Int J Stem Cells.  2025 Feb;18(1):59-71. doi.org/10.15283/ijsc24097.

The Effect of Nerve Growth Factor on Cartilage Fibrosis and Hypertrophy during In Vitro Chondrogenesis Using Induced Pluripotent Stem Cells

• Jung SI
• Choi SH
• Kim JW
• Lim J
• Rim YA
• Ju JH

• KMID: 2565197
• DOI: http://doi.org/doi.org/10.15283/ijsc24097

Abstract

Nerve growth factor (NGF) is a neurotrophic factor usually involved in the survival, differentiation, and growth of sensory neurons and nociceptive function. Yet, it has been suggested to play a role in the pathogenesis of osteoarthritis (OA). Previous studies suggested a possible relationship between NGF and OA; however, the underlying mechanisms remain unknown. Therefore, we investigated the impact of NGF in chondrogenesis using human induced pluripotent stem cells (hiPSCs)-derived chondrogenic pellets. To investigate how NGF affects the cartilage tissue, hiPSC-derived chondrogenic pellets were treated with NGF on day 3 of differentiation, expression of chondrogenic, hypertrophic, and fibrotic markers was confirmed. Also, inflammatory cytokine arrays were performed using the culture medium of the NGF treated chondrogenic pellets. As a result, NGF treatment decreased the expression of pro-chondrogenic markers by approximately 2∼4 times, and hypertrophic (pro-osteogenic) markers and fibrotic markers were increased by approximately 3-fold or more in the NGF-treated cartilaginous pellets. In addition, angiogenesis was upregulated by approximately 4-fold or more, bone formation by more than 2-fold, and matrix metalloproteinase induction by more than 2-fold. These inflammatory cytokine array were using the NGF-treated chondrogenic pellet cultured medium. Furthermore, it was confirmed by Western blot to be related to the induction of the glycogen synthase kinase-3 beta (GSK3β) pathway by NGF. In Conclusions, these findings provide valuable insights into the multifaceted role of NGF in cartilage hypertrophy and fibrosis, which might play a critical role in OA progression.

Keyword

Nerve growth factor; Induced pluripotent stem cells; Chondrogenesis; Hypertrophy; Chondrocalcinosis; Osteoarthritis

Figure

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  Fig. 1 Nerve growth factor (NGF) expression in osteoarthritis (OA) cartilage tissues and chondrogenic pellets treated with synovial fluid (SF) of OA patients. (A) Immunofluorescence staining was performed to determine NGF expression in the cartilage tissues of patients (n=3). It was confirmed that the damaged area is different in the same patient's articular cartilage tissue, and the less damaged area is shown in the left panel, and the severely damaged area is shown in the lower panel. Scale bar=40 μm. (B) Schematic of treatment of chondrogenic pellets with human SF (n=3). (C) Morphology of induced pluripotent stem cells (iPSCs), embryoid bodies (EBs), outgrowth cells (OGCs), and chondrogenic pellets during differentiation. Scale bar=100 μm. Images of EBs and OGCs are shown at 100 μm magnification. (D) Images of normal and OA-SF-treated pellets. (E) Relative gene expression of NGF and NTRK1 in normal group and SF-treated pellets. (F) Protein levels of NGF and TrkA confirmed through western blot analysis using pellet extracts. (G) Quantitative analysis of band intensity using western blotting. (H) NGF and TrkA expression determined via immunofluorescence (IFA) staining in OA-SF-treated pellets on days 7. (I) Quantitative measurements of IFA staining area by ImageJ software (National Institutes of Health). Scale bar=200 μm magnification. Expression of proteins was normalized to that of GAPDH. Statistical analysis was performed using the t-test (*p<0.05, **p<0.01) and Mann–Whitney U-test (#p<0.05). Data are represented as mean±SEM. NC: normal control.

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  Fig. 2 Characterization of nerve growth factor (NGF)-treated chondrogenic pellets at day 7 and 14. (A) Scheme of chondrogenic differentiation with NGF treatment. (B) Pellets morphology of normal control (NC) and NGF treated group. (C) Relative mRNA levels of NGF and NTRK1 in pellets. (D, E) Western blot image and quantitative evaluation of NGF and TrkA in normal and NGF-treated pellets. (F) Relative gene expression of chondrogenic markers SOX9, ACAN, and COL2A1 on days 7 and 14 along with COL10A1, VEGFA, and MMP13 as hypertrophic markers. Statistical analysis using t-test (*p<0.05, **p<0.01, ***p<0.001) and Mann–Whitney U-test (#p<0.05, ##p<0.01, ###p<0.001) are represented as mean±SEM. Gene expression was normalized to that of GAPDH. (G) Pellet images after Toluidine blue staining, Safranin O staining and Alcian blue staining. (H, I) Pellets images after immunohistological staining of Collagen Type II and Collagen Type X. Scale bar=200 μm magnification. hiPSCs: human induced pluripotent stem cells, EB: embryoid body, OG: outgrowth.

