Role of the Neurologic System in Fracture Healing: An Extensive Review

The intricate development of sensory and autonomic nerves in the skeletal system, as observed in embryonic and newborn mice, highlights the critical role of these nerve supplies in skeletal development, particularly in regions of high osteogenic activity, such as fracture sites [8].

The innervation of bone and reinnervation of fracture callus following injury have been well characterized using growth-associated protein-43 (GAP-43) and protein gene product 9.5 (PGP 9.5). GAP-43, a marker of new axonal growth and innervation, is highly expressed during the early stages of fracture healing, indicating active nerve regeneration or outgrowth, and is associated with nerve sprouting into the fracture callus before vascularization in rats [9]. GAP-43–positive nerve fibers persist in the periosteum, muscle, and callus of the fracture site after fully healing. This suggests that innervation is essential for bone growth and remodeling [9]. In addition, a time-dependent increase in the co-expression of GAP-43 and PGP-9.5, which tends to indicate only mature nerves, in the newly formed bone and connective tissue indicates the maturation of many nerves over the duration of fracture healing.

The proliferation of nerve fibers into the healing fracture callus is unsurprisingly attenuated by sciatic nerve resection, concomitant with significant impairment of fracture healing. The acceleration of rat tibial fracture union through the formation of bridging calluses is a common outcome of both spinal and sciatic denervation. However, these calluses, despite their rapid formation, are typically less dense and characterized by a reduced presence of collagenous matrix and minerals, suggestive of an osteoporotic transformation [10]. Radiographs support these findings, showing increased callus formation in fractured animals with sciatic nerve resection; however, there is a decrease in mechanical strength, suggesting a defective organization of the callus [11]. In contrast to primary fracture healing, which involves direct bone regeneration without a callus, secondary fracture healing is distinguished by the formation of a callus, followed by gradual bone remodeling. The PNS, particularly sensory innervation, plays a crucial role in recognizing movement in the fracture. In the presence of minimal movement, nerve signals initiate primary bone healing [11]. However, the resection of peripheral nerves changes the pattern of innervation of the fracture callus and adjacent bone, leading to a shift in the healing pattern [12, 13].

Periosteal stripping, which results in the removal of periosteal nerve terminals, also led to delays in osteotomy healing and the development of atrophic nonunions, which are fractures that fail to heal [14, 15]. Selective stripping of sensory neurons via local administration of excitotoxic capsaicin significantly affects fracture healing. Sensory denervation impacts the expression of collagen I, a major component of the extracellular matrix in bone, and influences the balance between bone formation and resorption during fracture healing. In bones with sensory denervation, the collagen fibrils in the fracture repair zone do not follow an organized neat pattern but are irregularly spaced throughout [16]. Moreover, a gradual reduction of collagen II along with the simultaneous increase of collagen I in the healing tissue at 2 weeks and 4 weeks post-fracture indicates a dynamic remodeling of the collagen matrix during the healing process [17]. Similarly to the callus trends seen in sciatic nerve resection, the fracture callus was found to be larger but less ossified in the sensory-denervated group than in the sensory-intact group, further highlighting the influence of sensory nerves on bone healing [16]. Altogether, these findings suggest that denervation of the fracture site, especially sensory denervation, compromises fracture healing.

The importance of intact sensory innervation is further highlighted when considering therapeutic interventions like low-intensity pulsed ultrasound. Ultrasound treatment to the fracture site significantly boosts the rate of union and the volumetric bone mineral density (BMD) in the fracture callus of rats with intact neural pathways, but its effectiveness diminishes in the absence of sensory innervation [18].

Dorsal root ganglia (DRG) contain the soma for sensory neurons, and significantly influence various physiological processes, including fracture healing. Electrical stimulation (ES) at the DRG has been shown to effectively promote fracture healing by increasing calcitonin gene related peptide (CGRP) expression in both DRG and the fracture callus [19••], suggesting a potential approach for promoting healing of limb fractures [20]. Upregulation of CGRP contributes to type-H vessel formation, a biological event coupling angiogenesis and osteogenesis, thereby enhancing osteoporotic fracture healing [19••]. These findings suggest that sensory nerve activation directly improves the rate and quality of fracture healing.

Research has shown that osteoblasts respond rapidly to mechanical loading, upregulating certain signaling pathways within an hour of loading, and this effect is dependent upon TrkA-expressing sensory neurons [21]. This suggests that the effects observed in sciatic nerve resection models might be influenced by these early sensory neuron responses to mechanical stimuli, signifying that this is an alternative site of action for sensory neurons that should be explored further. These findings further underscore the intricate role of the PNS in fracture healing, shedding light on the complex interplay between nerve growth, stimulation, and the process of bone healing and remodeling.

