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Sensory Innervation of Human Bone: An Immunohistochemical Study to Further Understand Bone Pain

Open AccessPublished:May 04, 2021DOI:https://doi.org/10.1016/j.jpain.2021.04.006

      Highlights

      • Innervation density across various human bones was studied by immunohistochemistry.
      • The periosteum was most densely innervated, followed by bone marrow and cortex.
      • Thoracic spine and parietal bones showed most and least innervation, respectively.
      • Innervation density decreased with age, in the elderly population studied.
      • The results can help explain the severe pain accompanying skeletal disease.

      Abstract

      Skeletal diseases and their surgical treatment induce severe pain. The innervation density of bone potentially explains the severe pain reported. Animal studies concluded that sensory myelinated A∂-fibers and unmyelinated C-fibers are mainly responsible for conducting bone pain, and that the innervation density of these nerve fibers was highest in periosteum. However, literature regarding sensory innervation of human bone is scarce. This observational study aimed to quantify sensory nerve fiber density in periosteum, cortical bone, and bone marrow of axial and appendicular human bones using immunohistochemistry and confocal microscopy. Multivariate Poisson regression analysis demonstrated that the total number of sensory and sympathetic nerve fibers was highest in periosteum, followed by bone marrow, and cortical bone for all bones studied. Bone from thoracic vertebral bodies contained most sensory nerve fibers, followed by the upper extremity, lower extremity, and parietal neurocranium. The number of nerve fibers declined with age and did not differ between male and female specimens. Sensory nerve fibers were organized as a branched network throughout the periosteum. The current results provide an explanation for the severe pain accompanying skeletal disease, fracture, or surgery. Further, the results could provide more insight into mechanisms that generate and maintain skeletal pain and might aid in developing new treatment strategies.

      Perspective

      This article presents the innervation of human bone and assesses the effect of age, gender, bone compartment and type of bone on innervation density. The presented data provide an explanation for the severity of bone pain arising from skeletal diseases and their surgical treatment.

      Key words

      Musculoskeletal diseases are highly prevalent in the general population and are a leading cause of disability among patients and medical costs every year.
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      Several mammalian studies identified two important types of sensory nerve fibers performing these functions: myelinated A∂-fibers and smaller diameter, unmyelinated C-fibers.
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      The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.
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      Size, neurochemistry, and segmental distribution of sensory neurons innervating the rat tibia.
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      Absence of large-diameter sensory fibres in a nerve to the cat humerus.
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      • Mantyh PW
      Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain.
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      • Mantyh WG
      • Bloom AP
      • Xu H
      • Ferng AS
      • Dussor G
      • Vanderah TW
      • Mantyh PW
      A phenotypically restricted set of primary afferent nerve fibers innervate the bone versus skin: therapeutic opportunity for treating skeletal pain.
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      • Mahns DA
      • Ivanusic JJ
      • Sahai V
      • Rowe MJ
      An intact peripheral nerve preparation for monitoring the activity of single, periosteal afferent nerve fibres.
      A∂-fibers are mainly responsible for conducting sharp, localized pain, while C-fibers transmit dull, diffuse pain.
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      The neurobiology of skeletal pain.
      Earlier mammalian studies concluded that innervation density of sensory nerve fibers is highest in the periosteum, followed by bone marrow and cortical bone.
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      • Jimenez-Andrade JM
      • Bloom AP
      • Taylor RN
      • Mantyh WG
      • Kaczmarska MJ
      • Ghilardi JR
      • Mantyh PW
      The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.
      ,
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      • Sabino MC
      • Luger NM
      • Schwei MJ
      • Pomonis JD
      • Keyser CP
      • Clohisy DR
      • Adams DJ
      • O'leary P
      • Mantyh PW
      Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur.
      The periosteum is therefore suggested to be a major contributor to pain arising from musculoskeletal diseases, but data in humans are lacking.
      The aim of the present study was to quantitatively explore the number and distribution of A∂-fibers, C-fibers, and sympathetic fibers in axial and appendicular human bones using immunohistochemistry, and fluorescent and confocal microscopy. Furthermore, this study set out to assess the distribution of nerve fibers between different bone-related compartments (ie periosteum, bone marrow, and cortical bone), between different anatomical locations (ie neurocranium, thoracic spine, upper extremity and lower extremity), and between genders. Lastly, the effect of age on bone innervation in the elderly population included in this study was determined.

      Methods

       Tissue Collection

      In order to represent the human skeleton as completely as possible, experiments were performed on available anatomical specimens from parietal bones of the neurocranium, thoracic vertebral bodies, midshaft humeri, midshaft radii, midshaft femora, and midshaft tibiae. Of all bones, a cross-section containing periosteum, cortical bone and bone marrow was retrieved (Fig. 1). To prevent interference of joint disease in innervation density, midshaft portions of long bones were used.
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      No approval was needed from the medical research ethics committee for this study. Tissues were collected from bodies that entered the department of anatomy of our institution through a donation program. These persons provided written consent during life, that allowed the use of their entire bodies for educational and research purposes and therefore, samples were collected based on availability.
      Figure 1
      Figure 1A cross-section of a radius stained with hematoxylin and eosin (H&E) displays the three bone-related compartments: bone marrow/trabecular bone (A), cortical bone (B) and periosteum (C) (50x magnification). The bone marrow consists of hematopoietic stem cells and adipose tissue (asterisk). In the cortical bone, Haversian canals run parallel to the longitudinal axis of the bone as part of an osteon and contain blood vessels and/or nerve fibers (arrow). The periosteum is a thin sheath of connective tissue that surrounds the outer surface of the cortical bone and consists of a cellular rich layer and a fibrous layer.
      Bodies were previously fixated in 4% formaldehyde for varying durations (6 mo – 4 y). Age and sex of the bodies was known. From the long bones, 0.5 cm transversal sections of the diaphysis were gently cut using a saw (Dremel 3000, Racine, WI, USA), and from the thoracic vertebrae 0.5 cm sagittal sections of the vertebral body. Sections measuring 1 × 1 cm from the parietal bone of the neurocranium were obtained using a saw. The anatomical specimens were stored until further processing in 4% buffered formaldehyde to preserve tissue characteristics and prevent decay.

