A Procedural Overview of the Involvement of Small Molecules in the Nervous System in the Regulation of Bone Healing

Introduction

Bone tissue injury caused by various factors such as inflammation, trauma, fracture, tumor, surgery, etc. is a prevalent clinical issue¹ that can significantly impact bodily function and diminish quality of life in severe cases.^2–4 Deficiency in bone mass resulting from injury at specific sites may also impact the prognosis of associated treatments.⁵ Various types of bone augmentation surgeries, such as autologous bone transplantation or allograft bone transplantation, are associated with certain drawbacks, including limited availability of autologous bone,⁶ extensive tissue damage, high complication rates,^7,8 and challenges in patient acceptance. Additionally, allograft bone carries the potential risk of disease transmission or adverse immune reactions.^7,9,10

The field of bone tissue engineering has emerged as a promising therapeutic approach, leveraging the principles of bionics to address the aforementioned limitations by incorporating diverse cell types, bioactive factors, and other components into materials for bone regeneration.^6,11 Although bone tissue engineering has achieved favorable outcomes in the treatment of conventional bone defects,^12,13 addressing large bone defects resulting from trauma and various bone diseases, specifically critical bone defects with a lesion length of at least 6 cm,^14–18 remains a challenging issue encountered by clinicians.¹⁹ This could be attributed to the oversight of the crucial role played by the nervous system in bone repair.²⁰

In recent years, the role of the nervous system in bone repair has garnered increasing attention and application from scientists.^21–25 Nerve tissue, which is primarily composed of neurons and glia, serves as the principal constituent of the nervous system. It has been widely observed to be distributed throughout the bone cortex²⁶ and bone trabeculae,²⁷ as well as in the bone marrow and periosteum.²⁸ Moreover, it plays a crucial role in facilitating bone tissue regeneration. Denervation experiments have directly demonstrated the influence of nerve tissue on bone resorption²⁹ and fracture healing.³⁰ Additionally, the presence of nerve fibers in the callus formed after bone injury³¹ further emphasizes the significant role played by nerve tissue in the process of bone healing. Small molecules of the nervous system are a series of small molecules including neurotrophic factors and neuropeptides in the nervous system, which play an important role in promoting the growth and development of neurons and repairing after injury. In addition to their role in the nervous system, numerous studies have also demonstrated^32–38 that following bone injury, there is an elevation in the levels of these small molecules within the bone defects compared to normal tissues. This suggests their significant involvement in the process of bone repair and regeneration.

The ideal material for bone tissue engineering should possess the dual functionality of replacing missing bone fragments and promoting autologous bone formation. However, small molecules derived from the nervous system, despite being bioactive, are not suitable for direct clinical application due to their instantaneous cell signaling effects and short half-life.^39–43 The utilization of bone tissue engineering carriers for the treatment of these active small molecules not only enables better preservation of their biological activity, but also facilitates direct programmed controlled release within bone defects, thereby exerting a long-term and efficient role in biological regulation. This represents one of the future directions for application development in the field of bone tissue engineering. Therefore, this paper provides a comprehensive review of the roles played by various small molecules in the nervous system on bone tissue, elucidating their specific mechanisms of action in different processes of bone regeneration. This will serve as a valuable reference for the systematic and rational design of vectors for bone tissue engineering and the targeted utilization of small molecules from the nervous system.

Bone Tissue and Bone Healing Process

The bone tissue possesses a complex three-dimensional architecture, primarily categorized into two structural forms: cancellous bone and cortical bone. Cancellous bone exhibits a porous structure composed of anisotropic trabeculae acting as pillars, while cortical bone is comparatively dense to provide support and withstand pressure. Furthermore, an intricate neurovascular network traverses the bone tissue, regulating its biological behavior and supplying it with nutrition and sensory support.^44–47

The process of bone healing is a complex and intricately coordinated physiological phenomenon that can be categorized into primary (direct) and secondary (indirect) bone healing.^48–50 Primary bone healing occurs under demanding circumstances, necessitating rigid fixation to prevent any movement of the bone fragments. This particular healing process seldom triggers inflammation but instead initiates a mechanism akin to normal bone remodeling. However, it should be noted that this recuperative process progresses at a sluggish pace, often spanning several months to years for complete recovery.

The secondary bone healing, which is a more commonly observed process, requires relatively simpler conditions for regeneration compared to primary bone healing. Therefore, it serves as the predominant form of bone tissue engineering materials’ healing. This intricate process involves the regulation of multiple signaling pathways and bioactive factors. The process of normal bone healing involves the following sequential stages:^7,51,52 When bone defects occur, rupture of blood vessels leads to the formation of local hematoma. Inflammatory cells infiltrate into the hematoma tissue and secrete pro-inflammatory cytokines, recruiting macrophages for debris removal from damaged tissues. Subsequently, under the regulation of bioactive factors such as glial cell line-derived neurotrophic factor (GDNF), recruited mesenchymal bone progenitor cells proliferate and differentiate into chondrocytes, secreting cartilage matrix and forming soft callus. The callus then undergoes mineralization while proliferative cells release growth factors like vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP), stimulating migration of osteogenic precursor cells and blood vessels to the site of bone defect. The soft callus is invaded by osteoclasts simultaneously, initiating the reabsorption of the mineralized cartilage matrix. The coordinated process of matrix absorption and bone tissue formation triggers the substitution of soft callus with hard bone callus, facilitating the subsequent formation and remodeling of woven bone. The process of bone healing can be described as a sequential progression through three partially overlapping stages:^49,53 inflammation, repair, and remodeling. Small molecules of the nervous system play diverse roles in each stage of bone healing, facilitating the promotion of bone regeneration. Bone tissue engineering suggests that bone replacement materials should possess appropriate osteoconductivity, osteoinductivity, and osteogenesis.⁴⁴ Incorporating suitable bioactive factors can better fulfill these requirements. Therefore, exploring the integration of neurotrophic small molecules with bone substitute materials to enhance their role in promoting bone healing represents a promising future direction for bone tissue engineering. The Figure 1 below shows how various small molecules in the nervous system play a role in each of the three stages of bone healing and induce different differentiation fates between osteoblasts and osteoclasts.

