Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis

Abstract

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain¹. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes², their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Na_v1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Na_v1.7 channels, with a density of 0.1 to 0.15 channels per µm² and 350 to 525 channels per cell. Serial genetic ablation of Na_v1.7 in multiple mouse models demonstrates that Na_v1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Na_v1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Na_v1.7 with selective or clinically used pan-Na_v channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Na_v1.7 blockers regulate intracellular Ca²⁺ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Na_v1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

Fig. 1. TTX-S currents are present in OA chondrocytes and are produced largely by Na_v1.7 ProTx II-S channels.

a, Representative current traces in control buffer (left), in the presence of 1 µM TTX (middle), and resulting traces of the TTX-S current (right). b, Current–voltage curves of peak current amplitudes in control solution () and in the presence of 1 µM TTX (), and the I-V curve for the TTX-S current (). c, G/G_max of the TTX-S current (mean ± s.e.m., n = 3) fitted with the Boltzmann equation. d, Single-exponential time constants of TTX-S current inactivation (mean ± s.e.m., n = 3). e, Time to peak of the TTX-S current (mean ± s.e.m., n = 3). f, Inhibition of fast-inactivating sodium current by 20 nM ProTx II. Averaged sodium current traces at 0 mV in control (black solid line) and in 20 nM ProTx II (black dotted line), and the resulting trace of their difference (ProTx II-S current, blue line). g, Inhibition of fast-inactivating sodium current by 1 µM TTX. Averaged traces of sodium currents evoked by 0 mV test voltage from −90 mV holding voltage in control solution (black solid line) and in the presence of 1 µM TTX (black dotted line), and the trace of their difference (TTX-S current, red trace). h, Overlays of TTX-S (red) and ProTx II-S (blue) current traces from f,g, normalized by peak current amplitudes. i, Left, inactivation time constants (mean ± s.e.m.) of TTX-S (n = 4) versus ProTx II-S (n = 5) currents. Right, time to peak of TTX-S (mean ± s.e.m., n = 4) versus ProTx II-S (mean ± s.e.m., n = 5) currents at 0 mV test voltage. j, Left, effect of 1 µM TTX on peak current amplitudes (mean ± s.e.m., n = 4) measured at 0 mV test voltage. Right, effect of 20 nM ProTx II on peak current amplitudes (mean ± s.e.m., n = 5) measured at 0 mV test voltage; averages of current amplitudes are shown for total, ProTx II-S and persistent sodium currents. n indicates cell number; P values by two-tailed Mann–Whitney test. NS, not significant.

Fig. 2. Ablation of chondrocyte Na_v1.7 protects against OA and reduces pain.

a, Safranin O and Fast Green-stained sections of knee joints of mice with the indicated genotype (n = 8). Scale bar, 50 µm. b–e, OARSI score (b), osteophyte development (c), subchondral bone plate (SBP) thickness (d) and synovitis score (e) in indicated mice 12 weeks after DMM (n = 8). f, Traces of open field testing at 12 weeks after DMM surgery. g,h, Two-minute travel distance (g) and von Frey testing (h) in DMM-operated mice at the indicated time points after surgery (n = 8). i, Safranin O and Fast Green-stained sections of knee joints (n = 8). Scale bars, 50 µm. j–m, OARSI score (j), osteophyte development (k), SBP thickness (l), and synovitis score (m) in indicated mice 12 weeks after DMM surgery (n = 8). n, Traces of open field testing at 12 weeks after DMM surgery. o,p, Two-minute travel distance (o) and von Frey testing (p) in DMM-operated mice at the indicated time points after surgery (n = 8). b–e,j–m, Data are mean ± s.d., P values by two-tailed unpaired Student’s t-test. g,h,o,p, Data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction.