       Tissue Processing

      The collected anatomical specimens were decalcified using 0.5 M ethylenediaminetetraacetic acid (EDTA). Every seven days the decalcification progress was radiographically monitored using a µ-CT scanner (PerkinElmer Quantum FX, Waltham, MA, USA). After each cycle of seven days, anatomical specimens were again fixated with 4% formaldehyde overnight, and placed in fresh EDTA the next day. Upon complete decalcification, anatomical specimens were stored in 15% sucrose in 0.1 M phosphate-buffered saline (pH 7.4) at 4ºC for further processing.
      Anatomical specimens were dehydrated in graded ethanol series, cleared in xylene and embedded in paraffin. 5 μm thick sections were cut and mounted on clean, positively charged microscopic slides (VWR Premium Printer Slides, Radnor, PA, USA). Cross-sections were dried overnight at room temperature (RT), placed for five hours on a 60ºC plate, and subsequently incubated at 60°C overnight to improve microscopic slide attachment.

       Immunohistochemistry on Bone Cross-Sections

      With immunohistochemistry, distinctive molecular markers can be used to label and visualize specific sensory and sympathetic nerve fibers. A∂-fibers were labeled with anti-neurofilament 200 kD (NF-200) antibodies (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). NF-200 is an intermediate filament part of the cytoskeleton of A∂-fibers.
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      ,
      • Lawson SN
      • Waddell PJ
      Soma neurofilament immunoreactivity is related to cell size and fibre conduction velocity in rat primary sensory neurons.
      C-fibers express calcitonin gene-related peptide (CGRP), a neurotransmitter involved in nociception and nerve injury, and were labeled with anti-CGRP (Sigma-Aldrich, St. Louis, MO, USA).
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      ,
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      • Bloom AP
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      • Delong D
      • Ghilardi JR
      • Kuskowski MA
      • Mantyh PW
      Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain.
      ,
      • Schou WS
      • Ashina S
      • Amin FM
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      • Ashina M
      Calcitonin gene-related peptide and pain: A systematic review.
      Anti-tyrosine hydroxylase (TH) antibodies (Pel-Freez, Rogers, AR, USA), an enzyme part of the catecholamine synthesis, were used to label sympathetic fibers.
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      • Aschrafi A
      • Gale J
      • Vohra SN
      • MacGibeny MA
      • Kar AN
      • Gioio AE
      • Kaplan BB
      The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.
      Anti-Protein gene product 9.5 (PGP9.5) (Dako, Carpinteria, CA, USA) antibodies were used as a pan-neuronal marker.
      • Doran JF
      • Jackson P
      • Kynoch PA
      • Thompson RJ
      Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis.
      ,
      • Lundberg LM
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      • Polak JM
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      ,
      • Thompson RJ
      • Doran JF
      • Jackson P
      • Dhillon AP
      Rode J: PGP 9.5–a new marker for vertebrate neurons and neuroendocrine cells.
      As positive controls, a human vagus nerve for NF-200, CGRP, PGP9.5 antibodies, and a human sympathetic trunk for TH and PGP9.5 antibodies were used. Primary antibodies were omitted in the negative controls. Details of the primary antibodies, including references to antibody characterization, are presented in Table 1.
      Table 1Background Information on Primary and Secondary Antibodies
      Primary/secondary antibodyImmunogenManufacturer, species raised in, mono/polyclonal, catalogue and lot numberDilutionReference
      PGP9.5Purified PGP9.5 isolated from bovine brainDako, rabbit, polyclonal, Cat# Z5116, Lot# 20043529Cross-sections: 1:2000 Whole-mount: 1:100Manufacturer's information (Bernal Sierra et al., 2017)
      • Benerini Gatta L
      • Cadei M
      • Balzarini P
      • Castriciano S
      • Paroni R
      • Verzeletti A
      • Cortellini V
      • De Ferrari F
      • Grigolato P
      Application of alternative fixatives to formalin in diagnostic pathology.


      (Cleypool et al., 2020)
      • Cimmino MA
      • Ferrone C
      • Cutolo M
      Epidemiology of chronic musculoskeletal pain.


      (Rots et al., 2019)
      • Rots ML
      • de Borst GJ
      • van der Toorn A
      • Moll FL
      • Pennekamp CWA
      • Dijkhuizen RM
      • Bleys RLAW
      Effect of bilateral carotid occlusion on cerebral hemodynamics and perivascular innervation: An experimental rat model.
      NF-200Semi-purified neurofilament 200 kD from rat brain homogenateDevelopmental Studies Hybridoma Bank (DSHB), mouse, monoclonal, Cat# RT97Cross-sections: 1:1000Manufacturer's information (Haskins et al., 2017)
      • Haskins W
      • Benitez S
      • Mercado JM
      • Acosta CG
      Cutaneous inflammation regulates THIK1 expression in small C-like nociceptor dorsal root ganglion neurons.
      (Lawson et al., 1993)
      • Lawson SN
      • Perry MJ
      • Prabhakar E
      • McCarthy PW
      Primary sensory neurones: neurofilament, neuropeptides, and conduction velocity.


