CGRP is widely distributed in bone tissue and promotes osteogenesis, inhibits osteolysis, induces angiogenesis, and regulates the immune microenvironment (Figure 1).

Bone tissue is rich in CGRP, with two types of nerve fibers—substance P (SP)-positive and CGRP-positive nerve fibers. They are mainly present in bone, bone marrow, periosteum, synovium, and adjacent soft tissues. The proportion of CGRP-positive nerve fibers is dominant [49], and CGRP is a critical node that cannot be bypassed and is a bridge between nerve and bone repair. The receptors of CGRP, CLR, and RAMP1 are widely distributed in vivo, and the co-localized expression of CLR and RAMP1 is also crucial for CGRP to exert its effects. In bone tissue, macrophages, osteoblasts, and vascular tissue, CLR and RAMP1 expression is abundant in macrophages, osteoblasts, and endothelial cells [50,51,52,53,54]. This regulates osteogenesis by affecting the immune microenvironment, vasculature, and accompanying nerves. Immune cells promote bone production by secreting certain osteogenic factors [55]. In contrast, blood vessels and nerve fibers are distributed throughout the bone tissue, providing oxygen, nutrients, and supporting cells to the bone tissue. Therefore, they are essential in bone growth, development, and fracture healing [56]. In short, sensory neurons are stimulated to continuously release CGRP into bone tissue to achieve neurological bone regulation.

Among the neuropeptides identified in bone tissue (CGRP, SP, NE, NPY), CGRP is most strongly associated with bone repair [57]. Mice with a systemic knockout of the CGRP gene have significantly reduced bone mass. In contrast, overexpression of the CGRP gene significantly enhances bone density [58,59]. Therefore, CGRP promotes bone repair accompanied by nerve fiber ingrowth. There were fewer CGRP-positive nerve fibers at inflammatory progression sites (larger defect sites) and more CGRP-positive nerve fibers at repair sites (smaller defects) during knee and ankle osteoarthritis. Thus, inflammation destroying bone may induce the ingrowth of CGRP-positive fibers, producing CGRP for local bone repair [60]. Sequencing results indicate that CGRP promotes extracellular matrix production, which may be an essential pathway for its bone repair effects [54]. Sensory nerves maintain extracellular matrix (ECM) homeostasis through the CGRP/CHSY1 axis, and the knockdown of sensory nerve CGRP induces similar disturbances in ECM metabolism [61].

CGRP can upregulate various osteogenic factors and promote osteoblast anabolism [62,63]. Haitao et al. observed that CGRP promotes elevated levels of cAMP, ATF4, and OCN expression in osteoblasts [64]. ATF4 is an ATF/CREB family member, a cell-specific CREB-related transcriptional essential for osteoblast differentiation and function factor [65]. ATF4 is identified as an osteoblast-specific transcription factor necessary for OCN transcription, an osteoblast-specific marker commonly used to indicate late osteoblast differentiation [66,67]. BMP2 is essential in osteogenesis induction and promotes ECM expression, mainly collagen production and calcium salt formation, using the Smad pathway [68,69,70]. In MG-63 cells, BMP2 can be involved in CGRP-induced osteogenic differentiation, and cAMP/p-CREB upregulation further promotes BMP2 expression [71]. Dental pulp stem cells (DPSCs) possess the qualities of bone marrow mesenchymal stem cells and are the primary source of dentin mineralization. CGRP directly stimulates a 1.8-fold increase in BMP2 mRNA expression in DPSCs, and a 2.8-fold increase in basal levels of cAMP, which promotes dentin formation. Liping et al. observed that CGRP stimulated bone marrow MSC proliferation, upregulated osteoblast gene expression, enhanced alkaline phosphatase activity, and increased calcium nodules in bone marrow mesenchymal stem cells (BMSCs) [72]. Direct action of CGRP on BMSCs elevated the migratory capacity and osteogenic differentiation and inhibited the bone marrow differentiation of MSCs to adipocytes [54,73]. This process involves molecular crosstalk between Wnt/β-catenin and CGRP signaling [74] other than enhanced cAMP response element binding protein 1 in periosteal-derived stem cells (CREB1) and SP7 (osterix, OSX) expression in periosteal-derived stem cells. Thus, osteogenic differentiation of periosteal-derived stem cells is promoted [75]. In vivo studies identified elevated CGRP levels in mice with femoral fractures [76]. Similar upregulatory effects could be seen when serum CGRP expression was examined in femoral neck fracture patients [77]. CGRP was used to transiently act on bone healing tissue and bone formation regulators (IL1b, Ccl7, MMP13, Mrc1), which increased the PPARγ pathway (Adipoq, Fabp4, Scd1, Cfd) members [76]. Thus, local bone defect repairing was induced by releasing CGRP from nerve endings within the periosteum [75]. Electrical stimulation (ES) of the spinal dorsal root ganglion directly enhances the biosynthesis and release of CGRP by activating Ca²⁺ and accelerating femur fracture healing in osteoporotic rats [15]. Therefore, CGRP is essential in the early generation of bone healing tissue by activating osteogenic effect-related pathways. CGRP also promotes the production of osteogenic factors, collagen, and extracellular matrix, facilitating bone repair.