Figure 1 Schematic summary of the stages of bone healing and temporal patterns of small molecule expression in the nervous system.

Effects of Small Molecules of Nervous System on Bone Healing

Neurotrophins and Their Receptors

Neurotrophins (NTs) are a group of closely related proteins initially believed to serve as survival factors for sensory and sympathetic neurons, but subsequent research has revealed their crucial role in regulating the survival, development, and functionality of both peripheral and central nervous system neurons.⁵⁴ The first neurotrophic factor to be identified was Nerve Growth Factor (NGF), followed by Brain-derived neurotrophic factor (BDNF) during the purification of pig brains.⁵⁵ Four neurotrophic factors, including NGF, BDNF, NT-3 and NT-4/5, have been isolated and purified in mammals. Furthermore, through the implementation of serum-free neuronal culture techniques, novel specific protein molecules have been discovered in various tissue fluids and extracellular matrix. These include Glial cell line-derived neurotrophic factor (GDNF) and Platelet-derived growth factor (PDGF), as well as Fibroblast growth factor (FGF), among others. It has been demonstrated that these particular protein molecules also play a crucial role in promoting neuronal proliferation, differentiation, and survival.

The four types of neurotrophic factors (NTs) in mammals, also referred to as neurosurvival factors, play a crucial role in regulating various processes such as synaptic plasticity, neurodegenerative diseases, demyelinating diseases, and inflammation.⁵⁶ Apart from their pivotal function in the nervous system, these NTs have been demonstrated to exert significant effects on bone tissue regeneration and skin wound healing,⁵⁷ as well as pregnancy and brain development,⁵⁸ inflammation, and allergies.⁵⁹

There are two distinct types of neurotrophic factor receptors, each exhibiting varying affinities towards neurotrophic factors. The initial receptor identified, known as the p75 neurotrophic factor receptor (p75^NTR), functions as a low-affinity receptor for NTs. The p75^NTR binds to all four NTs with equal affinity. Another group of receptors for NTs consists of three members belonging to the Trk receptor tyrosine kinase subfamily - TrkA, TrkB, and TrkC - which are recognized as high-affinity receptors for NTs. These receptors display specificity in their interactions with the four different types of NTs: NGF activates TrkA; BDNF and NT-4 activate TrkB; and NT-3 activates TrkC. Additionally, while NT-3 can still bind to the other two Trk receptors, its activation efficiency is relatively low.⁵⁴ In the context of bone healing, modulation of these distinct receptors’ functions can potentially enhance bone regeneration. Therefore, this paper provides a comprehensive overview of small molecule-related receptors within the nervous system.

During the development of the central nervous system, p75^NTR is extensively expressed in neurons and various glial cells, serving as a reliable marker for Schwann cells originating from either peripheral or central sources.⁶⁰ Apart from its distribution within the nervous system, p75^NTR is also present in diverse mesenchymal cells.⁶¹ Functionally, p75^NTR engages in signal transduction by interacting with different proteins to mediate distinct biological processes.^60,62,63

Xu et al⁶¹ conducted cross-breeding experiments between p75^fl/fl animals and PDGFR^αmT/mG lines to assess the necessity of p75^NTR in skull defect repair. The experimental findings revealed a decrease in bone volume (BV), BV fraction, and bone fraction area in the CT scans of p75^PDGFRα mice, along with an increase in the mean diameter of the bone defect area. H&E staining in Figure 2⁶¹ also showed significantly impaired healing between bone fronts in p75^NTR mutant mice. To investigate the role of p75^NTR in human skull osteoblasts, we observed p75^NTR knockdown mediated by small interfering RNA (siRNA), which resulted in impaired cell migration and osteogenic differentiation.

Figure 2 (A) Micro-CT reconstructions of the defect site in a top-down view (above) and coronal cross-sectional images (below) among p75fl/fl and p75PDGFRα animals, 28 days after injury. Margins of original defect are indicated by red dashed lines. (B) Hematoxylin and eosin (H&E) staining of coronal cross section of the healing defect site from p75fl/fl and p75PDGFRα mice at day 28 after injury. Black arrowheads indicate healing bone edges. Adapted from Xu J, Li Z, Tower RJ, et al. NGF-p75 signaling coordinates skeletal cell migration during bone repair. Sci Adv. 2022;8:eabl5716. Creative Commons.61

Wang et al’s study⁶⁴ also demonstrated that mice lacking thep75^NTR gene exhibited a reduction in alveolar bone mass compared to wild-type (WT) mice, and the mineralization induction process led to a decrease in osteogenic ability of Ecto Mesenchymal Stem cells (EMSCs). Furthermore, it was observed that the phosphatidylinositol 3-kinase/protein kinase B/β-catenin (PI3K/Akt/β-catenin) signaling pathway, which plays a positive role in regulating EMSCs’ differentiated mineralization potential, was down-regulated as well.⁶⁴ Experimental findings indicated that p75^NTR positively regulated the osteogenic differentiation of EMSCs by enhancing the PI3K/Akt/β-catenin pathway. Table 1 provides an overview of how neurotrophic factors affect bone through their respective receptors. In future research, if components responsible for regulating p75^NTR activity can be incorporated into osteogenic materials, it may enhance the healing process of bone defects.

Table 1 The Receptors and Effect of Neurotrophins on Bone

Nerve Growth Factor (NGF)

The role of NGF in bone pain conduction was initially identified within bone tissue. As the primary mediator of bone pain, NGF directly activates TrkA or indirectly transmits nociceptive signals to enhance the response of other pain pathways.^32,65,66

In recent years, the role of NGF in bone regeneration has gradually become elucidated. Vincent Fitzpatrick et al⁶⁷ incorporated NGF, BMP2, and VEGF into the 3D printed ink to fabricate a scaffold material with appropriate pore size, which effectively promotes neurovascularization and bone tissue regeneration in cases of bone defects. This study confirms that NGF can synergistically interact with other growth factors. For instance, the upregulation of BMP-2 induces an increase in Runt-related transcription factor 2(RUNX2), osteopontin (OPN), and bone sialoprotein (BSP) expression in human mesenchymal stem cells (HMSCs), thereby facilitating osteogenic differentiation.⁶⁷ The addition of NGF to the titanium surface coating of the implant has also been demonstrated to enhance the differentiation of bone marrow mesenchymal stem cells into osteoblasts.⁶⁸ Moreover, NGF can upregulate osteoblast activity, inhibit osteoclast activity, and stimulate in vivo osteogenesis to facilitate fracture healing.⁶⁹ Local injection of β-NGF further improves bone healing function by increasing bone volume and promoting the formation of newly formed bone with a higher number of trabeculae, connective tissue density, and bone density.⁷⁰