      (Viehöfer et al., 2015)
      • Woolf AD
      • Erwin J
      • March L
      The need to address the burden of musculoskeletal conditions.
      CGRPPurified rat α-CGRP peptide. The epitope recognized by the antibody resides within the C-terminal ten amino acids of rat α-CGRPSigma-Aldrich, mouse, monoclonal, Cat# C7113, Lot# 106M4836VCross-sections: 1:500Manufacturer's information (Yen et al., 2006)65
      THSDS-denatured rat tyrosine hydroxylase purified from pheochromocytomaPel-Freez, rabbit, polyclonal, Cat# P40101, Lot# AJO319OCross-sections: 1:500 Whole-mount: 1:100Manufacturer's information

      (Cleypool et al., 2020)
      • Cimmino MA
      • Ferrone C
      • Cutolo M
      Epidemiology of chronic musculoskeletal pain.
      (Rots et al., 2019)
      • Rots ML
      • de Borst GJ
      • van der Toorn A
      • Moll FL
      • Pennekamp CWA
      • Dijkhuizen RM
      • Bleys RLAW
      Effect of bilateral carotid occlusion on cerebral hemodynamics and perivascular innervation: An experimental rat model.
      BrightVision Poly-AP Anti-rabbitImmunoLogic (VWR), goat, Cat# VWRKDPVM110AP, Lot# 191217Used for cross-sections
      BrightVision Poly-AP Anti-mouseImmunoLogic (VWR), goat, Cat# VWRKDPVR110AP, Lot# 251018Used for cross-sections
      Alexa Fluor 594 Goat Anti-rabbitJackson ImmunoResearch, goat, polyclonal, Cat# 111-586-045, Lot# 112163Whole-mount: 1:200
      Bone cross-sections were deparaffinized in xylene (3 × 5 min), stepwise hydrated in an ethanol gradient (3 × 5 min in 100% and in 96%, 3 min in 80%, 70%, and 50%), and subsequently washed in de-ionized water. For antigen retrieval, cross-sections were incubated in 0.01M citrate buffer (pH 6.0) for five minutes at RT. Samples were then transferred to an 80ºC citrate buffer for 40 minutes, and subsequently cooled down to RT. Bone cross-sections were rinsed with wash buffer (0.05 M tris-buffered saline (TBS) (pH 7.6) and 0.05% Tween), and blocked with 5% normal human serum (Jackson Immuno Research, West Grove, PA, USA) in 0.05 M TBS (pH 7.6) for ten minutes at RT. The blocking buffer was removed, and primary antibodies were added in TBS with 3% bovine serum albumin (BSA) (GERBU Biotechnik GmbH, Heidelberg, BW, DE), and incubated overnight at 4ºC, except for the PGP9.5 antibodies, which were applied for 48 hours.
      After incubation in primary antiserum, bone cross-sections were washed with wash buffer, followed by incubation in secondary antibodies for 30 minutes at RT. BrightVision Poly AP-anti-Rabbit (ImmunoLogic VWR, Amsterdam, NH, NL) was used for TH and PGP 9.5 antibodies, and Poly AP-anti-Mouse for NF-200 and CGRP antibodies. Bone cross-sections were then rinsed with wash buffer, followed by 10 minutes incubation in Liquid Permanent Red (LPR) (Dako, Carpinteria, CA, USA). LPR was washed away with TBS and de-ionized water. To obtain sufficient contrast for tissue examinations, bone cross-sections were counterstained with hematoxylin. Finally, a cover slip with Entellan (Merck KGaA, Darmstadt, HE, DE) was applied, and bone cross-sections were air dried for 12 hours at RT before imaging.

       Immunohistochemistry on Whole-Mount Preparations of Periosteum

      Whole-mount preparations of periosteum were harvested from midshaft radii. Excess muscle was removed using a carbon steel surgical blade No. 20 (Swan-Morton, Sheffield, YSS, ENG). Sharpey's fibers were gently cut, and periosteum was taken off the bone by careful elevation. The periosteum was constantly irrigated with TBS during dissection to prevent tissue dehydration. Whole-mount samples were then frozen and thawed twice in TBS, and washed in whole-mount wash buffer (0.05 M TBS (pH 7.6) and 0.1% Saponin). Subsequently, whole-mount samples were incubated for 90 minutes at 37ºC in blocking solution containing 5% normal human serum and whole-mount wash buffer. Primary antibodies TH and PGP9.5 were applied for 12 hours and 48 hours, respectively, in whole-mount wash buffer with 3% BSA at RT. Primary antiserum was aspirated and whole-mount samples were rinsed with whole-mount wash buffer. Alexa Fluor 594 goat anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were applied for 30 minutes at RT in whole-mount wash buffer. BSA (3%) was added to minimize non-specific binding. Finally, samples were washed with whole-mount wash buffer, covered with FluorSave (Sigma-Aldrich, St. Louis, MO, USA), and dried for 12 hours at RT before imaging.