Fig. 3. Blockade of Na_v1.7 regulates chondrocyte biology through enhancing HSP70 and midkine secretion.

a, Peptide-spectrum match (PSM)-based abundance of proteins identified from the 30–100 kDa fraction of conditioned medium that are unique to (red dots) or increased with (blue dots) PF-04856264 and ProTx II treatment. b, PSM-based abundance of proteins (red dot) identified from the 10–30 kDa fraction of conditioned medium that are unique to PF-04856264 and ProTx II treatment. c, COL2 and ACAN mRNA levels in human C28I2 chondrocytes stimulated with conditioned medium collected from cells treated with ProTx II (Pro-CM) or PF-04856264 (PF-CM) in the absence or presence of IgG or anti-HSP70 antibodies (n = 4 biological replicates). d, MMP13 and ADAMTS5 mRNA levels in C28I2 chondrocytes stimulated with IL-1β and conditioned medium collected from cells treated with ProTx II (Pro-CM) or PF-04856264 (PF-CM) in the absence or presence of control IgG or anti-midkine antibodies (n = 4 biological replicates). e, Safranin O and Fast Green-stained knee joint sections (n = 8). Ver, VER 155008. Scale bar, 50 µm. f, OARSI score from images represented in e. g, Two-minute travel distance and von Frey testing at the indicated time points with indicated treatments after DMM surgery (n = 8). Data are mean ± 95% confidence interval, P values by two-way ANOVA with Bonferroni post hoc test. *Vehicle versus PF, P < 0.05; **vehicle versus PF, P < 0.01; ^#PF versus PF + Ver + iMDK, P < 0.05; ^##PF versus PF + Ver + iMDK, P < 0.01. c,d,f, Data are mean ± s.d., P values by one way ANOVA with Bonferroni post hoc test. Source Data

Fig. 4. Ca²⁺ signalling in chondrocytes.

a,b, F/F₀ (a) and area under the curve (AUC) of intracellular Ca²⁺ (b) in human OA chondrocytes following ATP stimulation, measured by plate reader. ATP present from red arrow. c,d, HSP70 (c) and midkine (d) levels in conditioned medium of C28I2 cells pre-treated with BAPTA-AM, followed by ProTx II or PF-04856264. e, F/F₀ of intracellular Ca²⁺ in KB-R7943 treated C28I2 chondrocytes following ATP stimulation, assayed by confocal fluorescence microscopy. f,g, HSP70 (f) and midkine (g) levels in conditioned medium of C28I2 cells treated with KB-R7943 in the presence or absence of PF-04856264. h, Expression of NCX isoforms in C28I2 cells. i, Knockdown efficiency of NCX1 in C28I2 cells. j,k, F/F₀ (j) and AUC of intracellular Ca²⁺ (k) following ATP stimulation in C28I2 chondrocytes transfected with scramble or NCX1 siRNA measured by plate reader. l, HSP70 and midkine levels in conditioned medium of chondrocytes transfected with scramble or NCX1 siRNA and treated with ProTx II or PF-04856264. m, Model of mechanisms of chondrocyte- and DRG-expressed Na_v1.7 in OA, and amelioration of OA and pain via Na_v1.7 blockade. b–d,f,g,k,l, Data are mean ± s.d. b,f,g, P values calculated by one way ANOVA with Bonferroni post hoc test. k, Two-tailed unpaired Student’s t-test. c,d,l, Two-way ANOVA with Bonferroni post hoc test. b,f–i,k, n = 3 biological replicates. c,d,l, n = 4 biological replicates. Source Data

Extended Data Fig. 1. Nav1.7 is expressed in chondrocytes and upregulated in OA cartilage.