CGRP inhibits osteoclasts, increases bone volume, and reduces bone resorption [58,78]. Previous studies have indicated that sympathetic and sensory nerves are cross-linked to the osteoclastic effect. In contrast, CGRP reverses the isoproterenol (Isp)-mediated osteoclastic effect [79] and inhibits the proliferation of granulocyte-macrophage lineage progenitor cells (precursor osteoclast cells) [80]. CGRP increases OCN and OPG in osteoblasts and inhibits RANKL expression [64]. RANKL is a RANK ligand, an activatable NF-κB pathway that promotes the proliferation and effects of osteoclasts. CGRP inhibits osteogenesis by suppressing RANKL expression, inhibiting the NF-κB pathway, and downregulating the expression of osteoclast TRAP and histone K [72]. However, NF-κB also has a vital role in senescence and apoptosis, and attention must be paid to whether CGRP plays a crucial role in their senescence and apoptosis.

CGRP is vital in blood vessel formation and growth [81] and can indirectly affect bone development and formation [82,83]. Bone development cannot be separated from the expression of CGRP, vascular endothelial growth factor (VEGF), a cluster of differentiation 31 (CD31), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) [84]. CGRP-positive nerve fibers during early embryonic development are present in blood vessels, developing muscles, or developing cartilage bones [85]. Angiogenesis and bone regeneration were observed by local CGRP injection into the defect site in rats [86]. CGRP promotes VEGF expression to enhance endothelial cell proliferation and migration [87,88], a mechanism validated in tumor-associated angiogenesis [89]. Moreover, CGRP promotes endothelial progenitor cell proliferation and restricts apoptosis by inhibiting MAPK signaling [90]. OCN upregulation, ALP gene expression, and increase in mineralized nodules in osteoblast (OB) monoculture and human umbilical vein endothelial cell (HUVEC) OB co-culture systems could be associated with particular cytokine expression regulation in HUVEC by CGRP [91]. Biodegradable biomagnesium implants can accelerate bone repair by upregulating CGRP and promoting angiogenesis [92]. BMSCs overexpressing CGRP can improve bone repair by promoting peripheral angiogenesis in diabetic rats having tibial defects [93]. CGRP release at the bone defect site can contribute to angiogenesis, enhancing repair [94].

Inflammation occurrence is essential for bone repair [95,96]. Mice with CGRP knockdown exhibit a higher degree of oxidative stress accompanied by reduced nitric oxide synthase (NOS) expression, increased phosphorylated p47 expression, elevated 4 hydroxynonenal (4HNE) levels, and macrophage infiltration [97]. CGRP inhibits LPS-induced TNFα production by macrophages [98] and osteoblasts [99]. Sensory nerve fibers secrete CGRP to inhibit inflammation by suppressing type 1 T helper cytokine production and leukocyte proliferation. CGRP affects the polarization of M0-type to M2-type macrophages, affecting bone tissue remodeling in the later stages of fracture repair [100]. Moreover, CGRP induces bone regeneration and differentiation by elevating M2-type macrophage proportion [101]. Lack of CGRP promotes M1 and inhibits M2 polarization in macrophages. Thus, CGRP knockdown mice hinder osseointegration in bone grafts, and its overexpression improves osseointegration by regulating the macrophage phenotype [102].

However, osteogenic factors (BMP2, BMP6, WNT10b, and OSM) secreted by M2 macrophages were reduced in the early stages and elevated at later stages of CGRP action [103]. Pajarinen et al. indicated that proper inflammation is beneficial in the early stages of bone repair. M1 macrophages may exert their effects in the early and middle stages of osteogenesis. In contrast, M2 macrophages later affect bone matrix mineralization [55], which aligns with the prevailing view of immune regulation of bone repair. Thus, CGRP plays a vital role in regulating the immune microenvironment for bone repair [96,104].