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  Fig. 3 Inflammatory cytokine expression in nerve growth factor (NGF)-treated chondrogenic pellet culture medium. (A) Cytokine array results showing the most altered expression of markers related to (B) angiogenesis (angiogenin, RBP-4, VCAM-1), (C) osteogenesis (IGFBP-2, IGFBP-3, IP-10, HGF), (D) MMP induction (MIF, EMMRPIN), and (E) Osteopontin (OPN), which has showed a highly significant reduction. NC: normal control. *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 4 Induction of cartilage hypertrophy and fibrosis by nerve growth factor (NGF) treatment in chondrogenic pellets. (A) Relative gene expression of hypertrophy markers, ALP, OCN, and RUNX2. (B) Relative gene expression of fibrosis markers, COL1A1, COL3A1, and α-SMA. (C) The protein expression levels of alkaline phosphatase (ALP), RUNX2, Collagen Type III (COL3), and α-SMA. (D) Quantitative evaluation of Western blot. (E) Alizarin red S and Von Kossa staining images of normal control (NC) and NGF-treated pellets. Scale bar=100 μm magnification. (F) Protein expression of fibrotic marker Collagen Type I and hypertrophic marker osteocalcin in NGF-treated pellets on day 14 and (G) quantitative evaluation. Image scale bar=200 μm magnification. Statistical significance is shown as t-test (*p<0.05, **p<0.01, ***p<0.001) and Mann–Whitney U-test (#p<0.05, ##p<0.01, ###p<0.001). (H) Western blot image of GSK3β and p-GSK3β. (I) Quantitative analysis of the western blot image of GSK3β and p-GSK3β.

Reference

References

1. Rim YA, Nam Y, Ju JH. 2020; The role of chondrocyte hypertrophy and senescence in osteoarthritis initiation and progression. Int J Mol Sci. 21:2358. DOI: 10.3390/ijms21072358. PMID: 32235300. PMCID: PMC7177949.

2. O'Brien MS, McDougall JJ. 2019; Age and frailty as risk factors for the development of osteoarthritis. Mech Ageing Dev. 180:21–28. DOI: 10.1016/j.mad.2019.03.003. PMID: 30898635.

3. Jang S, Lee K, Ju JH. 2021; Recent updates of diagnosis, pathophysiology, and treatment on osteoarthritis of the knee. Int J Mol Sci. 22:2619. DOI: 10.3390/ijms22052619. PMID: 33807695. PMCID: PMC7961389.

4. Enomoto M, Mantyh PW, Murrell J, Innes JF, Lascelles BDX. 2019; Anti-nerve growth factor monoclonal antibodies for the control of pain in dogs and cats. Vet Rec. 184:23. DOI: 10.1136/vr.104590. PMID: 30368458. PMCID: PMC6326241.

5. Barker PA, Mantyh P, Arendt-Nielsen L, Viktrup L, Tive L. 2020; Nerve growth factor signaling and its contribution to pain. J Pain Res. 13:1223–1241. DOI: 10.2147/JPR.S247472. PMID: 32547184. PMCID: PMC7266393.

6. Eitner A, Hofmann GO, Schaible HG. 2017; Mechanisms of osteoarthritic pain. Studies in humans and experimental models. Front Mol Neurosci. 10:349. DOI: 10.3389/fnmol.2017.00349. PMID: 29163027. PMCID: PMC5675866.

7. Wild KD, Bian D, Zhu D, et al. 2007; Antibodies to nerve growth factor reverse established tactile allodynia in rodent models of neuropathic pain without tolerance. J Pharmacol Exp Ther. 322:282–287. DOI: 10.1124/jpet.106.116236. PMID: 17431136.

8. Neogi T, Hunter DJ, Churchill M, et al. 2022; Observed efficacy and clinically important improvements in participants with osteoarthritis treated with subcutaneous tanezumab: results from a 56-week randomized NSAID-controlled study. Arthritis Res Ther. 24:78. DOI: 10.1186/s13075-022-02759-0. PMID: 35351194. PMCID: PMC8966257.