       Microscopy and Nerve Fiber Quantification

      High-power field (HPF, 400x magnification) images of bone cross-sections were captured with a Leica DM6 B (Wetzlar, HE, DE) fluorescent microscope using LAS X imaging software. A 400x magnification (311 × 233 μm in dimension) was used to distinguish between individual axons. The I3 filter cube (excitation range: blue, excitation filter: BP 450 - 490 nm, dichromatic mirror: 510 nm, and suppression filter: LP 515 nm) was set. Three HPFs were obtained per bone compartment per histology slide. The imaging location was chosen so that each set of three HPFs would adequately resemble the overall bone-related compartment. Fluorescent images of bone cross-sections were analyzed, and nerve fibers were quantified manually in ImageJ (NIH, Bethesda, MD, USA) by two observers (J.S. and D.O.). A fiber was counted if it was identifiable as a vividly fluorescent solid, solitary, round (in case of transverse cross-section) or elongated (in case of longitudinal cross-section) structure. No minimal dimensions were pre-specified. In case one fiber yielded more than one cross-section due to a sinuous course, this was counted as a single fiber. Mean fiber count of the three HPFs and of both observations was used for analysis. Brightness and contrast were adjusted to enhance image quality, no other image corrections or filters were used.
      For whole-mount preparations of periosteum, images were captured with a ZEISS LSM 800 (Oberkochen, BW, DE) laser confocal microscope using a 5x objective, a 180 µm pinhole and ZEN imaging software. A 594 nm excitation beam was used, and emission was detected using a 620 nm emission filter. Z-stacks were generated to allow for three-dimensional assessment of nerve fiber distribution through the full-thickness periosteum using a 25 µm step size. Acquired images of whole-mount preparations were displayed as a maximal intensity projection in ImageJ. Background was subtracted using the Subtract Background function in ImageJ, and brightness and contrast were adjusted in Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA).

       Statistical Analysis

      Data was subsequently analyzed in RStudio version 1.2.5033 (RStudio Inc., Boston, MA, USA). Data distribution was checked for normality graphically, using Q-Q plots, and confirmed using Shapiro-Wilk testing. Spearman's Rho and an Intra-Class Correlation coefficient for absolute agreement were calculated to assess inter-observer variation. Comparisons were performed using a Kruskal-Wallis test and a post hoc Wilcoxon rank-sum test. P-values were corrected for multiple testing with a Bonferroni correction. A multivariate regression was performed using a generalized linear model with Poisson residuals and a log link function allowing for identification of interaction effects between variables. Residuals were checked for normality using Q-Q plots. If interaction effects were statistically significant, the effect was embedded into the generalized linear model. Variables studied were age, gender, bone-related compartment (ie periosteum, bone marrow, and cortical bone), immunohistochemical staining, anatomical location, and nerve fiber count. As nerve fiber count functioned as the outcome variable, ten unique interaction effects were possible and thus analyzed. Data were presented as median number of nerve fibers with interquartile range (IQR). Results from the multivariate regression were reported as incident rate ratios (IRR) calculated from Poisson coefficients (ePoisson coefficient), and represent a multiplicative factor. In addition, estimated marginal means (EMM) with standard error (SE) were presented. The level of significance was set at P < .05.

      Results

       Demographics of Collected Tissue

      A total of 54 anatomical specimens were harvested from 29 human fixated cadavers. Four parietal bones (7%), six thoracic vertebrae (11%), five midshaft humeri (9%), seven midshaft radii (13%), 17 midshaft femora (32%), and 15 midshaft tibiae (28%) were available and studied after harvesting. Sixteen (55%) cadavers were female, and overall mean age was 84.0 ± 8.2 years (range 66–99). Anatomical specimen characteristics are displayed in Table 2. Complete decalcification of anatomical specimens was achieved after a maximum of 23 days. A total of 3 NF-200, 5 CGRP, 4 TH, and 1 PGP9.5-stained bone cross-sections failed during the immunohistochemical staining process and were thus discarded, despite protocol changes to improve microscopic slide attachment.
      Table 2Bone Sample Characteristics
      CharacteristicsValue
      Gender, n (%)
       Female16 (55%)
       Male13 (45%)
      Age, mean (SD) (years)84.0 (±8.2)
      Bones, n (%)N = 54
       Neurocranium4 (7%)
       Thoracic vertebra6 (11%)
       Midshaft humerus5 (9%)
       Midshaft radius7 (13%)
       Midshaft femur17 (32%)
       Midshaft tibia15 (28%)

       Innervation of the Periosteum, Cortical Bone, and Bone Marrow

      Overall, periosteum contained most nerve fibers, as compared to bone marrow, and cortical bone (Fig 2). The amount of A∂-fibers was largest in the periosteum (median 7 (IQR 4–12) fibers/HPF; P < .001 versus cortical bone, P < .001 versus bone marrow), followed by bone marrow (2 (1–4) fibers/HPF; P < .001 vs cortical bone), and cortical bone (0 (0–1) fibers/HPF) (Fig2A1-A3, E). The number of C-fibers in periosteum was higher (6 (3–10) fibers/HPF; P < .001 vs cortical bone, P = .075 vs bone marrow), compared with bone marrow (5 (2.75–8) fibers/HPF; P < .001 vs cortical bone), and cortical bone (1 (1–2) fibers/HPF) (Fig 2B1-B3,2E). The number of sympathetic nerve fibers was greatest in periosteum (9 (6–13) fibers/HPF; P < .001 vs cortical bone, P < .001 vs bone marrow), followed by bone marrow (5 (3–9) fibers/HPF; P < .001 vs cortical bone), and cortical bone (2 (1–4) fibers/HPF) (Fig 2C1-C3, E). PGP9.5-positive nerve fibers were mostly present in periosteum (9 (6–16.2) fibers/HPF; P < .001 vs cortical bone, P < .001 vs bone marrow), followed by bone marrow (3 (2–5.75) fibers/HPF; P < .001 vs cortical bone), and cortical bone (1 (1–3) fibers/HPF) (Fig 2D1-D3, 2E). Spearman's Rho was 0.890 and the intra-class correlation coefficient for absolute agreement was 0.851 (0.751–0.904), indicating good to excellent agreement between observers.
      • Koo TK
      • Li MY
      A guideline of selecting and reporting intraclass correlation coefficients for reliability research.
      ,
      • Schober P
      • Boer C
      • Schwarte LA
      Correlation coefficients: Appropriate use and interpretation.
      Figure 2
      Figure 2Fluorescent images showing NF-200-positive myelinated A∂-fibers (A, A1-A3), CGRP-positive unmyelinated C-fibers (B, B1-B3), TH-positive sympathetic nerve fibers (C, C1-C3), and PGP9.5-positive nerve fibers (D, D1-D3) in red. In periosteum and bone marrow, sensory nerve fibers were organized in large bundles (A1, A3, B1, B3), or were branched off as single nerve fibers (A3, D1). TH-positive sympathetic nerve fibers were co-localized with blood vessels (C1, C3). Cortical bone only contained sensory and sympathetic nerve fibers in Haversian canals (C2, D2), but not every canal was innervated (A2). Immunohistochemical stainings were quantified and presented as median (IQR) number of nerve fibers per HPF for periosteum (light blue), cortical bone (marine blue), and bone marrow (dark blue) (E). Fiber counts from all locations were combined. For every immunohistochemical staining, periosteum contained most nerve fibers, followed by the bone marrow, and cortical bone (color version of figure is available online.)

