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. 2019 Mar 1;129(3):1076-1093.
doi: 10.1172/JCI121561. Epub 2019 Feb 4.

Subchondral Bone Osteoclasts Induce Sensory Innervation and Osteoarthritis Pain

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Free PMC article

Subchondral Bone Osteoclasts Induce Sensory Innervation and Osteoarthritis Pain

Shouan Zhu et al. J Clin Invest. .
Free PMC article

Abstract

Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete netrin-1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, dorsal root ganglion neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for netrin-1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g., netrin-1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.

Keywords: Bone Biology; Innervation; Neuroscience; Osteoarthritis; Pain.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. CGRP+ sensory nerves in subchondral bone increased along with an increase in osteoclast activity and DRG neuron hypersensitivity during OA progression.
(A) Safranin orange/fast green (SO/FG) staining (first row), TRAP staining (second row, magenta), and immunofluorescence analysis of CGRP+ sensory nerve fibers (third row, green) in mouse tibial subchondral bone after ACLT surgery at different time points. Scale bars: 500 μm (first row), 100 μm (second row), and 50 μm (third row). Excitability (fourth row) of L4 DRG in Pirt-GCaMP3 mice at different time points after surgery. Scale bar: 100 μm. n = 7 per time point (see neuronal hyperactivity in Supplemental Videos 1–6). (B and C) Quantitative analysis of density of TRAP+ and CGRP+ sensory nerves in subchondral bone marrow. *P < 0.05 compared with the sham-operated group at the corresponding time points. n = 7 per time point. (D) Quantification of activated DRG neurons. *P < 0.05, **P < 0.01 compared with the sham-operated group at the corresponding time points. n = 7 per group. (E) Representative photomicrographs of CGRP and Dil double-labeled neurons in L4 DRG. Scale bar: 50 μm. n = 6 per group. (F) Percentage of L4 DRG neurons retrogradely labeled with Dil in all CGRP+ neurons 10 weeks after sham or ACLT surgery. **P < 0.01 compared with the sham-operated group at the corresponding time points. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations.
Figure 2
Figure 2. In OA, most DRG neurons responding to knee pinch are nociceptive neurons.
(A) Relative frequency distributions of the areas of neurons responding to 20-g knee pinch in ACLT mice at different time points. Mean ± standard deviation. (B) Excitability of L4 DRG in Pirt-GCaMP3 mice responding to knee pinch or direct drop of 1 μM capsaicin. White arrows indicate neurons responding only to capsaicin; yellow arrows indicate neurons responding to both knee pinch and capsaicin. Scale bar: 250 μm. (C) Number of DRG neurons responding to knee pinch or capsaicin.
Figure 3
Figure 3. Decreased sprouting of CGRP+ sensory nerves in the subchondral bone and pain amelioration in Dmp1-Ranklfl/fl mice.
(A) TRAP staining (first row, magenta) and immunofluorescence analysis of CGRP+ sensory nerve fibers (second row, green) in mouse tibial subchondral bone after ACLT surgery. Scale bars: 100 μm. Third row: Safranin orange and fast green (SO/FG) staining of articular cartilage in sagittal sections of tibial medial compartment of Ranklfl/fl and Dmp1-Ranklfl/fl mice with or without ACLT surgery. Scale bar: 500 μm. Fourth row: 3-Dimensional μCT image of tibial subchondral bone medial compartment (sagittal view) of Ranklfl/fl and Dmp1-Ranklfl/fl mice with or without ACLT surgery. Scale bar: 1 mm. n = 9/group. (B and C) Quantitative analysis of the density of TRAP+ osteoclasts and CGRP+ nerve fibers in subchondral bone marrow. (D and E) Quantitative analysis of total tissue volume (TV) (D) and OARSI scores 8 weeks after surgery (E). n = 9/group. (F) In vivo calcium imaging in whole L4 DRG primary sensory neurons after mechanical press to knees of Ranklfl/fl;Pirt-GCaMP3 