The diverse functions of NGF in bone regeneration are intricately linked to the cellular signaling pathways involved. NGF exerts its crucial role through two receptors, namely the high-affinity TrkA and the low-affinity P75^NTR. Studies have demonstrated that TrkA signaling plays a pivotal role in stress fracture repair.³² In cases of bone defects, increased innervation occurs prior to vascularization, ossification, and mineralization of the fracture site. Inhibition of TrkA signaling results in reduced innervation, angiogenesis, and osteoblast activity within the callus at the site of stress fracture. Activation of TrkA receptors by NGF can induce reinnervation of skeletal muscle nerves, thereby promoting bone repair including revascularization and deposition of bone matrix.^32,71

NGF may also play a role in bone repair through its low-affinity receptor P75^NTR. Studies have demonstrated that NGF-expressing macrophages stimulate the migration of bone cells and promote bone formation during the early stages of bone repair.^61,72 The combination of NGF and P75^NTR can induce cell migration and initiate recruitment of various aptamers, including C-Jun N-terminal kinase signaling. KDM4B serves as a crucial epigenetic regulator in NGF-mediated osteogenesis.⁷² Activated NGF-c-Jun binds to the promoter region of KDM4B and directly upregulates its expression. Subsequently, KDM4B actively demethylates the H3K9me3 marker on DLX5, which is a master osteogenic gene. Moreover, both KDM4B and c-Jun within the JNK signaling pathway collaborate to regulate NGF-mediated osteogenic differentiation by simultaneously recruiting the promoter region of DLX5.

Brain-Derived Neurotrophic Factor (BDNF)

BDNF is extensively distributed and extensively researched in the mammalian brain as a neurotrophic factor. BDNF signals are transmitted through the high-affinity TrkB receptor and the low-affinity p75^NTR receptor. BDNF plays a crucial role in synaptic development and plasticity,⁷³ thereby highlighting its significant therapeutic potential in neurosystem-related diseases, depression, and neurodegenerative disorders such as Alzheimer’s disease.⁷⁴

BDNF is also expressed in human osteoblasts and mediates the process of fracture healing.⁷⁵ Nanoparticles loaded with BDNF have the potential to enhance the proliferation and differentiation of mesenchymal stem cells into osteoblasts, thereby promoting osteogenesis.⁷⁶ Consistent with NGF’s role, BDNF regulates new bone production by up-regulating osteoblast activity, promoting their differentiation and mineralization, as well as facilitating fracture healing.^69,77 Furthermore, BDNF can improve trabecular microstructure, tibial biomechanical properties, and bone biochemical indices. It exhibits a certain therapeutic effect on postmenopausal osteoporosis.^75,78

The osteogenic effect of BDNF is primarily mediated through TrKB receptors. FanXue et al⁷⁸ utilized 7, 8-dihydroxyflavone (7, 8-DHF), a plant-derived small molecule agonist for TrkB that mimics the functionality of BDNF, to demonstrate the activation of the Wnt/β-catenin signaling pathway by BDNF via TrkB receptors. This activation resulted in up-regulation of cyclin D1, phosphorylated glycogen synthetase kinase-3β (p-GSK3β), β-catenin, Runx2, Osterix and osteoprotegerin expression in MC3T3-E1 cells. The increased expression of osteoprotegerin promoted cell proliferation, osteogenic differentiation and mineralization processes. Furthermore, upon binding to the TrkB receptor, BDNF also exhibited inhibitory effects on osteoclast differentiation and bone resorption by suppressing transcription factor c-fos as well as key genes involved in osteoclast function such as tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9) and Adamts5.

The BDNF/TrkB signaling pathway also activates Akt, leading to phosphorylation and inhibition of asparagine peptidase (AEP), which plays a regulatory role in the differentiation of human bone marrow stromal cells (hBMSC). 7, 8-DHF effectively inhibits the C/EBP-β/AEP pathway. Furthermore, the inhibition of Rank-L-induced RAW264.7 osteoclasts⁷⁵ provides additional evidence for the crucial involvement of BDNF in bone healing.

Neurotrophin-4/5 (NT-4/5)

NT-4/5, similar to BDNF, exhibits specific binding affinity for the TrkB receptor. When both BDNF and NT-4/5 coexist within the same environment, their effects vary depending on the sequence of exposure: BDNF does not influence the actions of NT-4/5; however, NT-4/5 inhibits the activity of BDNF.⁷⁹ During tooth development, prenatal oral and internal tooth epithelium have been found to express NT-4 mRNA,⁸⁰ suggesting a potential role for NT-4 in tooth morphogenesis. Furthermore, through activation of the ERK1/2 signaling pathway, NT-4/5 can regulate periodontal ligament cell function and induce expression of ALPase, OPN, and BMP-2 mRNA in HPL cells.⁷⁷ These findings may guide future research directions in oral studies.

Neurotrophin-3 (NT-3)

NT-3 is a neurotrophic protein that plays a crucial role in the survival and axonal regeneration of neurons in the spinal cord.⁸¹ NT-3 specifically binds to the TrkC receptor, and under certain cellular conditions, it can also activate TrkA and TrkB with reduced efficiency.⁵⁴ Studies have demonstrated that NT-3 has the potential to mitigate brain damage,⁸² as well as promote neuronal survival and differentiation. Additionally, the involvement of NT-3 in the process of fracture repair has garnered attention from relevant scholars. Su et al,³³ discovered that NT-3 and its receptor TrkC were significantly upregulated at the site of bone injury using a proximal tibial drilling injury repair model in young mice, indicating their potential to promote bone repair. In vitro experiments revealed that NT-3 can enhance osteogenic differentiation and mineralization, stimulate BMP-2 secretion, and augment its activity during the osteogenic process.

