network within the nerve fibers, which led to an increase in osteogenesis.
Given the roles of nerve-vascular interactions and functions in bone repair and regeneration, therapeutic targeting of neural and vascular signals may be a promising therapeutic option for bone regeneration.

THE ROLE OF NEUROVASCULAR COUPLING IN ECTOPIC BONE FORMATION Nerve signals during the ectopic bone formation process
In the context of HO, the observation of a coregulation of nerves and bone implicates nerves as a critical mediator in HO pathogenesis. HO has been frequently observed in those who have paroxysmal sympathetic hyperactivity 92,93 . Moreover, the incidence of HO is dramatically increased in patients with traumatic CNS injury 94 . This evidence suggests an association between HO and peripheral nerves. Peripheral nerves enable HO through the release of molecules such as SP and the induction of neuroinflammation 95 . In addition, Nguyen et al. reported BMP2 as a dual-function cytokine that promotes HO via osteoinductive action and neuroinflammation induction 96 . Salisbury et al. demonstrated that sensory nerves contribute to HO development and that the inhibition of sensory nerves led to reduced HO formation by suppressing BMP2-mediated SP and CGRP 97 . Altogether, these findings suggest that HO formation depends on peripheral innervation.
In addition to the nociceptor potential of sensory nerves, a large body of literature has demonstrated the role of sensory nerves in osteoanabolic potential, as well as in regulating bone regeneration and formation. In the context of HO, our recent studies demonstrated that vascular smooth muscle cell-and pericyteinduced NGF-mediated axon innervation accompanied posttraumatic HO in a murine traumatic HO model 9 . Notably, in this study, surgical denervation blunted axonal invasion and reduced chondrogenesis and ectopic bone formation. Similarly, either NGF deletion or TrkA inhibition phenocopied impaired axonal ingrowth and heterotopic bone formation.
Vascular signals during the ectopic bone formation process It has long been known that angiogenesis and vascularization make a remarkable contribution to ectopic bone formation. The representative angiogenic factor VEGFA, for instance, influences vascularity in the form of ectopic bone formation 98,99 . Although this is not fully understood, the activity of a tissue ischemia marker, hypoxia-inducible factor (HIF), induced by a local hypoxic microenvironment, initiates the pathogenesis of HO 100,101 , which induces the expression of VEGF, thereby enhancing angiogenesis, cartilage differentiation, and ultimately HO formation 99,102,103 . Furthermore, vascular histomorphometric analysis in human HO samples demonstrated that the pathophysiological processes of osteogenesis and angiogenesis are coupled in a time-and space-dependent manner 104 . Macrophage-and brown adipocytederived VEGF promoted the occurrence and pathogenesis of HO 99,105 . More recently, by using a murine posttraumatic HO model, our group demonstrated that traumatic HO is highly vascular and identified the critical role of HIF1α in traumatic HO 102 . Moreover, the release of VEGFA from mesenchymal cells drives HO formation 106 . The effects of angiogenic factors can be recapitulated in vivo with a transgenic mouse model of HO. Dilling et al. reported an increase in endothelial cells and a corresponding increase in VEGF mRNA expression in the area of HO, which preceded the inception of mesenchymal contribution and cartilaginous tissue formation 99 . This suggests that vascularization plays a pivotal role in MSC condensation and chondrogenesis during HO progression.

Neurovascular crosstalk and potential sources of HO progression
Although neurovascular coupling is well demonstrated during skeletal development, until recently, it has remained largely unknown in the context of HO. Our studies revealed that experimental models of HO are hyperinnervated, and sensory nerve innervation is an early and necessary feature for HO 9 . Furthermore, by using the same posttraumatic HO model, our group identified neural-derived angiogenic factors and demonstrated a unifying mechanism for neurovascular crosstalk in the context of HO 106 . Hwang et al. demonstrated that HO lesions are highly vascularized and that mesenchymal-derived VEGFA drives ectopic bone formation 106 . These findings implicate the interaction between nerves and blood vessels during HO formation. More recently, by using the traumatic HO model, we demonstrated that progressive neurovascular infiltration accompanies posttraumatic HO 107 . Qin et al. demonstrated progressive ingrowth in overall Tubb3 + nerve fibers and CD31 + blood vessels by 1883% and 3468%, respectively, over the early period (up to 3 weeks) of HO progression. These findings suggest that increased nerve fiber innervation mirrors the upregulation of endothelial vascularity during the early phase of HO development 107 . The authors further investigated neurovascular coupling by using sciatic neurectomy, which greatly inhibited axonal ingrowth and reduced vascular ingrowth into the injury site. This observation was phenocopied by independent mechanisms, and chemical or genetic inhibition of axonal ingrowth led to similar deficits in injury site angiogenesis, suggesting that neuronal perturbation impairs nerve and vessel infiltration and potential neurovascular crosstalk 107 . Singlecell transcriptomic analysis identified that vascular and perivascular cell clusters were present at the injury site, whereas surgical denervation led to a reduction in the proliferative index and a reduction in overall VEGF signaling activity in vascular and perivascular cell clusters 107 . Furthermore, a combination of single-cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neurovascular coupling 107 .
Reciprocally, the potential mutual impact of vessels on nerves during HO was investigated by the same group. By using a previously reported animal model, vascular ingrowth and HO were inhibited by conditional deletion of Vegfa in Prrx1-Cre animals 106 . The deletion of Vegfa had significant effects on vascular density, as expected. In addition, a significant reduction in TUBB3 + axons was also found within the HO site (unpublished data). Taken together, these findings demonstrate that it is plausible to assert that nerves and blood vessels are closely interrelated and promote ectopic bone formation interdependently.

SUMMARY
Nerve and blood vessel ingrowth are spatially coordinated and functionally interdependent during skeletal development and regeneration. The mechanisms of neurovascular coupling are complex, and a variety of cells and molecules participate in neurovascular crosstalk. Many regulatory effects remain unknown. In this review, we discussed the role of nerves and blood vessels in the regulation of bone remodeling and repair and explored the interaction between nerves and vessels during physiological and pathological scenarios. Nerves densely innervate bone, and increased nerve density coincides with bone remodeling and regeneration 11 . Moreover, nerve innervation is normally followed by vascularization during bone fracture repair. In contrast, the inhibition of nerve signaling is accompanied by blunted revascularization and delayed fracture healing 7 . The secretion of VEGF by sensory nerves is the major factor influencing vascular growth and differentiation 108 . NGF-TrkA signaling promotes vascularization by inducing sensory innervation during embryonic development 22 . Furthermore, NGF leads to endothelial cell proliferation and inflammation 109 . NGF-expressing cells may play a key role in neurovascular coupling, which remains to be further investigated. On the other hand, VEGF promotes reinnervation and stimulates axonal outgrowth in the peripheral nervous system 110 . In this regard, further research on the neurotrophic activity of VEGF in bone remodeling and fracture healing is needed.
A better understanding of neurovascular coupling and underlying cellular and molecular drivers could advance the identification of novel targets for the treatment and clinical prevention of diseases such as heterotopic bone formation, osteoarthritis, and even bone tumors. Research focusing on neurovascular coupling during bone regeneration could also contribute to the development of novel differentiation factors and biomaterials for bone repair.