a, Relative expression of 9 distinct VGSCs in human C28I2 chondrocytes, assayed by PCR. Expression of GAPDH mRNA serves as internal control. b, Relative expressions of VGSCs in human OA (n = 4) versus healthy (n = 3) cartilage, in RNA-sequencing data of human cartilage. c, The expression levels of chondrocytes expressed VGSCs in human C28I2 cells treated with 10 ng/ml TNFα or IL-1β for 24 h, assayed by qRT-PCR (n = 4 biological replicates). d, qPCR analysis of SCN9A in human cartilage from healthy individuals (n = 11) and from individuals with early stage (KL grade 1-2, n = 14) or late stage OA (KL grade 3-4, n = 22). e, Expression of Nav1.7 in cartilage from healthy individuals and OA patients with KL grade 1-2 or 3-4, assayed by Immunoblotting (n = 3 for each group). f, Membrane location of Nav1.7 in human chondrocytes detected by Immunoblotting (n = 3 biological replicates). g, Immunostaining of Nav1.7 in cartilage from healthy individuals and from individuals with late stage OA (n = 4 for each group). Scale bar = 100 µm. h, Immunostaining of Nav1.7 in cartilage collected from mice subjected to sham or DMM surgery (n = 4 mice per group). Scale bar = 100 µm. Data are means ± SD, P values are calculated by two-tailed unpaired Student’s t-test in c and one way ANOVA with Bonferroni post-hoc test in d and e. DMM, destabilization of medial meniscus; OA, osteoarthritis.

Extended Data Fig. 2. Generation of conditional Nav1.7 knockout mouse.

a, Schematic of mouse breeding strategy to generate conditional knockout mice lacking Nav1.7 in chondrocyte (Nav1.7^chondrocyte), DRG neurons (Nav1.7^DRG) or both chondrocyte and DRG neurons (Nav1.7^{DRG;chondrocyte}). b-d, Relative Nav1.7 mRNA level in chondrocyte and DRG neurons from Nav1.7^chondrocyte (b), Nav1.7^DRG (c), and Nav1.7^{DRG;chondrocyte} (d) mice (n = 3 biological replicates). 10-week-old mice Agc1-Cre^ERT2;Nav1.7^flox/flox and Agc1-Cre^ERT2;Nav1.8-Cre;Nav1.7^flox/flox were intraperitoneally injected with tamoxifen at a dose of 150 µg per gram of body weight, administered daily for 5 consecutive days. Basal levels of Nav1.7 in DRG neurons and chondrocytes from Nav1.7^flox mice were set as 1, respectively. Data are means ± SD, P values are calculated by two-tailed unpaired Student’s t-test. Source Data

Extended Data Fig. 3. Genetic ablation of Nav1.7 in both chondrocyte and DRG neuron ameliorates OA progression and alleviates pain in chemically induced OA model.

a, Experimental scheme showing establishment of MIA model in mice harboring tamoxifen inducible Nav1.7 deletion in chondrocyte and DRG neuron. b, Representative Safranin O/fast green stained knee joint sections of Nav1.7^flox and Nav1.7^{DRG;chondrocyte} male after saline or MIA injection at day 28 (n = 8 mice for each group). Scale bar = 50 µm. c, Quantification of OARSI score shown in b. d, Representative traces of open field testing at day 28 post MIA injection in Nav1.7^flox and Nav1.7^{DRG;chondrocyte} mice. e, f, Quantitation of 2 min travel distance (e) and von Frey testing (f) in Nav1.7^flox and Nav1.7^DRG mice at the indicated time-points post MIA injection (n = 8 mice per group). g, Immunohistochemical staining and corresponding quantification for COL2, Mmp13, Aggrecan neoepitope, and Comp fragment in knee joint sections of Nav1.7^flox and Nav1.7^{DRG;chondrocyte} mice at day 28 post MIA injection (n = 6). Scale bar = 50 µm. h, (related to Fig. 2a–h) Immunohistochemical staining and corresponding quantification of COL2, Mmp13, aggrecan neoepitope, and Comp fragment in knee joint sections of Nav1.7^flox and Nav1.7^{DRG; chondrocyte} mice at 12 weeks post DMM surgery (n = 8). Scale bar = 50 µm. Data are means ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (c) and two-tailed unpaired Student’s t-test (g, h). Data are means ± 95% confidence interval (CI), P values are calculated by two-tailed multiple unpaired Student’s t-test with Welch correction (e, f). * P < 0.05; ** P < 0.01. Source Data