The expression of NT-3 and TrkC receptors was observed in both normal bone and osteoclasts at the site of injury repair. Osteoblast-derived NT-3 can potentially function as a regulatory signal for osteoclast mineral uptake during bone injury.^83,84 Activation of the downstream kinase Erk1/2 of TrkC is involved in mediating the effect of NT-3 on osteoclast formation. Upon activation by TGF-β, NT-3 acts on TrkC to induce endothelial-mesenchymal transformation (EndMT) and enhance MSC production, leading to Heterotopic ossification (HO). Furthermore, NT-3 promotes the differentiation of MSCs into chondrocytes and osteoblasts by upregulating Sox9, OCN, RUNX2, and other bone markers.⁸⁴ Neovascularization promotion also contributes to HO formation mediated by NT-3.⁸⁴ Additionally, macrophage-derived NT-3 has been demonstrated to stimulate osteogenic differentiation of mesenchymal lineage tendon stem cells (TDSCs) through activating ERK1/2 and PI3K/Akt signaling pathways.⁸⁵

Glial Cell Line-Derived Neurotrophic Factor (GDNF)

GDNF was identified in 1993 as a crucial neurotrophic factor for the survival of embryonic dopaminergic neurons in the midbrain.⁸⁶ It belongs to the TGF-β ligand superfamily.⁸⁷ Members of the GDNF family encompass GDNF, artemisinin, neurourea, and Persian peptide. All ligands within the GDNF family bind to various complexes through GFRα1-4. Among them, GDNF exhibits a strong affinity for GFRα-1 but typically utilizes the receptor tyrosine kinase RET as a signaling coreceptor.⁸⁷ Additionally, GDNF can also interact with other complexes via GFRα2.

Through the GDNF/GFRα1 (GDNF-GFRα1-RET-ERK) and neuroprotein/GFRα2 signaling pathways, GDNF exerts a significant influence on bone pathology or disease-related pain.^88,89 Additionally, GDNF is involved in the regulation of bone metabolism, with pre-osteoclast (poc)-derived GDNF promoting migration of bone marrow mesenchymal stem cells and enhancing osteogenic differentiation through GDNF-GFRα1-RET signaling.⁸⁷ Moreover, GDNF induces migration of Schwann cell precursors which accumulate in damaged periodontal tissue and play a crucial role in wound healing, particularly in alveolar bone tissue.⁹⁰ Furthermore, Schwann cells are vital for peripheral tissue regeneration including bone tissue regeneration; thus suggesting that GDNF indirectly influences the process of bone regeneration by regulating Schwann cell precursors. In the NRTN-GFRα2 neurotrophic axis, IL-6-induced skeletal sympathetic cholinergic nerve fibers sustain survival and function of bone cells during postnatal growth and development as well as physical activity during adolescence.⁹¹

Platelet-Derived Growth Factor (PDGF)

PDGF family comprises five dimeric ligands: PDGF-AA, -AB, -BB, -CC, and -DD.^9,92,93 Among its receptors are PDGFRα and PDGFRβ. Notably, PDGFRα is predominantly expressed in megakaryocytes while PDGFRβ is almost exclusively expressed in fibroblasts.⁹³ PDGF-BB, a chemotactic and mitotic factor within the PDGF family, facilitates the migration, proliferation and differentiation of various mesenchymal cell types such as endothelial progenitor cells and mesenchymal stem cells through signaling via PDGFRβ. This process promotes the establishment of a complete vascular network during bone healing,^94–96 thereby inducing vascularization into bone formation. It plays a pivotal role in promoting tissue repair and regeneration.⁹ Following temporomandibular arthritis, the bone microenvironment exhibits an elevated presence of platelet-derived growth factor (PDGF). Furthermore, there is an upregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9). The formation of H-type blood vessels not only facilitates subchondral bone remodeling but also induces a bone pain response.¹⁰⁸ Compared to conventional bone repair methods that solely focus on new bone quantity, vascularization osteogenesis places greater emphasis on constructing a blood vessel network throughout the bone healing process. Studies have demonstrated that this mode of vascularization osteogenesis can generate new bone with superior quality; thus making it more suitable for healing large bone defects.

In a clinical study,⁹⁷ various biomaterials were introduced into the extraction wound after tooth removal to investigate alveolar ridge preservation and bone regeneration. The findings demonstrated that the addition of rhPDGF-BB enhanced the biological properties of newly formed bone and reduced bone resorption. This could be attributed to the differentiation of macrophage/non-absorbent osteoclast lineage cells into bone-resorbing osteoclasts during bone remodeling, as well as the secretion of PDGF-BB for recruiting endothelial cells and osteoblast progenitors, thereby forming a vaso-osteoblastic unit.⁹⁵ In one experiment, it was observed that older mice and mice on a high-fat diet (HFD) exhibited excessive secretion of PDGF-BB from preosteoclasts in their bones/bone marrow compared to younger mice and those on a normal diet. Consequently, elevated serum levels of PDGF-BB led to both bone loss and arteriosclerosis.⁹⁸ This phenomenon further supports the association between PDGF-BB derived from osteoclasts and the process of bone formation. Also, Gao et al⁹⁹ also discovered that macrophage/monocyte-derived PDGF-BB could induce periosteal derived cells (PDC) to express periosteal bone proteins while attracting them for supporting osteogenesis along with H-type blood vessel formation (a vascular subtype characterized by high expression levels of CD31 and Emcn).