The autonomic nervous system (ANS), encompassing the sympathetic (SNS) and parasympathetic nervous systems, also plays a pivotal role in bone metabolism and fracture healing. Study of the autonomic nervous system is wrought with complexity, as the sympathetic nervous system influences multiple aspects of physiology and pathology. Preclinical evaluation of the role of the sympathetic nervous system can be achieved by several methods. Systemic administration of 6-hydroxydopamine (6-OHDA) induces a peripheral chemical sympathectomy; thus, effects of 6-OHDA are wide ranging and include the immune system [22]. Alternatively, sympathetic nerve bundles can be transected to examine a more limited effect of sympathetic denervation on fracture healing. In preclinical models of fracture, systemic sympathectomy reduces the mechanical stability of the bone, suggesting a role in fracture healing and mechanical strength [22, 23]. It is important to note that the effects on mechanical stability may be due to the homeostatic effect of the SNS on BMD or via the regulation of immune responses to fracture.

Sympathetic denervation can also impact the maturity of new bone formation in distraction osteogenesis (DO) models, providing additional evidence for the importance of the SNS in bone regeneration [24]. In cases of trophic disturbances in the lower extremities, the SNS plays a crucial role, with the first sacral ganglion being particularly significant [25]. Moreover, the SNS can influence the growth rate of an extremity, as evidenced by the effects of a surgical sympathectomy. A successful lumbar sympathectomy performed in children with polio, which results in a sustained increase in circulation, can cause an acceleration of the rate of growth of a leg affected by conditions such as infantile paralysis [26]. This procedure can also halt the progression of shortness in the affected leg, suggesting that the SNS plays a role in the regulation of bone growth through regulation of blood flow [26].

The SNS also influences bone remodeling via release of norepinephrine (NE) and activation of cognate β2-adrenergic receptors on osteoblasts, cells responsible for bone formation. Activation of sympathetic signaling is associated with low BMD in humans and animal models [27, 28], and beta-blockers reverse this suggesting that beta-blockers might be used as bone anabolic and anti-catabolic drugs and that persistent exposure of osteoblasts to norepinephrine leads to decreased bone formation and increased bone resorption [29, 30]. Indeed, there are ongoing clinical trials investigating this possibility.

Fracture healing is a complex process that involves a multitude of molecular factors and signaling pathways that originate from the nervous system. Recent research has highlighted the role of various neuropeptides and signaling molecules in this process, providing new insights into the mechanisms of bone regeneration and the potential for therapeutic interventions. Specifically, peptidergic neuropeptides such as CGRP, SP, and vasoactive-intestinal peptide (VIP) have been found to accelerate fracture healing. Neurotrophins like nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) uniformly stimulate osteogenesis, thereby promoting fracture healing [31]. These findings highlight the intricate interplay between neural regulation and bone healing, opening new avenues for therapeutic strategies. Figure 1 shows the effects CGRP, SP, neuropeptide Y (NPY), VIP, BDNF, NGF, epidermal growth factor (EGF), and NE on fracture healing. These factors will all be discussed with respect to their roles in connecting the nervous system to fracture healing.

Neuropeptides released by sensory nerve fibers via axonal transport may exert paracrine trophic effects on bone, such as CGRP, which has been implicated in the regulation of bone remodeling [13]. CGRP is a neuropeptide distributed throughout the CNS and PNS, playing a significant role in processes like vasodilation, immune cell modulation, and pain transmission. Research indicates that CGRP serves as a pivotal orchestrator, interconnecting the neural, immunological, and skeletal systems throughout the process of bone repair [32]. In humans, an elevation of CGRP and SP in the serum of patients was noted with femur fractures [33]. In a rabbit mandibular injury model, CGRP expression was found to increase over a period of 14 days after injury, reaching its maximum at this point [34]. This timepoint coincided with the transition from the inflammatory stage to new bone regeneration in a rabbit bone-tendon interface injury model [35, 36]. These findings indicate that CGRP expression may correlate to the transition in stages of fracture healing in mammalian models. Moreover, CGRPα-deficient mice displayed impaired bone regeneration, suggesting the essential role of CGRPα in bone healing [37]. Comprehensive genomic studies reveal that CGRP stimulates specific genes related to ossification, bone remodeling, and adipogenesis, indicating a crucial role for CGRP receptor-dependent peroxisome proliferator-activated receptor gamma (PPARγ) signaling in fracture healing [37]. These data also suggests that CGRP may promote the proliferation of periosteal progenitor cells, crucial for new bone formation during fracture healing. Research has shown that inhibiting CGRP signaling by blocking receptor binding or deleting the receptor resulted in reduced callus volume, bone mass, and bone strength [38•]. Furthermore, CGRP was found to modulate M2 macrophages, influencing the secretion of osteogenic factors such as bone morphogenetic protein-2 (BMP-2), BMP-6, WNT10b, and oncostatin M [32]. Macrophages, which have been reported to play a functional role during bone development and regeneration, were found to aggregate in the fracture area during the healing process, suggesting a potential relationship between CGRP and macrophage localization during fracture repair, further underscoring the multifaceted role of CGRP in the healing process [39].