Extended Data Fig. 4. Genetic ablation of Nav1.7 in DRG neuron contributes to reduce OA associated pain without affecting OA pathological progression in surgically induced OA model.

a, b, (related to Fig. 2i–p) Immunohistochemical staining (a) and corresponding quantification (b) of Col2, Mmp13, Aggrecan neoepitope, and Comp fragment in knee joint sections of Nav1.7^flox and Nav1.7^Chondrocyte mice at 12 weeks post DMM surgery (n = 8). Scale bar = 50 µm in a. c, Representative Safranin O/fast green stained sections of knee joints from DMM operated Nav1.7^flox and Nav1.7^DRG mice (n = 8 mice per group). Scale bar = 50 µm. d-g, Quantitation of OARSI score (d), osteophyte development (e), SBP thickness (f), and synovitis score (g) in Nav1.7^flox and Nav1.7^DRG mice subjected to DMM surgery at week 12 (n = 8 mice per group). h, Representative traces of open field testing at 12 weeks post DMM surgery in Nav1.7^flox and Nav1.7^DRG mice. i, j, Quantitation of 2 min travel distance (i) and von Frey testing (j) in DMM operated Nav1.7^flox and Nav1.7^DRG mice at the indicated time-points after surgery (n = 8 mice per group). k, l, Immunohistochemical staining (k) and corresponding quantification (l) for Col2, Mmp13, Aggrecan neoepitope, and Comp fragment in knee joint sections of Nav1.7^flox and Nav1.7^DRG mice at 12 weeks post DMM surgery (n = 6). Scale bar = 50 µm in k. Data are means ± SD, P values are calculated by two-tailed unpaired Student’s t-test in b, d-g and l; data are means ± 95% confidence interval (CI), P values are calculated by two-tailed multiple unpaired Student’s t-test with Welch correction in i and j (** P < 0.01). Source Data

Extended Data Fig. 5. Pharmacological blockade of Nav1.7 through locally is therapeutic against OA and OA-related pain in surgically induced mouse models.

a, Schematic of the experimental design to determine the effects of intra-articular administrated PF-04856264 (the selective Nav1.7 inhibitor) on OA progression and pain-related behaviors in the surgically induced DMM mouse model. b, Representative Safranin O/fast green stained knee joint sections of DMM operated WT male mice treated with or without PF-04856264 for 8 weeks (n = 8 mice for each group). Scale bar = 50 µm. c-e, Quantitation of OARSI score (c), osteophyte development (d) and SBP thickness (e) in DMM operated WT male mice treated with or without PF-04856264 for 8 weeks (n = 8 mice per group). f, g, Quantitation of 2 min travel distance (f) and von Frey testing (g) in DMM-operated WT male mice treated with or without PF-04856264 at the indicated time-points after surgery (n = 8 mice per group). h, i, Immunohistochemical staining (h) and corresponding quantification (i) of Col2, Mmp13, and Aggrecan neoepitope in knee joint sections of DMM operated WT male mice treated with or without PF-04856264 for 8 weeks (n = 6). Scale bar = 50 µm in h. j, Representative Safranin O/fast green stained knee joint sections of DMM operated WT female mice treated with or without PF-04856264 for 8 weeks (n = 8 mice for each group). Scale bar = 50 µm. k-m, Quantitation of OARSI score (k), osteophyte development (l) and SBP thickness (m) in DMM operated WT female mice treated with or without PF-04856264 for 8 weeks (n = 8 mice per group). n, o, Quantitation of 2 min travel distance (n) and von Frey testing (o) in DMM-operated WT female mice treated with or without PF-04856264 at the indicated time-points after surgery (n = 8 mice per group). Data are means ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (c, d, e, k, l and m) and two-tailed unpaired Student’s t-test (i). Data are means ± 95% confidence interval (CI), P values are calculated by two-tailed multiple unpaired Student’s t-test with Welch correction in f, g, n and o (** P < 0.01). Source Data