The PDGF-B derived from endothelial cells (EC) also serves as a crucial niche factor for BMSC, facilitating the osteogenic differentiation of bone marrow mesenchymal stem cells.⁹⁴ It plays a role in promoting osteogenesis and triggering the activation of bone stem and progenitor cells during bone repair through PDGFRβ signaling.¹⁰⁰

Fibroblast Growth Factor (FGF)

FGF belongs to a superfamily of genes that exert pleiotropic effects on various biological processes by activating the FGF receptor tyrosine kinase (FGFR).¹⁰¹ The FGF family comprises 22 members, out of which 18 are secreted and possess the ability to bind to one or more of the four FGF receptors 1–4 (FGFR1-4), thereby forming FGF signaling pathways.¹⁰² These pathways regulate intricate cellular behaviors in vertebrates, including migration, proliferation, self-renewal, lineage commitment, aging, and survival.¹⁰³

Many members of the FGF family are expressed in and around the membranous and endochondroskeletal elements, including FGF1, 2, 6–10, 17, 18, and 21–23.¹⁰³ FGF-2, FGF-9, and FGF-18 play a role in osteogenesis and may present a novel option for bone regeneration.¹⁰⁴ The bone-derived hormone FGF23 acts synergistically with parathyroid hormone (PTH) and the active vitamin D metabolite calcitriol to regulate calcium (Ca) and phosphate (P) homeostasis.^101,105 Deficiency of FGF23 results in hyperphosphatemia and ectopic calcification while excessive levels of FGF23 lead to hypophosphatemia and skeletal abnormalities.¹⁰⁶

Another type, fibroblast growth factor 8 (FGF-8), also known as androgen-induced growth factor (AIGF), plays a crucial role in limb development. Within the bone microenvironment, FGF-8 produced or received by precursor chondrocyte cells binds to fibroblast growth factor receptor (FGFR), thereby inducing activation of downstream signaling pathways to varying extents. These signaling pathways include phospholipase Cγ (PLCγ)/Ca2+, RAS/mitogen-activated protein kinase-extracellular regulatory protein kinase (RAS/MAPK-MEK-ERK), and Wnt-β-catenin-Axin2, which ultimately govern chondrocyte proliferation, differentiation, cell survival, and migration.¹⁰⁷

Neuropeptide

Neuropeptides, such as CGRP, SP, VIP, PACAP and NPY, are synthesized and secreted by neuronal cells to regulate various biological processes including bone homeostasis.¹⁰⁹ Table 2 summarizes the effects of different neuropeptides on bone healing.

Table 2 The Effect of Neuropeptides on Bone

Calcitonin Gene-Related Peptide (CGRP)

CGRP, a 37-amino acid neuropeptide belonging to the calcitonin family,¹¹⁰ exhibits sensitivity to mechanical, chemical, and electrical stimulation,^111–115 and interacts with a receptor similar to the calcitonin receptor (CLR). In humans, CGRP is classified into two types: alpha-CGRP and β-CGRP. Alpha-CGRP is encoded by the Calca gene and predominantly expressed in both central and peripheral nervous systems. On the other hand, β-CGRP is encoded by the Calcb gene and primarily found in the enteric nervous system.^116,117 Apart from its distribution in neural tissues such as cardiovascular system, respiratory system, thyroid gland; CGRP also shows equal presence in bone marrow and periosteal tissue.¹¹⁸ Furthermore, it can exert its effects on various non-neuronal tissues including bone tissue itself.^{115,119–122}

CGRP and its receptor CLR, along with the receptor active Modification protein 1 (RAMP1) complex, have been demonstrated to play a crucial role in the pathogenesis of migraine.^123–128 Moreover, CGRP’s involvement in bone turnover has also been confirmed.^129,130 Specifically, CGRP promotes bone formation and contributes significantly to α-CGRP’s function.¹²⁹ Studies on mice lacking alpha-CGRP have revealed reduced bone formation and osteopenia, indicating that alpha-CGRP serves as a physiological activator of bone formation.

CGRP promotes osteoblast differentiation and function while inhibiting osteoclast development and activity.^131,132 Overexpression of CGRP in osteoblasts specifically upregulates insulin-like growth factor I (IGF-I) secretion and downregulates tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) secretion. These cytokines, including IGF-I, TNF-α, and IL-6, are produced by various cell types including osteoblasts. IGF-I has been shown to promote positive bone balance while TNF-α and IL-6 have been implicated in the pathogenesis of osteoporosis. This finding suggests that CGRP protects bones through these pathways. Furthermore, CGRP receptors on the plasma membrane of periosteoblasts and osteoclasts bind to CGRP, inhibiting osteoclast formation and bone resorption by blocking RANKL-mediated NF-κB activation. As a result, this leads to positive bone balance and increased bone mineral density.¹³³

The CGRP receptor has also been identified on the plasma membrane of bone marrow mesenchymal stem cells, and CGRP has the ability to stimulate the proliferation of these cells.¹³¹ Additionally, it can induce osteoblast gene expression and synergize with Wnt signaling to enhance bone formation.^{131,134–136}

The role of CGRP involves pain transmission and sensitization, with its pain intensity positively correlated to serum CGRP levels.^137,138 Following a fracture, there is an increase in CGRP content at the site of bone injury, indicating its potential involvement in the process of fracture pain transmission. Furthermore, CGRP plays a role in bone regeneration after a fracture. It reaches peak levels during the early post-fracture period and promotes bone formation by facilitating larger callus formation. Over time, CGRP gradually decreases to allow for normal or physiological bone remodeling.¹³⁹ In terms of bone healing, the expression and activity of NOS are closely associated with CGRP expression.¹⁴⁰ By inducing NOS-mediated vasodilation, local blood flow increases due to CGRP’s promotion of fracture callus formation.¹⁴¹ Additionally, nerve fibers containing sensory neuropeptide CGRP proliferate within and around the callus.¹⁴² Apart from the NOS pathway, local administration of CGRP may also contribute to rapid recovery from bone defects by promoting positive bone balance. Moreover, it can assist in fracture healing through ERK phosphorylation stimulation.¹⁴³

The bone tissue is innervated by a dense sensory neural network, with the CGRP-positive nerve being the most commonly distributed sensory nerve.¹⁴⁴ During fracture healing, CGRP-positive nerves exhibit regenerative properties.^22,23,145 Following neuronal depolarization at their endings, CGRP is released and exerts its biological functions in bone regeneration, including angiogenesis and osteogenesis.^146,147 Furthermore, researchers have discovered that loading CGRP into porous microspheres can provide protection for BMSCs against inflammatory environments and promote bone regeneration.¹⁴⁸ To some extent, this suggests the protective effect of CGRP on BMSCs in an inflammatory environment.