       Nerve Fiber Distribution in Bone-Related Compartments

      The periosteum and bone marrow cavity were innervated with large bundles of multiple sensory nerve fibers, while in the cortical bone only single fibers confined to Haversian canals were observed (Fig 2A1, A3, B1, D2 and 3A-3B). Sympathetic fibers were also present in all three bone-related compartments, but always co-localized with blood vessels (Fig 2C1-C3). Confocal images of periosteal whole-mount samples were prepared to allow assessment of coherence between nerve fibers, and illustrated that PGP9.5-positive nerve fibers (pan-neuronal marker) formed a branched network throughout the periosteum (Fig 3A-3B). Periosteal whole-mount preparations confirmed the co-localization of sympathetic fibers and blood vessels, and indirectly illustrated the abundancy of blood vessels in the periosteum (Fig 3C).
      Figure 3
      Figure 3Confocal images of whole-mount preparations of periosteum from a radius (female, 83 ) demonstrating PGP9.5-positive nerve fibers (A-B), and TH-positive sympathetic nerve fibers (C) in white. PGP9.5-positive nerve fibers were organized in a branched network throughout the periosteum. TH-positive sympathetic nerve fibers were co-localized with blood vessels, which indirectly showed the abundancy of blood vessels in the periosteum.
      Differences between A∂-fibers, C-fibers, and sympathetic nerve fibers within bone-related compartments were analyzed. See previous section and Fig 2E for median nerve fiber count. In periosteum, no difference between the number of A∂-fibers and C-fibers was observed (P = .71). However, the number of C-fibers was significantly higher compared to the amount of A∂-fibers in cortical bone, and bone marrow (both P < .001). Nerve fiber count for sympathetic nerve fibers was significantly higher compared with C-fibers in periosteum (P < .001). This difference was not observed in bone marrow or cortical bone (P = 1.000 and P = .59, respectively). The number of sympathetic nerve fibers was significantly higher compared with A∂-fibers in cortical bone (P < .001), bone marrow (P < .001), and in periosteum (P = .043). These results indicate that in bone marrow and cortical bone, C-fibers are slightly more abundant than A∂-fibers, while in periosteum both C-fibers and A∂-fibers are present to the same extent. In addition, sympathetic fibers are large in number in every bone-related compartment.