9. Ugolini G, Marinelli S, Covaceuszach S, Cattaneo A, Pavone F. 2007; The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain. Proc Natl Acad Sci U S A. 104:2985–2990. DOI: 10.1073/pnas.0611253104. PMID: 17301229. PMCID: PMC1815293.

10. Berenbaum F, Blanco FJ, Guermazi A, et al. 2020; Subcutaneous tanezumab for osteoarthritis of the hip or knee: efficacy and safety results from a 24-week randomised phase III study with a 24-week follow-up period. Ann Rheum Dis. 79:800–810. DOI: 10.1136/annrheumdis-2019-216296. PMID: 32234715. PMCID: PMC7286052.

11. Robinson WH, Lepus CM, Wang Q, et al. 2016; Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat Rev Rheumatol. 12:580–592. DOI: 10.1038/nrrheum.2016.136. PMID: 27539668. PMCID: PMC5500215.

12. Chawla S, Mainardi A, Majumder N, et al. 2022; Chondrocyte hypertrophy in osteoarthritis: mechanistic studies and models for the identification of new therapeutic strategies. Cells. 11:4034. DOI: 10.3390/cells11244034. PMID: 36552796. PMCID: PMC9777397.

13. Dreier R. 2010; Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther. 12:216. DOI: 10.1186/ar3117. PMID: 20959023. PMCID: PMC2990991.

14. Jiang Y, Hu C, Yu S, et al. 2015; Cartilage stem/progenitor cells are activated in osteoarthritis via interleukin-1β/nerve growth factor signaling. Arthritis Res Ther. 17:327. DOI: 10.1186/s13075-015-0840-x. PMID: 26577823. PMCID: PMC4650403.

15. Park N, Rim YA, Jung H, Nam Y, Ju JH. 2022; Lupus heart disease modeling with combination of induced pluripotent stem cell-derived cardiomyocytes and lupus patient serum. Int J Stem Cells. 15:233–246. DOI: 10.15283/ijsc21158. PMID: 34966002. PMCID: PMC9396017.

16. Mo H, Kim J, Kim JY, et al. 2023; Intranasal administration of induced pluripotent stem cell-derived cortical neural stem cell-secretome as a treatment option for Alzheimer's disease. Transl Neurodegener. 12:50. DOI: 10.1186/s40035-023-00384-8. PMID: 37946307. PMCID: PMC10634159.

17. Sayegh S, El Atat O, Diallo K, et al. 2019; Rheumatoid synovial fluids regulate the immunomodulatory potential of adipose-derived mesenchymal stem cells through a TNF/NF-κB-dependent mechanism. Front Immunol. 10:1482. DOI: 10.3389/fimmu.2019.01482. PMID: 31316519. PMCID: PMC6611153.

18. Nam Y, Rim YA, Jung SM, Ju JH. 2017; Cord blood cell-derived iPSCs as a new candidate for chondrogenic differentiation and cartilage regeneration. Stem Cell Res Ther. 8:16. DOI: 10.1186/s13287-017-0477-6. PMID: 28129782. PMCID: PMC5273802.

19. Pecchi E, Priam S, Gosset M, et al. 2014; Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain. Arthritis Res Ther. 16:R16. DOI: 10.1186/ar4443. PMID: 24438745. PMCID: PMC3978639.

20. Martel-Pelletier J, Barr AJ, Cicuttini FM, et al. 2016; Osteoarthritis. Nat Rev Dis Primers. 2:16072. DOI: 10.1038/nrdp.2016.72. PMID: 27734845.

21. Berenbaum F, Langford R, Perrot S, et al. 2021; Subcutaneous tanezumab for osteoarthritis: is the early improvement in pain and function meaningful and sustained? Eur J Pain. 25:1525–1539. DOI: 10.1002/ejp.1764. PMID: 33728717. PMCID: PMC8360021.

22. Mapp PI, Walsh DA. 2012; Mechanisms and targets of angiogenesis and nerve growth in osteoarthritis. Nat Rev Rheumatol. 8:390–398. DOI: 10.1038/nrrheum.2012.80. PMID: 22641138.

23. Segarra-Queralt M, Neidlin M, Tio L, et al. 2022; Regulatory network-based model to simulate the biochemical regulation of chondrocytes in healthy and osteoarthritic environments. Sci Rep. 12:3856. DOI: 10.1038/s41598-022-07776-2. PMID: 35264634. PMCID: PMC8907219.