Substance P (SP)

SP is a tachykinin-like peptide composed of 11 amino acids that triggers a signal transduction cascade via the neurokinin-1 receptor (NK1-R).¹⁴⁹ Additionally, SP can be produced by non-neuronal cells and exert its function. SP+ nerves are widely distributed in periosteum, subchondral bone, bone marrow, ligaments, synovium, and other bone tissues.¹⁵⁰

Delayed mobilization of mesenchymal stem cells may result in delayed bone healing following fracture.¹⁵¹ Substance P can serve as a stimulating factor to facilitate the migration of CD29 mesenchymal stem cells towards the site of bone defect, thereby expediting the process of bone healing.^152–154 Furthermore, Substance P not only promotes mobilization and migration of mesenchymal cells but also enhances their proliferation and osteogenic differentiation.^155,156 Additionally, Substance P exerts an influence on bone metabolism and is considered an osteoneutronic mediator that regulates bone remodeling through increased neuro-bone crosstalk activity such as Ca2+ signaling.^157,158 Studies have reported that Substance P has been observed to augment the expression and mineralization of osteogenic markers in bone marrow stromal cells.^156,159 Moreover, by promoting the secretion of angiogenic cytokines to stimulate angiogenesis and enhance local blood flow, Substance P indirectly affects bone metabolism while participating in callus development and facilitating bone healing processes.¹⁴¹

The hydrogel experiment on skull regeneration revealed that chitin-PLGA-calcium sulfate hydrogel scaffolds, incorporating both SP and lactoferrin (LF) for dual regulation, exhibited superior osteogenic induction potential compared to single-component hydrogel scaffolds. This suggests that the synergistic combination of bone-inducing biomolecules and SP can effectively enhance bone regeneration.¹⁶⁰

Jun-Kyu Lee et al¹⁶¹ also discovered that the combined treatment of 4-hexylresorcinol (4HR) and substance P (SP) synergistically induces the phosphorylation of p38 MAPK and ERK in human umbilical vein endothelial cells (HUVECs), thereby upregulating the expression level of vascular endothelial growth factor (VEGF). This, in turn, promotes the migration and tubular ability of endothelial cells, ultimately accelerating bone formation and maturation. Furthermore, SP expression enhances HUVEC migration and angiogenesis while inhibiting osteoclast formation. Based on this synergistic effect, it is possible to achieve bone regeneration by inhibiting the RANKL pathway both in vitro and in vivo to counteract osteoporosis.

Vasoactive Intestinal Peptide (VIP)

VIP is a 28-amino acid prelysate product of VIP,¹⁶² and it acts on three receptor subtypes: VPAC1, VPAC2, and PAC1.¹⁶³ Immunoreactive nerve fibers containing VIP were identified in the periosteum and bone of animals.²⁸ VIP plays a crucial role in bone metabolism and remodeling processes.¹⁶⁴ By activating specific VIP receptors in osteoclasts and osteoblasts, VIP can regulate bone resorption activity and promote osteoblast differentiation.^165–167 Specifically, VIP can enhance osteoblast activity to facilitate the production of bone tubercle alkaline phosphatase (ALP) and augment calcium accumulation,^168,169 while concurrently inhibiting osteoclast activation to reduce bone resorption.¹⁶⁶ Another study revealed that VIP can stimulate bone formation by activating the Wnt/beta-catenin signaling pathway in bone marrow stromal cells (BMSC).¹⁷⁰ In a mouse model of 6-hydroxydopamine-induced sympathectomy, local injection of VIP improved both the quality and mechanical properties of bones, leading to an upregulation in the expression of osteogenic markers.¹⁶⁴

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)

PACAP is a neuropeptide¹⁷¹ present in the hypothalamic-pituitary system with a significantly short half-life¹⁷² in vivo. It exists in two bioactive forms, PACAP 1–38 and PACAP 1–27. PACAP exhibits three major G-protein-coupled receptors, namely PAC1, VPAC1, and VPAC2, among which PAC1 shows the highest affinity towards PACAP.^172,173 Through these receptors, PACAP can stimulate adenylate cyclase activation, elevate cellular cAMP concentration and initiate PKA phosphorylation activity. Consequently, it may activate the CREB transcription factor or Runx2 gene downstream of this kinase in osteoblasts. Moreover, PACAP demonstrates cross-talk effects with multiple pathways including Runx2-, BMP-, or WNT-associated pathways,^174,175 all of which have regulatory effects on osteogenesis. This suggests that PACAP plays an osteogenic role. Studies have shown that PACAP can modulate bone differentiation processes,¹⁷⁶ its absence can alter the microstructure of long bones,¹⁷⁷ and it has a protective effect against mechanical forces during cartilage differentiation as well.¹⁷⁵ In preclinical models of osteoarthritis (OA), it has been observed that PACAP possesses anti-inflammatory properties and protects cartilage integrity,¹⁷⁸ Therefore, PACAP exerts positive effects during both the inflammatory stage and bone remodeling stage of bone healing. Utilizing this property to enhance bone regeneration is one of the challenges we need to address.

Neuropeptide Y (NPY)

NPY is a highly conserved neurotransmitter peptide, predominantly released by sympathetic nerves in the central or peripheral nervous system.¹⁷⁹ Its immunoreactive fibers are primarily distributed in periosteal tissues, bone marrow, cortical bone, and blood vessels.^{118,119,180–182} NPY receptors encompass Y1, Y2, Y4, Y5, and Y6 subtypes.¹⁸³

NPY is involved in bone biology through various pathways. Initially, it was believed that NPY indirectly regulates bone by influencing blood vessels.^182,184 However, subsequent findings revealed the expression of NPY in MC3T3-E1 osteoblasts and bone marrow mesenchymal stem cells, as well as their role in osteogenic differentiation.¹⁸⁵ Additionally, the presence of NPY receptors in bone cells has been identified,^150,186 indicating a direct involvement of NPY in regulating osteoblast activity. Studies have demonstrated that cultured bone marrow stromal cells (BMSC) and osteoblasts express only the Y1 receptor while lacking expression of Y2, Y4, Y5 or Y6 receptors.¹⁸⁷ Nevertheless, further investigation confirmed the expression of Y2 receptor in MC3T3-E1 preosteoblasts derived from mouse skulls,¹⁸⁸ with its mRNA expression occurring under conditions promoting osteoblast differentiation.¹⁸⁹ In studies involving Y2 ^−/− knockout mice, an increase in both the number of bone progenitor cells and osteoblastic activity was observed. This led to elevated trabecular bone volume and thickness along with enhanced bone formation within these mice lacking functional Y2 ^−/− receptors.¹⁹⁰ The anatomical structure of the NPY/NPY receptor is illustrated in Figure 3 below.