In addition to CGRP, SP has been implicated in aspects of bone healing, including angiogenesis. Models of fracture nonunion have revealed a substantial reduction in the mRNA expression of both CGRP and SP [40]. Both CGRP and SP are increased in the first 24 h after fracture [41]. Inhibition of the receptor for SP (neurokinin-1-tachykinin receptor) has been linked to decreased gene expression and biomechanical strength in bone healing, suggesting a potential therapeutic role for SP in disrupted fracture repair [42]. Mechanistically, SP was found to promote osteogenesis and mitigate bone resorption via activated Wnt signaling [43].

NPY also has been implicated in fracture healing, with several studies highlighting its significant role in this process. An increase in autonomic NPY-positive nerve fibers was observed using immunohistochemical staining at the fracture site, suggesting a significant role of NPY in fracture healing [44]. Acting via its Y1 receptor, NPY has been found to play a crucial role in bone repair. Global deletion of the Y1 receptor has been shown to hinder fracture healing. Specifically, in Y1 receptor-deficient mice, there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Additionally, the histopathological characteristics revealed an increase in the avascular and cartilaginous regions, leading to a subsequent delay in cartilage resorption, thereby resulting in hindered bone union [45]. NPY has also been found to be associated with macrophage aggregation during the fracture repair process [39]. As mentioned above, macrophages play a significant role in the immune response and tissue repair, including bone regeneration. Altogether, these findings suggest that NPY may directly contribute to bone formation and may also indirectly facilitate healing by influencing the immune response [39].

BDNF plays a significant role in fracture healing, with high levels of BDNF and its receptor, TrkB, found at fracture sites [46]. BDNF and TrkB immunoreactivity and mRNA analyses show that the ligand is expressed predominantly by endothelial cells and the receptor is expressed on osteoblastic lineage cells within the fracture microenvironment [47]. BDNF promotes osteoblast migration and increases integrin β1 expression, a protein involved in cell adhesion and migration, through TrkB receptor activation of ERK1/2 and AKT signaling pathways [48]. While BDNF does not significantly affect the expression of receptor activator of nuclear factor kappa beta (RANKL) or osteoprotegerin (OPG), key regulators of osteoclastogenesis and bone resorption in MLO-Y4 osteocytes [49], it does increase OPG expression in earlier osteoblast progenitor MC3T3 cells in a TrkB-dependent manner [50]. BDNF also has been shown to enhance endothelial cell movement, growth, and the creation of new vasculature, primarily through the augmentation of vascular endothelial growth factor (VEGF) production [51]. Furthermore, BDNF administration has been found to elevate the levels of alkaline phosphatase (ALP) and BMP-2 in cementoblasts, cells that share similarities with osteoblasts [52]. Despite all of these putative bone-anabolic functions of BDNF, the TrkB agonist, 7,8-DHF, impaired fracture healing in mice, suggesting that TrkB activation and possibly BDNF’s downstream effects may have varying impacts on bone health and fracture healing [46].