       Multivariate Regression

      Possible effects of bone-related compartment, gender, age, anatomical location, and immunohistochemical staining on nerve fiber count were analyzed using a Poisson multivariate regression with interaction effects. Interaction tables and estimated marginal means (EMM) are provided in the supplementary information. The multivariate regression model was based on 1836 individual nerve fiber counts. In the elderly individuals studied (age range 66–99 years), the number of nerve fibers significantly declined per year of age (IRR = 0.985, P < .001), especially in the periosteum (Table 3, S1). No overall effect of sex on innervation density was present (male N = 738 counts, female N = 1098 counts, IRR = 1.016, P = .881), however the interaction effect between gender and anatomical location demonstrated higher nerve fiber counts for lower extremity bones of females (Table 3, S3, S8). The overall number of C-fibers was significantly lower compared to PGP9.5-positive nerve fibers (IRR = 0.650, P = .003) (Table 3, S2). No differences were observed in the number of A∂-fibers (IRR = 1.225, P = .132) and sympathetic nerve fibers (IRR = 1.052, P = .704) compared to PGP9.5-positive nerve fibers (Table 3). The thoracic spine (IRR = 2.513, P < .001), upper extremity (IRR = 1.595, P < .001), and lower extremity (IRR = 1.540, P < .001) contained significantly more nerve fibers compared to neurocranium (Table 3, S5, S8, Fig 4). Nerve fiber count was significantly lower for bone marrow compared to periosteum (IRR = 0.189, P < .001), and lowest for cortical bone compared to periosteum (IRR = 0.094, P < .001). The low number of nerve fibers in cortical bone was mainly visible in female bones and lower extremity (Table S4, S7, S8).
      Table 3Multivariate Regression Analyzing Factors that Influence Nerve Fiber Count Per High Power Field
      Fiber countn=1836GLM Poisson LogLink with interactionsGLM Poisson LogLink without interactions
      IRREMM
      Results are averaged over the levels of other categorical variables. Estimated Marginal Means (EMM) represent the mean fiber count for each factor, adjusted for any other variables in the model. In the GLM Poisson LogLink without interactions, multiplying the factor's reference EMM (eg male in Gender) by the incident rate ratio (IRR) of non-reference categories gives the EMM for the non-reference category. Due to the involvement in interactions, this multiplication does not work in the GLM Poisson LogLink with interactions.
      ,
      Results may be misleading due to involvement in interactions
      EMM SEP-valueIRREMM
      Results are averaged over the levels of other categorical variables. Estimated Marginal Means (EMM) represent the mean fiber count for each factor, adjusted for any other variables in the model. In the GLM Poisson LogLink without interactions, multiplying the factor's reference EMM (eg male in Gender) by the incident rate ratio (IRR) of non-reference categories gives the EMM for the non-reference category. Due to the involvement in interactions, this multiplication does not work in the GLM Poisson LogLink with interactions.
      ,
      Results may be misleading due to involvement in interactions
      EMM SEP-value
      Intercept25.1534.320.080<.00123.8714.420.073<.001
      AgePer year0.9854.320.080<.0010.9874.420.072<.001
      GenderMaleReference4.320.114N/AReference3.970.084N/A
      Female1.0164.330.112.8811.2404.920.094<.001
      StainingPGP9.5Reference4.880.151N/AReference5.470.118N/A
      NF-2001.2252.780.125.1320.6353.480.089<.001
      CGRP0.6504.220.149.0030.7063.860.097<.001
      TH1.0526.110.181.7040.9505.200.116<.001
      LocationNeurocraniumReference3.320.184N/AReference3.060.145N/A
      Thoracic spine2.5135.730.191<.0012.0576.280.163<.001
      Upper extremity1.5954.830.123<.0011.5674.790.103<.001
      Lower extremity1.5403.810.072<.0011.3594.150.065<.001
      CompartmentPeriosteumReference9.300.188N/AReference9.900.166N/A
      Cortical bone0.0941.760.078<.0010.1831.810.057<.001
      Bone marrow0.1894.950.131<.0010.4864.810.101<.001
      NOTE. All significant P-values are reported in bold.
      low asterisk Results are averaged over the levels of other categorical variables. Estimated Marginal Means (EMM) represent the mean fiber count for each factor, adjusted for any other variables in the model. In the GLM Poisson LogLink without interactions, multiplying the factor's reference EMM (eg male in Gender) by the incident rate ratio (IRR) of non-reference categories gives the EMM for the non-reference category. Due to the involvement in interactions, this multiplication does not work in the GLM Poisson LogLink with interactions.
      Results may be misleading due to involvement in interactions
      Figure 4
      Figure 4The median number (IQR) of CGRP-positive unmyelinated C-fibers, NF-200-positive myelinated A∂-fibers, TH-positive sympathetic nerve fibers, and PGP9.5-positive nerve fibers separately displayed for the neurocranium, thoracic vertebra, upper extremity, and lower extremity. Compared to the neurocranium, the number of nerve fibers was higher in all three anatomical locations, especially in the thoracic vertebra. Skeleton adjusted from sciepro/shutterstock.com, used under license of Shutterstock.com.