Figure 3 Anatomical structures with NPY/NPY receptors. Peripheral nerve fibers derived from basal, dorsal root and sympathetic ganglia innervate the bone and release NPY in the sites of innervation. Besides peripheral innervation, bone biology is also centrally regulated by NPY (highly expressed in the hypothalamus) and probably also by autocrine mechanisms, as osteoblasts (expressing Y1 and possibly Y2) are themselves capable of producing and secreting NPY. Reprinted from Franquinho F, Liz MA, Nunes AF, et al. Neuropeptide Y and osteoblast differentiation – the balance between the neuro-osteogenic network and local control. FEBS J. 2010;277(18):3664–3674. © 2010 The Authors Journal compilation © 2010 FEBS.192

NPY functions as a negative regulator of Y1 receptor expression.¹⁸⁹ Stimulation of bone progenitor cells with NPY leads to significant downregulation of the Y1 receptor, thereby enhancing osteoblast phenotypic markers and demonstrating the osteogenic promoting effect of NPY. Furthermore, during the early stages of osteoblast differentiation, NPY inhibits the transcriptional activity of RANKL promoter in osteoblasts, resulting in increased expression of OPG.^189,191 When directly acting on osteoblasts, the NPY pathway plays a crucial role in maintaining bone homeostasis.

Programmed Regulatory Role of Nervous System Small Molecules in Bone Regeneration

Stage of Bone Injury

In the initial stage following bone defect occurrence, blood vessels rupture, leading to local hematoma formation, inflammation formation, and pain sensation. NGF,¹⁹³ BDNF,⁶⁵ CGRP,¹³⁷ SP,^138,194,195 VIP¹⁹⁶ and PACAP are believed to be involved in the initiation, maintenance and sensitization of peripheral pain. This process is indirectly regulated by recruiting sensory nerves during bone regeneration. In the early inflammatory response phase, there may be a variety of small molecules acting together. Functional NGF receptors have been identified in monocytes,¹⁹⁷ B cell or T cell clones.¹⁹⁸ In stressful situations, NGF functions as a broad “alarm” molecule that can recruit and activate local and systemic defense systems. It also causes monocyte toxicity, basophil differentiation, mast cell formation and degranulation.¹⁹⁹ Apart from the immune response triggered by NGF, microglia - the main innate immune cells of the central nervous system - participate in early immune responses by releasing BDNF. The effects of microglia may also be regulated by BDNF.²⁰⁰ Additionally GDNF and NT-3 are involved in immune responses but their effects are not prominent.²⁰¹ FGF, CGRP and SP act as anti-inflammatory factors during the inflammatory stage of bone repair to prevent excessive inflammation. Specifically,FGR-21 inhibits IL-1 expression in lipopolysaccharide-induced THP-1 cells in vivo.Additionally,FGR-21 is associated with an increase in IL-10 through ERK1/2 and NF-kB pathways.²⁰² CGRP participates in validation processes by inducing overexpression of IL-10 in innate immune cells while inhibiting NF-kB activity.²⁰³ As a superior anti-inflammatory agent. SP can inhibit expression of inflammatory cytokines around injury sites.¹⁶¹

Stage of Fibrovascular Healing

During the fibrovascular healing phase of bone regeneration, blood vessels and mesenchymal stem cells/progenitor cells (MSCS) are recruited into the temporary callus to re-establish the vascular network at the bone defect. Small molecules in the nervous system promote vascular regeneration and subsequently osteogenesis through various mechanisms, forming a complex neuro-vascular-bone regulatory network. NGF acts as a chemical attractant for ECs, facilitating their migration in human and pig aorta^204,205 and faster recruitment to bone defect sites. Additionally, NGF promotes VEGF production and induces angiogenesis by binding to TrkA receptors.^206,207 In a study²⁰⁸ examining patients with both clavicular fractures and TBI, elevated levels of NGF, CD31 (an endothelial cell marker), and VEGF were also observed to enhance VEGF-mediated angiogenesis for accelerated fracture healing. BDNF can bind to TrkB receptors to stimulate VEGF production;²⁰⁹ however, unlike NGF, its pro-angiogenic effect is not inhibited by VEGF neutralization. NT-4/5 and NT-3 also possess angiogenic properties.²⁰⁹ In human bone marrow mesenchymal stem cells, NT-3 not only significantly increases the release of vasoactive substances such as VEGF and NGF in vitro²¹⁰ but also enhances blood circulation recovery after ischemia or ischemia-induced injury.²¹¹ GDNFs promote BMSC migration while controlling microvascular networks independently of VEGFs activity,²¹² thereby promoting vascular network growth.²¹³ PDGF acts as a chemoattractant and mitogen for mesenchymal cells promoting angiogenesis along with recruiting bone progenitor cells.²¹⁴ FGFs and VEGFs influence survival, promotion, migration and differentiation of endothelial cells.²¹⁵ CGRP itself exhibits potent vasodilation and angiogenesis-inducing effects,²¹⁶ while CGRP-positive nerves with both vascular and non-vascular terminations have been identified in rat long bones¹⁵⁰ thereby highlighting the vasogenic role of CGRP in peribone tissues. Nitric oxide produced by SP can act as an autocrine regulator of microvascular events essential for neovascularization.²¹⁷