NGF plays a significant role in bone fracture healing. In general, NGF promotes local regulation of nerves leading to increased neuropeptide expression, cytokine levels, and sensory innervation density to promote the bone healing process [53]. NGF is found to be expressed in cells such as osteoclasts, osteocytes, chondrocytes, synovial fibroblasts, and immune cells [54, 55]. NGF mediates osteoblast differentiation, a critical process in bone formation and fracture healing, via the BMP-2/Smad pathway. This pathway regulates the expression of runt-related transcription factor 2 (Runx2), a transcription factor crucial for osteoblast differentiation and skeletal morphogenesis. Observations indicate an elevation in Runx2 protein levels when exposed to NGF, implying a potential enhancement of the BMP-2/Smads/Runx2 signaling cascade by NGF [54]. NGF in the callus following a femur fracture was observed in aged mice to be increased relative to their younger counterparts, implying a heightened demand for neural restoration and skeletal restructuring in the elderly [56•]. Further, Li et al. [57] reported that NGF expression was upregulated in the fracture callus, peaking at day 3 post-fracture. NGF also plays a role in the transition from cartilage to bone during the healing process. Indeed, local injections of β-NGF resulted in a gene expression profile favoring endochondral bone formation and enhancing the structural integrity of the healing bone [58].

The neuropeptide VIP, released by the SNS, promotes in vitro osteogenesis of bone marrow mesenchymal stem cells (BMSCs) and in vivo bone defect repair [23]. The SNS also influences endochondral ossification, a crucial process in fracture healing. The absence of sympathetic neurotransmitters delays the differentiation of mesenchymal callus tissue toward a cartilaginous matrix, suggesting a role in callus formation and differentiation [22]. Fracture chondrocytes produce SP and its receptor NK1, indicating an endogenous callus signaling loop [59]. This suggests that sensory and sympathetic neurotransmitters play a crucial role in the regulation of callus formation and differentiation during fracture healing [22].

Sympathetic denervation has also been shown to reduce NE levels in DO, leading to a decrease in adrenergic receptor beta 3 (adrb3) in bone marrow stromal cells and promoting their migration to bone-forming areas [60]. While NE typically inhibits mesenchymal stem cell migration and osteogenic differentiation, this effect can be reversed by using siRNA to knockdown adrb3 [60].

In summary, the molecular factors influencing fracture healing are diverse and interconnected, involving various neuropeptides, neurotransmitters, and signaling pathways. Although further research is needed to fully elucidate their complex interactions with other biological systems, understanding these molecular mechanisms can provide valuable insights into the process of bone regeneration and offer potential targets for therapeutic interventions to enhance fracture healing.

Traumatic injuries to the CNS can have profound and multifaceted impacts on the body, extending beyond the immediate neurological implications. Many retrospective cohort studies have been conducted to examine the effects of TBI on fracture healing. TBI including its mild form (mTBI) can significantly impact bone health, leading to growth hormone deficiency and disrupted bone mass accrual, which increases the risk of fractures and osteoporosis [61, 62]. TBI can also induce sympathetic hyperactivity, further affecting bone health [63]. Clinical studies have shown a relationship between TBI and bone-related abnormalities such as osteoporosis, osteopenia, and increased risk of fractures [62]. Regardless of mobility restrictions placed on animals, TBI notably diminishes cortical bone and leads to a reduction in the mass of trabecular bone in the tibia of murine models [62, 64]. Osteoporosis treatments like bisphosphonates can help reverse bone damage associated with inflammation-related bone loss [65]. Therefore, understanding these mechanisms and strategies to mitigate TBI’s secondary skeletal effects can improve patient outcomes.

The interplay between TBI and bone fracture healing presents a complex picture. Preclinical research suggests that TBI may accelerate callus formation and alter its composition and strength, as evidenced by mice with TBI exhibiting earlier bridging callus formation and greater final mean callus thickness than those without TBI [66]. This acceleration may be due to the release of osteoinductive factors, such as BMPs [62], into the systemic blood circulation and increased hematoma formation associated with TBI in patients [67‐69]. The effect of TBI on fracture healing also may be site-specific, as the association of head injury did not increase the rate of union in forearm fractures [70]. Clinically, rapid union of fractures in association with severe head injury may lead to malunion, particularly in patients where treatment of cerebral and other injuries takes priority over their fractures [71]. The quantity of callus formation in fracture patients who have sustained head injuries notably surpasses that observed in a control (no head trauma) group, implying a direct influence of the head trauma on the fracture healing trajectory [72]. The interpretation of these head trauma effects as either an acceleration in fracture healing or a manifestation of local heterotopic ossification (HO) continues to be unclear [73, 74]. Of note, there are important caveats to consider. Specifically, patients sustaining a TBI and femoral fracture who were treated non-operatively did not develop excessive callus formation [75]. Furthermore, when TBI is of a repeated mild nature (r-mTBI), it may impair the healing process, leading to a significant reduction in the bone volume/tissue volume (BV/TV) phenotype and a decrease in the mechanical strength of the newly formed bone in mice [76]. These contrasting findings underscore the complexity of TBI’s impact on fracture healing, which may be influenced by the severity and frequency of the injury.