Extended Data Fig. 6. Oral delivery of PF-04756264 attenuates OA progression and alleviates pain in both chemically and surgically induced OA models.

a, A schematic representation highlights the experimental outline to determine the effects of PF-04856264 through oral delivery on OA progression and behavior changes in WT male and female mice with MIA model. b, Representative Safranin O/fast green stained knee joint sections of WT male and female mice treated with or without PF-04856264 after saline or MIA injection at day 28 (n = 6 mice for each group). Scale bar = 50 µm. c, d, Quantification of OARSI score in male (c) and female (d) mice as shown in b. e, f, Quantitation of 2 min travel distance and von Frey testing in WT male (e) and female (f) mice treated with or without PF-04856264 at the indicated time-points post MIA injection (n = 6 mice per group). g, h, Immunohistochemical staining (g) and corresponding quantification (h) for Col2, Mmp13, and Aggrecan neoepitope in knee joint sections of WT male mice treated with or without PF-04856264 at day 28 post MIA injection (n = 6). Scale bar = 50 µm in g. i, Representative Safranin O/fast green stained knee joint sections of DMM operated WT male mice orally treated without or with PF-04856264 for 8 weeks (n = 8 mice for each group). Scale bar = 50 µm. j, Quantitation of OARSI score for i. Data are means ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (c, d and j) and two-tailed unpaired Student’s t-test (h). Data are means ± 95% confidence interval (CI), P values are calculated by two-tailed multiple unpaired Student’s t-test with Welch correction in e and f (* P < 0.05; ** P < 0.01). Source Data

Extended Data Fig. 7. Carbamazepine (CBZ), a clinically used sodium chancel blocker, attenuates OA progression and pain in chemically and surgically induced OA models.

a, Schematic of timeline to analyze the effects of systemic oral delivery carbamazepine (CBZ) on OA progression and pain in MIA model. b, Representative Safranin O/fast green stained knee joint sections of WT male mice treated with or without CBZ after saline or MIA injection at day 28 (n = 6 mice for each group). Scale bar = 50 µm. c, Quantification of OARSI score as shown in b. d, Representative traces of open field testing at day 28 post MIA injection in WT male mice with indicated treatment at day 28 post MIA injection. e, f, Quantitation of 2 min travel distance (e) and von Frey testing (f) in WT male mice at the indicated time-points post MIA injection (n = 6 mice per group). g, h, Immunohistochemical staining (g) and corresponding quantification (h) for Col2, Mmp13, and Aggrecan neoepitope in knee joint sections of WT mice with indicated treatment at day 28 post MIA injection (n = 6). Scale bar = 50 µm in g. i, Schematic of timeline to analyze the effects of systemic oral delivery of various dosages of CBZ on OA progression and pain in surgically induced DMM model. j, Representative Safranin O/fast green stained knee joint sections of DMM operated WT male mice treated without or with 10 mg/kg body weight, 50 mg/kg body weight or 250 mg/kg body weight CBZ for 8 weeks (n = 8 mice for each group). Scale bar = 50 µm. k, Quantitation of OARSI score for j. l, m, Quantitation of 2 min travel distance (l) and von Frey testing (m) in DMM-operated WT male mice treated without or with various dosage of CBZ, as indicated, at specified time-points after surgery (n = 8 mice per group). Data are means ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (c, k) and two-tailed unpaired Student’s t-test (h). Data are means ± 95% confidence interval (CI), P values are calculated by two-tailed multiple unpaired Student’s t-test with Welch correction in (e and f) and two way ANOVA with Bonferroni post-hoc test (l, m). * P < 0.05; ** P < 0.01.