Stage of Bone Healing

After the completion of vascular network construction, bone progenitor cells recruited to the bone defect site initiate osteoblast and chondrocyte development, thereby promoting bone formation through intrachondral and intramembranous ossification. This is followed by cartilage mineralization, woven bone formation, and remodeling. As a crucial participant in bone healing,²¹⁸ NGF regulates the survival and differentiation of osteoblast cell lines by activating the BMP2/Runx2 signaling pathway.²¹⁹ Additionally, it can induce osteoclast generation independently of RANKL,²²⁰ thus regulating both osteogenesis and bone resorption processes. NGF plays a direct role in bone repair and remodeling during bone regeneration. When stimulated by mechanical load, osteoblasts produce NGF which activates TrkA receptors in the periosteum to enhance Wnt/β-catenin signaling in both osteoblasts and osteocytes through NGF-TRKA signaling.²²¹ BDNF shares similar effects on osteogenesis as NGF does; it positively regulates new bone formation by inducing proliferation, differentiation, and mineralization of osteoblasts²²² while also contributing to RANKL secretion from human BMSC for enhanced osteoclast generation,²²³ ultimately achieving effective bone remodeling. However, BDNF and its receptor TrkB are localized to the fracture tissue during the inflammatory and early stages of bone formation, where BDNF is exclusively present in the granulation tissue along the periphery of woven bone, while being absent in chondrocytes and mature bone. NT-3 promotes osteoblast differentiation and contributes to bone regeneration. By regulating endothelium-interstitial transition (EndMT), NT-3 may induce ectopic ossification. Proosteoclast (poc)-derived GDNF also enhances osteogenic differentiation of BMSC through GDNF-GFRα1-RET signaling pathway. In developing rats, moderate bFGF promotes endochondral osteogenesis but inhibits endoperiosteal osteogenesis.²²⁴ CGRP and SP can also induce osteoblast differentiation of BMSC via Wnt/β-catenin signaling pathway. Additionally, CGRP blocks RANKL-mediated NF-κB activation in bone marrow-derived macrophages (BMM), thereby suppressing osteoclast generation and bone resorption.^131,132 In BMSCs, SP appears to stimulate BMP-2 expression and migration, thus influencing bone formation.¹⁵⁵ VIP and PACAP attenuate the stimulatory effect of vitamin D3 on RANKL and RANK expression by reversing vitamin D3’s inhibitory effect on osteophorin expression—a RANK antagonist—thereby inhibiting bone resorption.²²⁵

Conclusions

Numerous studies have confirmed that nerve tissue within bone tissue regulates the behavior of bone tissue through signaling pathways, and there is also an interaction between bone repair and nerve growth.^226,227 Currently, when constructing certain bone tissue engineering scaffolds, consideration has been given to the role of nerve tissue, leading to the development of relevant scaffolds based on bionics principles.²⁰ Through specific signaling pathways, various small molecules from the nervous system can promote osteogenic differentiation of bone progenitor cells, inhibit osteoclastic differentiation, and coordinate migration of bone cells. This direct promotion facilitates bone healing. Additionally, these molecules indirectly contribute to promoting bone regeneration by facilitating migration of vascular endothelial cells, reconstruction of vascular networks, and establishment of a neuro-vascular-bone regulatory network. Figure 4 below shows how neuro-bone tissue engineering is used.

Figure 4 Schematic representation of neuro-bone tissue engineering strategies. (a) Regulate cell differentiation by controlling the surface morphology and structures of scaffolds; (b) regulate cellular behavior by modifying scaffolds with neural growth factor/neuropeptides. (c) Incorporate bioactive ions that can influence cellular behavior into scaffolds. (d) Scaffolds containing electroactive nanoparticles. (e) Coculture systems mimic the crosstalk between different cells during bone regeneration. (f) External field stimulation activates endogenous neurogenic repair signals, including skeletal interoception (left) and neuropeptide secretion (right).Reprinted from Sun W, Ye B, Chen S, et al. Neuro-bone tissue engineering: emerging mechanisms, potential strategies, and current challenges. Bone Res. 2023;11(1):65. Creative Commons.228

Due to the short half-life of small molecules in the nervous system, their bioavailability is limited or their direct application is ineffective. When developing bone replacement materials, one approach involves encapsulating small molecules from the nervous system within a carrier to achieve controlled release. For instance, Liang et al²²⁹ loaded naringenin (NG) and CGRP into a hydroxyapatite-sodium alginate composite hydrogel scaffold, enabling gradual release of CGRP during bone regeneration to sustain its role in promoting bone growth. However, finding suitable carrier materials as an in vivo platform for these small molecules remains a key challenge in future bone tissue engineering endeavors. Currently, certain biocomposite scaffolds have demonstrated the ability to enhance local bone healing^230,231 and exhibit stem cell-promoting effects,²³² indicating the potential for these scaffolds to be modified with neurogenic factors to facilitate bone regeneration. This approach, however, introduces additional design challenges, including the efficient loading of these small molecules and the creation of an optimal environment for their controlled release, thereby ensuring the effective functioning of all stages of bone healing.²²⁸

Additionally, exploring appropriate substitutes for nervous system small molecules that can exert similar osteogenic effects is also a promising direction for bone tissue engineering research. Indeed, utilizing Trk receptor agonists like 7, 8- DHF to activate osteogenic signaling pathways associated with neurotrophins has been shown to be feasible; Considering the role of various small molecules at various stages, the synthesis of endogenous bone peptides with osteogenic activity²³³ can regulate bone metabolism and promote bone homeostasis, which is a further application of biomaterials.

While this paper has summarized the role of numerous neuropeptide small molecules in bone regeneration, many members of the extensive neuropeptide family do not directly engage in bone tissue healing. Instead, they exert an indirect regulatory influence via the endocrine system, a field referred to as bone neuroendocrinology.²³⁴ Moreover, sensory neurons within the nervous system collaborate synergistically with these small molecules to enhance their effects.²³⁵ The human body maintains a complex and intricately regulated internal environment; for instance, menopausal women may experience decreased bone mineral density due to estrogen deficiency, resulting in osteoporosis.²³⁶ It is evident that each physiological system interacts and influences one another. Further in-depth research is required to fully understand the various regulatory substances involved in bone healing.

Programmed regulation of bone regeneration has emerged as a prominent research area in recent years, with various small molecules within the nervous system playing crucial roles throughout the entire process. However, an urgent challenge that remains is how to design materials capable of controlled release and optimal functionality at specific time points. Therefore, this paper aims to provide a comprehensive review on the involvement of small molecules from the nervous system in bone regeneration, integrating their biological behaviors across different stages. The ultimate goal is to offer theoretical support for future advancements in designing bone replacement materials.