and Dmp1-Ranklfl/fl;Pirt-GCaMP3 mice. Scale bar: 50 μm. n = 10/group. (G) Number of neurons activated by mechanical press. (H) ΔF/Fo for neurons in a representative DRG responding to an approximately 20-g paw pinch in Ranklfl/fl (black) and Dmp1-Ranklfl/fl (red) mice after ACLT. (I) Paw withdrawal threshold (PWT) was tested at the right hind paw of Ranklfl/fl-sham, Ranklfl/fl-ACLT, Dmp1-Ranklfl/fl-sham, and Dmp1-Ranklfl/fl-ACLT mice. (J) Representative images of ink blotting trial of Ranklfl/fl and Dmp1-Ranklfl/fl mice after ACLT surgery on right knees. RH, right hind (orange); LH, left hind (orange); RF, right front (black); LF, left front (black). (K and L) Percentage RH ipsilateral intensity (K) and percentage RH ipsilateral contact area (L) were calculated using ImageJ software. n = 10/group. All data are shown as means ± standard deviations. *P < 0.05, **P < 0.01 by multifactorial ANOVA. NS, no significant difference.
Figure 4
Figure 4. Netrin-1 from osteoclasts induces axonal growth.
(A) Microfluidics assay of osteoclast-conditioned medium promoting DRG neuron axonal growth with treatment of functional blocking antibodies. Mono-CM, monocyte-conditioned medium; OC-CM, osteoclast-conditioned medium; ab, antibody. Scale bar: 100 μm. (B) Quantification of the length of axons that protruded into axonal side. **P < 0.01 compared with mono-CM group; #P < 0.05 compared with OC-CM group. n = 3/group. (C) Microfluidics assay of recombinant mouse netrin-1 promoting DRG neuron axonal growth. Scale bar: 100 μm. (D) Quantification of the length of axons that protruded into axonal side. **P < 0.01 compared with BSA control group. n = 3/group. (E) Western blots of the phosphorylation of FAK and AKT in DRG neurons treated with netrin-1 for 0–150 minutes (m). (F) Western blots of netrin-1 expression in monocytes, preosteoclasts, and osteoclasts. (G) ELISA analysis of netrin-1 concentration in conditioned media during osteoclast differentiation. **P < 0.01 compared with mono-CM group. (H) Immunohistochemical staining of netrin-1 and costaining of netrin-1 and TRAP in subchondral bone of WT mice at different time points after surgery. Scale bar: 100 μm. (I) Quantitative analysis of density of netrin-1 in subchondral bone marrow. *P < 0.05 compared with the sham-operated group. (J) ELISA analysis of netrin-1 concentration in subchondral bone marrow of Ranklfl/fl and Dmp1-Ranklfl/fl with or without ACLT surgery. *P < 0.05. All data are shown as means ± standard deviations. Statistical significance was determined by multifactorial ANOVA. NS, no significant difference.
Figure 5
Figure 5. Osteoclast-derived netrin-1 is elevated in human OA subchondral bone.
(A) Top: Safranin orange staining of human normal and OA cartilage and subchondral bone. Scale bar: 100 μm. Bottom: Immunofluorescence staining of TRAP and netrin-1 in human subchondral bone. Scale bar: 50 μm. (B) Quantitative analysis of relative intensity of TRAP and netrin-1 double-positive cells in human subchondral bone. *P < 0.05, compared with healthy control by unpaired, 2-tailed Student’s t test.
Figure 6
Figure 6. Knockout of Netrin1 in osteoclast-lineage cells reduces sprouting of CGRP+ sensory nerves in subchondral bone and ameliorates OA pain.
(A) ELISA analysis of netrin-1 concentration in subchondral bone marrow of Ntnfl/fl and Trap-Ntnfl/fl mice with or without ACLT surgery. n = 5/group. (B) Left: 3-Dimensional μCT image of the tibial subchondral bone medial compartment (sagittal view) of Ntnfl/fl and Trap-Ntnfl/fl with or without ACLT surgery. Middle and right: Safranin orange and fast green staining of articular cartilage in sagittal sections of tibial medial compartment of mice. Scale bars: 1 mm (left), 500 μm (middle), and 100 μm (right). (C) OARSI scores 8 weeks after surgery. n = 8/group. (D) Quantitative analysis of total tissue volume (TV) in subchondral bone determined by μCT. n = 8/group. (E) TRAP staining (top, magenta) and immunofluorescence analysis