Extended Data Fig. 8. Pharmacological blockade or deletion of Nav1.7 enhances anabolic and inhibits catabolic metabolism in chondrocytes.

a-d, mRNA levels of catabolism markers MMP13 (a), ADAMTS5 (b), COX2 (c) and NOS2 (d) in human C28I2 chondrocytes treated with or without 10 ng/ml IL-1β in the absence or presence of 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 h, assayed by qRT-PCR (n = 4 biological replicates). e-h, mRNA levels of catabolism markers MMP13 (e), ADAMTS5 (f), COX2 (g) and NOS2 (h) in human C28I2 chondrocytes treated with or without 10 ng/ml TNFα in the absence or presence of 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 h, assayed by qRT-PCR (n = 3 biological replicates). i-l, mRNA levels of catabolism markers MMP13 (i), ADAMTS5 (j), COX2 (k) and NOS2 (l) in human C28I2 chondrocytes treated with or without 500 ng/ml poly(I:C) in the absence or presence of 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 h, assayed by qRT-PCR (n = 3 biological replicates). m, n, mRNA levels of anabolic markers COL2 (m) and ACAN (n) in human C28I2 chondrocytes treated with 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 h, assayed by qRT-PCR (n = 4 biological replicates). o, p, mRNA levels of Col2 (o) and Acan (p) in chondrocytes isolated from Nav1.7^flox and Nav1.7^chondrocyte mice at P6 (n = 4 biological replicates). q-t, mRNA levels of Mmp13 (q), Adamts5 (r), Cox2 (s) and Nos2 (t) in chondrocytes isolated from Nav1.7^flox and Nav1.7^chondrocyte mice at P6 which are treated with or without IL-1β for 24 h (n = 4 biological replicates). Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (a-n) and two-tailed unpaired Student’s t-test (o-t).

Extended Data Fig. 9. Blocking Nav1.7 pharmacologically inhibit catabolism and enhance anabolism through regulating the chondrocyte secretion.

a-d, mRNA levels of MMP13 (a), ADAMTS5 (b), COX2 (c) and NOS2 (d) in primary human chondrocytes isolated from patients with last stage OA which are treated with or without 10 ng/ml IL-1β along with a serial doses of ProTx II (Pro) or PF-04856264 (PF) for 24 h, assayed by qRT-PCR (n = 6 donors). e, f, mRNA levels of COL2 (e) and ACAN (f) in primary human chondrocytes isolated from patients with late stage OA which are treated with a serial doses of ProTX II (Pro) or PF-04856264 (PF) for 24 h, assayed by qRT-PCR (n = 6 donors). g, h, Proteins levels of MMP13 (g) and PRG4 (h) in the supernatants of full-thickness human OA cartilage explants, as determined by ELISA (n = 8 donors). Cartilage explant was cultured with 10 ng/ml IL-1β in the absence or presence of 25 nM ProTx II or 1 µM PF-04856264 for 5 days. i-l, mRNA levels of MMP13 (i), ADAMTS5 (j), COL2 (k), and ACAN (l) in the human OA cartilage explants cultured with 10 ng/ml IL-1β in the absence or presence of 25 nM ProTx II or 1 µM PF-04856264 for 5 days, assayed by qRT-PCR (n = 8 donors). m, n, mRNA levels of COL2 (m) and ACAN (n) in human C28I2 cells treated with conditioned medium collected from 25 nM Pro or 1 µM PF treated C28I2 cells (n = 4 biological replicates). o-r, mRNA levels of MMP13 (o), ADAMTS5 (p), COX2 (q) and NOS2 (r) in human C28I2 cells treated with conditioned medium collected from 25 nM Pro or 1 µM PF treated C28I2 cells as well as 10 ng/ml IL-1β for 24 h (n = 4 biological replicates). Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test.