      Discussion

      The present study set out to study the sensory innervation of human bone. The results demonstrate that the highest sensory innervation density can be found in the periosteum, followed by bone marrow and cortical bone. Of the locations studied, the thoracic vertebra received most sensory innervation. The neurocranium received the least sensory innervation. Innervation density decreased with age. In the present study, relative differences in innervation between periosteum, cortical bone and bone marrow were smaller compared to earlier mammalian studies.
      • Castaneda-Corral G
      • Jimenez-Andrade JM
      • Bloom AP
      • Taylor RN
      • Mantyh WG
      • Kaczmarska MJ
      • Ghilardi JR
      • Mantyh PW
      The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.
      ,
      • Mach DB
      • Rogers SD
      • Sabino MC
      • Luger NM
      • Schwei MJ
      • Pomonis JD
      • Keyser CP
      • Clohisy DR
      • Adams DJ
      • O'leary P
      • Mantyh PW
      Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur.
      One study reported a ratio of summed A∂-fibers and C-fibers between periosteum, bone marrow and cortical bone of 100:2:0.1, while in the current study a ratio of 100:54:8 was found.
      • Castaneda-Corral G
      • Jimenez-Andrade JM
      • Bloom AP
      • Taylor RN
      • Mantyh WG
      • Kaczmarska MJ
      • Ghilardi JR
      • Mantyh PW
      The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.
      The use of a different species and calculations based on medians instead of means might partially account for these differences. Remarkably, not all osteons were innervated, which partially explained the low number of both sensory and sympathetic nerve fibers in cortical bone as presented in section 3.3 (Fig 2A2). The number and distribution of sensory nerve fibers varied between different anatomical locations (ie neurocranium, thoracic spine, upper, and lower extremity). Possibly, skeletal injury in more densely innervated locations is more painful than in locations receiving less sensory innervation. Previous reports have shown correlations between innervation of the intervertebral disc and severity of low back pain. However, a causal relationship between innervation density and pain severity cannot be established, as tissue injury (inflicting pain) and the subsequent inflammatory processes have been shown to lead to hyperinnervation.
      • Amaya F
      • Izumi Y
      • Matsuda M
      • Sasaki M
      Tissue injury and related mediators of pain exacerbation.
      Gerbershagen et al. studied pain scores on the first postoperative day for any type of surgery. The top ten of most painful interventions included seven skeletal surgeries, of which three were in the spinal column.
      • Gerbershagen HJ
      • Aduckathil S
      • van Wijck AJM
      • Peelen LM
      • Kalkman CJ
      • Meissner W
      Pain intensity on the first day after surgery: a prospective cohort study comparing 179 surgical procedures.
      Although pain is a multidimensional phenomenon and not necessarily equal to sensory innervation density, the high nerve fiber count in thoracic spine as demonstrated in the current study reflects these clinical findings, supporting a correlation between innervation density and pain severity.
      We demonstrated that sensory nerve fibers in periosteum were organized in a network of large bundles or were branched off as single nerve fibers forming a net-like structure. The typical symptom of a bone fracture is acute, stabbing pain localized near or at the fracture site.
      • Santy J
      • Mackintosh C
      A phenomenological study of pain following fractured shaft of femur.
      Mechanical distortion of the periosteum might help explain the severe pain experienced by these patients. In addition, skeletal pain can be caused by pathology in the bone marrow (eg in bone tumors), and is typically described as a dull and aching pain.
      • Ivanusic JJ
      Molecular mechanisms that contribute to bone marrow pain.
      ,
      • Luger NM
      • Mach DB
      • Sevcik MA
      • Mantyh PW
      Bone cancer pain: from model to mechanism to therapy.
      ,
      • Nencini S
      • Ivanusic J
      Mechanically sensitive Adelta nociceptors that innervate bone marrow respond to changes in intra-osseous pressure.
      ,
      • Nencini S
      • Ivanusic JJ
      The physiology of bone pain. How much do we really know?.
      In the present study we demonstrated that the number of C-fibers was slightly higher compared to the number of A∂-fibers in bone marrow. This finding is in line with the clinical picture of patients experiencing different types of pain in different types of pathology. The here presented data may provide new treatment targets for these various types of pain arising from skeletal pathology.
      A strength of this study was the use of four immunohistochemical stains to visualize A∂-fibers, C-fibers, and sympathetic fibers. Subsequently, we were able to draw conclusions about their relative number in different bone-related compartments. It should be noted that the total volume of bone marrow is greater than cortical bone and periosteum.
      • Mach DB
      • Rogers SD
      • Sabino MC
      • Luger NM
      • Schwei MJ
      • Pomonis JD
      • Keyser CP
      • Clohisy DR
      • Adams DJ
      • O'leary P
      • Mantyh PW
      Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur.
      Therefore, the absolute number of sensory and sympathetic nerve fibers might be higher in bone marrow. Use of multivariate regression enabled us to draw conclusions on the complete data set. For example, to analyze the effect of gender on bone innervation, all 1836 observations were taken into account. Another strong element of the present study was the use of bones from multiple anatomical locations, to adequately represent human axial and appendicular innervation. The use of bones from both men and women of ages ranging from 66 to 99 were used to account for potential differences in bone innervation based on sex or age. Our findings demonstrated no overall differences in innervation between female and male bones. Collecting all four bone sites studied from the same individual would have provided paired samples and thus might have further strengthened our conclusions, but was not possible due to incompleteness of the skeletal material available. Furthermore, experiments were performed on macroscopically healthy bones, making the results relevant to the general population. As mentioned, average donor age was relatively high (84 ± 8.2 y) and ranged from 66 until 99 years. We found that the number of sensory and sympathetic nerve fibers declined with age, which is consistent with a previous animal study.
      • Chartier SR
      • Mitchell SAT
      • Majuta LA
      • Mantyh PW
      The changing sensory and sympathetic innervation of the young, adult and aging mouse femur.
      Nevertheless, a wider age range of subjects is necessary to determine whether our findings are applicable for a younger population. A drawback of using anonymous human cadavers, is that the cause of death and its possible effects on bone innervation remains unknown. Previous studies on human innervation have reported on the effects of osteoarthritis and degenerative disc disease on bone innervation. To avoid influence of joint diseases on nerve fiber density, we collected samples from midshaft regions of bone only.
      • Brown MF
      • Hukkanen M V
      • McCarthy ID
      • Redfern DR
      • Batten JJ
      • Crock H V
      • Hughes SP
      • Polak JM
      Sensory and sympathetic innervation of the vertebral endplate in patients with degenerative disc disease.
      ,
      • Koeck FX
      • Schmitt M
      • Baier C
      • Stangl H
      • Beckmann J
      • Grifka J
      • Straub RH
      Predominance of synovial sensory nerve fibers in arthrofibrosis following total knee arthroplasty compared to osteoarthritis of the knee.
      CGRP-positive fibers have been reported to have a punctuated morphology, while the NF200 signal has a continuous morphology. To avoid overestimation of CGRP-positive fibers due to their morphology, bone cross-sections were used for nerve fiber quantification. As nerve fibers run parallel to the longitudinal axis of long bones, risks of overcounting are minimized. Another possible limitation is the increased EDTA bone decalcification time when compared to previously conducted mammalian studies (>3 wk vs 2 wk).
      • Castaneda-Corral G
      • Jimenez-Andrade JM
      • Bloom AP
      • Taylor RN
      • Mantyh WG
      • Kaczmarska MJ
      • Ghilardi JR
      • Mantyh PW
      The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.
      ,
      • Chartier SR
      • Mitchell SAT
      • Majuta LA
      • Mantyh PW
      The changing sensory and sympathetic innervation of the young, adult and aging mouse femur.
      ,
      • Mach DB
      • Rogers SD
      • Sabino MC
      • Luger NM
      • Schwei MJ
      • Pomonis JD
      • Keyser CP
      • Clohisy DR
      • Adams DJ
      • O'leary P
      • Mantyh PW
      Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur.
      The extended decalcification was necessary to demineralize cortical bone, which is thicker in humans than in rodents. Literature described that EDTA negatively affects the antigenicity of A∂-fibers, C-fibers, and sympathetic fibers.
      • Chartier SR
      • Mitchell SAT
      • Majuta LA
      • Mantyh PW
      The changing sensory and sympathetic innervation of the young, adult and aging mouse femur.
      ,
      • Mach DB
      • Rogers SD
      • Sabino MC
      • Luger NM
      • Schwei MJ
      • Pomonis JD
      • Keyser CP
      • Clohisy DR
      • Adams DJ
      • O'leary P
      • Mantyh PW
      Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur.
      This might have resulted in a lower nerve fiber count in the present study and thus underestimation of overall innervation density, although this is expected to equally affect all bone compartments and thus does not necessarily impact the here presented ratios. Contradictory reports on the effect of formaldehyde on immunoreactivity of tissue exist.
      • Benerini Gatta L
      • Cadei M
      • Balzarini P
      • Castriciano S
      • Paroni R
      • Verzeletti A
      • Cortellini V
      • De Ferrari F
      • Grigolato P
      Application of alternative fixatives to formalin in diagnostic pathology.
      ,
      • Shi S-R
      • Liu C
      • Pootrakul L
      • Tang L
      • Young A
      • Chen R
      • Cote RJ
      • Taylor CR
      Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry.
      The specimens have been fixated in formaldehyde for various amounts of time, which might have led to variation in immunoreactivity. Our and others’ experience is that no large differences regarding immunoreactivity between fresh-frozen samples and formaldehyde-fixated samples were present.
      • Shi S-R
      • Liu C
      • Pootrakul L
      • Tang L
      • Young A
      • Chen R
      • Cote RJ
      • Taylor CR
      Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry.
      Fourth, since PGP9.5 is a pan-neuronal marker, the number of PGP9.5-positive nerve fibers should theoretically equal the sum of A∂-fibers, C-fibers, and sympathetic fibers. However, in the present study we found that the sum of A∂-fibers, C-fibers, and sympathetic fibers was approximately 4 times the number of PGP9.5-positive nerve fibers in bone marrow. For periosteum and cortical bone, similar computations resulted in a factorial difference of about 2.4 and 3, respectively. This finding was remarkable as previous studies reported adequate specificity of the immunohistochemical markers used, and showed a lack of other sensory nerve fibers innervating the bone such as Aβ-fibers.
      • Bernal Sierra YA
      • Haseleu J
      • Kozlenkov A
      • Begay V
      • Lewin GR
      Genetic tracing of Cav3.2 T-type calcium channel expression in the peripheral nervous system.
      ,
      • Haskins W
      • Benitez S
      • Mercado JM
      • Acosta CG
      Cutaneous inflammation regulates THIK1 expression in small C-like nociceptor dorsal root ganglion neurons.
      ,
      • Ivanusic JJ
      • Mahns DA
      • Sahai V
      • Rowe MJ
      Absence of large-diameter sensory fibres in a nerve to the cat humerus.
      ,
      • Lawson SN
      • Perry MJ
      • Prabhakar E
      • McCarthy PW
      Primary sensory neurones: neurofilament, neuropeptides, and conduction velocity.
      ,
      • Rots ML
      • de Borst GJ
      • van der Toorn A
      • Moll FL
      • Pennekamp CWA
      • Dijkhuizen RM
      • Bleys RLAW
      Effect of bilateral carotid occlusion on cerebral hemodynamics and perivascular innervation: An experimental rat model.
      ,
      • Yen LD
      • Bennett GJ
      • Ribeiro-da-Silva A
      Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury.
      Although CGRP has been used as a marker for non-myelinated fibers in similar experiments,
      • Eftekhari S
      • Warfvinge K
      • Blixt FW
      • Edvinsson L
      Differentiation of nerve fibers storing CGRP and CGRP receptors in the peripheral trigeminovascular system.
      ,
      • Mach DB
      • Rogers SD
      • Sabino MC
      • Luger NM
      • Schwei MJ
      • Pomonis JD
      • Keyser CP
      • Clohisy DR
      • Adams DJ
      • O'leary P
      • Mantyh PW
      Origins of skeletal pain: Sensory and sympathetic innervation of the mouse femur.
      other studies have shown CGRP to be expressed by A∂-fibers.
      • Iyengar S
      • Ossipov MH
      • Johnson KW
      The role of calcitonin gene-related peptide in peripheral and central pain mechanisms including migraine.
      Further, calculations with median nerve fiber counts and usage of separate microscopic slides for each neuronal marker might have contributed to this discrepancy. These factors might explain the observation that the sum of A∂-fibers, C-fibers, and sympathetic fibers exceeded PGP9.5 fiber count and highlights the need for a more specific C-fiber marker. However, as these limitations were present in all bones and bone compartments, the authors suggest that these phenomena do not affect the overall trends observed in this study nor the final conclusions.