of CGRP+ sensory nerve fibers (bottom, green) in mouse tibial subchondral bone after ACLT surgery. Scale bars: 50 μm. (F and G) Quantitative analysis of relative density of TRAP+ osteoclasts and CGRP+ nerve fibers in subchondral bone marrow. (H) In vivo calcium imaging in whole L4 DRG primary sensory neurons after mechanical press to knees of Ntnfl/fl;Pirt-GCaMP3 and Trap-Ntnfl/–;Pirt-GCaMP3 ACLT mice. Scale bars: 50 μm. (I) Number of neurons activated by mechanical press. (J) ΔF/Fo for neurons in a representative DRG responding to approximately 20-g knee pinch in Ntnfl/fl (black) and Trap-Ntnfl/– (red) mice after ACLT. (K) Paw withdrawal threshold (PWT) was tested at the right hind paw of Ntnfl/fl and Trap-Ntnfl/fl mice with or without ACLT. (L) Representative copies of ink blotting trial of Ntnfl/fl and Trap-Ntnfl/fl mice after ACLT surgery on right knees. RH, right hind (orange); LH, left hind (orange); RF, right front (black); LF, left front (black). (M and N) Percentage RH ipsilateral intensity (M) and percentage RH ipsilateral contact area (N) determined by ImageJ software. n = 10/group. All data are shown as means ± standard deviations. *P < 0.05 by multifactorial ANOVA. NS, no significant difference.
Figure 7
Figure 7. In vivo silencing of murine Dcc mRNA by siRNA systemic administration reduced CGRP+ sensory nerve subchondral bone innervation and OA pain.
(A) Microfluidics assay of osteoclast-conditioned medium promoting DRG neuron axonal growth with treatment of siDcc and siUnc5. Scale bar: 100 μm. *P < 0.05. (B) Top: Safranin orange and fast green staining of articular cartilage in sagittal sections of the tibial medial compartment of mice. Scale bar: 100 μm. Immunofluorescence analysis of DCC+ (middle, red) and CGRP+ (bottom, green) sensory nerve fibers in mouse tibial subchondral bone 4 weeks after surgery. Scale bars: 50 μm. (C) Quantitative analysis of OARSI score (top), relative density of DCC+ (middle), and CGRP+ (bottom) nerve fibers in subchondral bone marrow. *P < 0.05. NS, no significant difference. (D) Paw withdrawal threshold (PWT) was tested at the left hind paw of sham, scramble ACLT, and siDcc ACLT mice at different time points after surgery. *P < 0.05, compared with sham mice; #P < 0.05, compared with ACLT-operated and scramble siRNA–administered mice. (E) Variations in the ipsilateral and contralateral hind limbs of gait parameters obtained from CatWalk analysis. *P < 0.05, compared with sham mice; #P < 0.05, compared with ACLT-operated and scramble siRNA–administered mice. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations.
Figure 8
Figure 8. Effect of alendronate on DMM-induced OA pain.
(A) Top: Safranin orange and fast green staining of articular cartilage in sagittal sections of the tibial medial compartment of mice. Scale bar: 100 μm. Middle and bottom: Immunohistochemical analysis of TRAP+ (middle) and immunofluorescence analysis of CGRP+ (bottom, green) sensory nerve fibers in mouse tibial subchondral bone after DMM surgery. Scale bars: 50 μm. (B) Quantitative analysis of OARSI score (top), relative density of TRAP+ osteoclasts (middle), and CGRP+ (bottom) nerve fibers in subchondral bone marrow. *P < 0.05. (C) Immunohistochemical staining and quantification of netrin-1 in subchondral bone of sham-operated mice and DMM-operated mice treated with either vehicle or ALN. Scale bar: 50 μm. *P < 0.05. (D) Paw withdrawal threshold (PWT) was tested at the left hind paw of sham, vehicle DMM, and ALN ACLT mice at different time points after surgery. *P < 0.05, compared with sham mice; #P < 0.05, compared with DMM-operated and vehicle-administered mice. (E) Variations in the ipsilateral and contralateral hind limbs of gait parameters obtained from CatWalk analysis. *P < 0.05, compared with sham mice; #P < 0.05, compared with DMM-operated and vehicle-administered mice. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations.

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