Extended Data Fig. 10. Effects of conditioned medium on human chondrocyte anabolism and catabolism.

a, Schematic of the experimental strategy of conditioned medium production, fraction and characterization. Human C28I2 chondrocytes are expanded in DMEM supplemented with FBS until they are 90% confluent. The growth medium is them exchanged with medium supplemented with ITS and 25 nM ProTX II (Pro) or 1 µM PF-04856264 (PF) for 2 days. The medium is then collected and separated into 4 fractions based on sized exclusion: <10 kDa, 10−30 kDa, 30−100 kDa, and >100 kDa using the centrifugal filter device. b, mRNA levels of COL2 and ACAN in human C28I2 cells treated with 30–100 kDa fraction of the conditioned medium for 24hrs (n = 4 biological replicates). c, mRNA levels of MMP13 and ADAMTS5 in human C28I2 cells treated with 30–100 kDa fraction of the conditioned medium in the absence or presence of 10 ng/ml IL-1β for 24hrs (n = 4 biological replicates). d, mRNA levels of COL2 and ACAN in human C28I2 cells treated with 10–30 kDa fraction of the conditioned medium for 24hrs (n = 4 biological replicates). e, mRNA levels of MMP13, ADAMTS5, COX2 and NOS2 in human C28I2 cells treated with 10–30 kDa fraction of the conditioned medium in the absence or presence of 10 ng/ml IL-1β for 24hrs (n = 4 biological replicates). Data are means ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (b-e). Source Data

Extended Data Fig. 11. HSP70 enhances anabolism and midkine inhibits IL-1β induced catabolism in human chondrocytes.

a, b, ELISA quantification of HSP70 in conditioned medium (a) and cell lysate (b) of human C28I2 cells treated with 25 nM ProTx II (Pro), 1 µM PF-04856264 (PF), or 10 µM CBZ for 48 h. c, d, ELISA quantification of Midkine in conditioned medium (c) and cell lysate (d) of human C28I2 cells treated with 25 nM Pro or 1 µM PF for 48 h. e, mRNA levels of COL2 and ACAN in human C28I2 cells treated with serial doses of HSP70 for 24 h. f, mRNA levels of MMP13 and ADAMTS5 in human C28I2 cells treated with serial doses of HSP70 and 10 ng/ml IL-1β for 24 h. g, mRNA levels of COL2 and ACAN in human C28I2 cells treated with serial doses of midkine for 24 h. h, mRNA levels of MMP13 and ADAMTS5 in human C28I2 cells treated with serial doses of midkine and 10 ng/ml IL-1β for 24 h. n = 4 biological replicates; Data are mean ± SD, P values are calculated by. i, j, ELISA quantification of HSP70 (i) and midkine (j) in conditioned medium of primary chondrocytes isolated from Nav1.7^flox and Nav1.7^chondrocye mice at 12 weeks post DMM surgery (n = 4 biological replicates). k, l, Levels of HSP70 (k) and midkine (l) in mouse sera collected from sham surgery control and from DMM surgery WT mice at 12 weeks after surgery (n = 24), assayed by ELISA. m, o, Levels of HSP70 (m) and midkine (o) in human sera collected from healthy individuals (n = 22) and from patients with OA (n = 165), assayed by ELISA. n, p, Correlation analysis (Pearson R and two-tailed P value) of HSP70 (n) and midkine (p) between matched sera and synovium fluids isolated from OA patients (n = 35). Data are means ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (a-h) and two-tailed unpaired Student’s t-test (i-l, m, o).

Extended Data Fig. 12. Intracellular Ca²⁺ signals are essential for the enhanced secretion of HSP70 and midkine following Nav1.7 blockade.

a, b, Traces (F/F0) of intracellular Na⁺ levels over time in human OA chondrocytes (a) and C28I2 cells (b) following ATP stimulation, measured by confocal microscopy. ATP present from red arrows. c, d, Traces (F/F0) (c) and quantification of AUC (d) of intracellular Ca²⁺ levels in C28I2 chondrocytes following ATP stimulation, measured by plate reader (n = 3 biological replicates). e, f, Traces (F/F0) of intracellular Ca²⁺ levels in human OA (e) and C28I2 (f) chondrocytes following ATP stimulation, assayed by confocal fluorescence microscopy. ATP present from red arrows. g, h, ELISA quantification of HSP70 (g) and midkine (h) in conditioned medium of human C28I2 cells treated with or without Ionomycin in the absence or presence of 1 µM PF-04856264 (PF) for 48 h. n = 3 biological replicates; Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test in d, g and h.