      Conclusion

      To our knowledge, the present study is the first to demonstrate the sensory innervation in the human axial and appendicular skeleton. The current results provide an explanation for the severe pain experienced by patients suffering from musculoskeletal diseases or following skeletal surgery. The number of sensory and sympathetic nerve fibers was highest in periosteum, followed by bone marrow and cortical bone. Of all locations studied, bone from thoracic vertebral bodies was most densely innervated. In the periosteum, sensory nerve fibers were organized in a net-like structure that may allow for detection of mechanical distortion such as experienced during traumatic events. The number of nerve fibers declined with age. Since the periosteum is the most densely innervated bone-related compartment in human bones, treatment strategies that locally target the periosteum might aid in attenuating pain after skeletal surgery. Future studies are needed to understand how bone diseases affect density, sensitivity, and morphology of sensory and sympathetic nerve fibers.

      Acknowledgments

      We would like to thank prof. dr. Ronald Bleys, Simon Plomp, and Marco Rondhuis from the Department of Anatomy (UMC Utrecht) for their help with tissue collection, designing immunohistochemical protocols, and for using their microscope facility. Peter Zuithoff from the Julius Center (Utrecht) is thanked for valuable input on statistical analysis. The Developmental Studies Hybridoma Bank is gratefully acknowledged for supplying the NF-200 antibody. No financial arrangements that may represent a possible conflict of interest are present. The authors declare that they have no conflict of interest.
      For the purpose of replicating procedures and reproducing results, data and program codes are available upon request. Requests can be sent to the corresponding author.

      Appendix. Supplementary data

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