Indeed, the severity of TBI, as measured by the Glasgow Coma Scale (GCS), has been found to correlate with the volume of callus formed [77]; however, clinical correlations are inconsistent between elements like the intensity of the cerebral trauma, the nature of the intracranial bleed, and patient sex with the pace of bone recuperation or the magnitude of ultimate callus development [78]. Humoral factors, including growth hormone, IL-6, and prolactin, are elevated in patients with TBI and may contribute to enhanced osteogenesis and more pronounced callus formation [77]. These factors could be potential targets for therapeutic interventions to enhance fracture healing in patients with TBI.

HO is a negative side effect observed in high prevalence following TBI [79]. HO is a pathological process characterized by the formation of ectopic bone in non-skeletal tissues, including muscles, tendons, and other soft tissues. This aberrant bone formation can occur in response to various stimuli, including trauma, neurological injury, specific genetic disorders, or as a side effect of certain surgical procedures. Management of HO aims to limit its progression and maximize joint function, with surgical excision considered in severe cases [80]. The complex interplay between hyperadrenergic activity, HO, and fracture healing following TBI or draws attention to the need for further research to develop effective management strategies [62, 81‐83]. The management of complications such as HO also remains a challenge.

Animal models of combined TBI and standard closed fracture have demonstrated a systemic response that enhances fracture healing, particularly through increased stiffness and the stimulation of mesenchymal stem cell proliferation in rats [84]. These models, which may mirror high-energy trauma scenarios, provide a platform for exploring the interplay between neuronal, endocrine, and metabolic consequences of TBI and the individual fracture healing response [84]. Overall, while compelling evidence suggests that TBI may influence fracture healing, affecting callus volume, composition, and strength, further research is needed to elucidate the precise mechanisms underlying this phenomenon in patients with TBI.

Although the effects of TBI on fracture healing are inconsistent, it is still informative to highlight several signaling molecules that are affected by TBI that have fracture-healing effects. NPY contributes to post-fracture bone healing, especially in patients with TBI-fracture combined injuries. NPY levels were found to be increased in patients with combined injuries, accompanied by a rise in bone healing markers such as ALP, osteocalcin, type I procollagen peptide, and carboxy-terminal telopeptide of type I collagen compared to those with fractures only [85••].

Studies have shown that when a fracture is combined with peripheral nerve injury, the healing process can be slowed down [86]. In contrast, a fracture combined with cerebral cortex injury will accelerate the healing process. The concentration of CGRP in the DRG varied depending on the site of injury [86]: spinal or cerebral cortex injuries increased CGRP and positively affected fracture healing. Additionally, research has demonstrated that CGRP augments the process of DO through the amplification of angiogenesis, which in turn facilitates the healing of bones [87]. As may be expected, CGRP expression significantly escalates near the fracture location in the group with both TBI and fractures [88, 89].

EGF has been identified as a critical factor in neuronal differentiation and its potential implications for bone healing are noteworthy. EGF has been found to be closely related to cell growth, proliferation, differentiation, and regeneration of the CNS. EGF may promote the formation of bone tissue and accelerate the synthesis and deposition of the matrix in patients with TBI and limb fracture [90]. Moreover, EGF can act as a neurotrophic factor in the CNS, promoting neurite outgrowth and cell survival of cultured cerebral, subneocortical telencephalic, glial cells, and cerebellar neurons [91].

NGF plays a critical role in fracture healing, particularly in the context of TBI. As detailed earlier, NGF is essential for the survival and development of central and peripheral neurons. Its synthesis and release increase following TBI or bone injury, leading to elevated serum NGF levels. This elevation is particularly noticeable in patients who have experienced TBI in conjunction with limb fractures, implying a crucial role of NGF in facilitating the healing of fractures in these individuals [90, 92]. NGF’s role in TBI and fracture healing is significant, promoting neuron development and enhancing osteogenic capacity, potentially accelerating the fracture healing process.

Together, the intricate interplay between neuropeptides and other growth factors plays a pivotal role in the process of fracture healing associated with CNS injuries. Understanding these mechanisms can provide valuable insights into the development of therapeutic strategies for enhancing fracture healing. However, further research is needed to fully elucidate the complex series of signals from hormones, growth factors, and mechanical and neuronal changes that lead to this clinical phenomenon.