24. Han H, Kim Y, Mo H, et al. 2022; Preferential stimulation of melanocytes by M2 macrophages to produce melanin through vascular endothelial growth factor. Sci Rep. 12:6416. DOI: 10.1038/s41598-022-08163-7. PMID: 35440608. PMCID: PMC9019043.

25. Moraes-Vieira PM, Yore MM, Dwyer PM, Syed I, Aryal P, Kahn BB. 2014; RBP4 activates antigen-presenting cells, leading to adipose tissue inflammation and systemic insulin resistance. Cell Metab. 19:512–526. DOI: 10.1016/j.cmet.2014.01.018. PMID: 24606904. PMCID: PMC4078000.

26. Wei Z, Li HH. 2015; IGFBP-3 may trigger osteoarthritis by inducing apoptosis of chondrocytes through Nur77 translocation. Int J Clin Exp Pathol. 8:15599–610.

27. Furman BD, Kent CL, Huebner JL, et al. 2018; CXCL10 is upregulated in synovium and cartilage following articular fracture. J Orthop Res. 36:1220–1227. DOI: 10.1002/jor.23735. PMID: 28906016. PMCID: PMC5851826.

28. Abed E, Bouvard B, Martineau X, Jouzeau JY, Reboul P, Lajeunesse D. 2015; Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity. Bone. 75:111–119. DOI: 10.1016/j.bone.2015.02.001. PMID: 25667190.

29. Liu M, Hu C. 2012; Association of MIF in serum and synovial fluid with severity of knee osteoarthritis. Clin Biochem. 45:737–739. DOI: 10.1016/j.clinbiochem.2012.03.012. PMID: 22449335.

30. Tomita T, Nakase T, Kaneko M, et al. 2002; Expression of extracellular matrix metalloproteinase inducer and enhancement of the production of matrix metalloproteinases in rheumatoid arthritis. Arthritis Rheum. 46:373–378. DOI: 10.1002/art.10050. PMID: 11840439.

31. Luo W, Lin Z, Yuan Y, Wu Z, Zhong W, Liu Q. 2022; Osteopontin (OPN) alleviates the progression of osteoarthritis by promoting the anabolism of chondrocytes. Genes Dis. 10:1714–1725. DOI: 10.1016/j.gendis.2022.08.010. PMID: 37397527. PMCID: PMC10311054.

32. Mimpen JY, Baldwin MJ, Cribbs AP, et al. 2021; Interleukin-17A causes osteoarthritis-like transcriptional changes in human osteoarthritis-derived chondrocytes and synovial fibroblasts in vitro. Front Immunol. 12:676173. DOI: 10.3389/fimmu.2021.676173. PMID: 34054865. PMCID: PMC8153485.

33. Choi SH, Lee K, Han H, et al. 2023; Prochondrogenic effect of decellularized extracellular matrix secreted from human induced pluripotent stem cell-derived chondrocytes. Acta Biomater. 167:234–248. DOI: 10.1016/j.actbio.2023.05.052. PMID: 37295627.

34. Guidotti S, Minguzzi M, Platano D, et al. 2017; Glycogen synthase kinase-3β inhibition links mitochondrial dysfunction, extracellular matrix remodelling and terminal differentiation in chondrocytes. Sci Rep. 7:12059. DOI: 10.1038/s41598-017-12129-5. PMID: 28935982. PMCID: PMC5608843.

35. Wang X, Cornelis FMF, Lories RJ, Monteagudo S. 2019; Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation. Osteoarthritis Cartilage. 27:1702–1710. DOI: 10.1016/j.joca.2019.07.007. PMID: 31330188.

36. Boland GM, Perkins G, Hall DJ, Tuan RS. 2004; Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells. J Cell Biochem. 93:1210–1230. DOI: 10.1002/jcb.20284. PMID: 15486964.

37. Choi SH, Kim H, Lee HG, et al. 2017; Dickkopf-1 induces angiogenesis via VEGF receptor 2 regulation independent of the Wnt signaling pathway. Oncotarget. 8:58974–58984. DOI: 10.18632/oncotarget.19769. PMID: 28938611. PMCID: PMC5601707.

38. Akoumianakis I, Polkinghorne M, Antoniades C. 2022; Non-canonical WNT signalling in cardiovascular disease: mechanisms and therapeutic implications. Nat Rev Cardiol. 19:783–797. DOI: 10.1038/s41569-022-00718-5. PMID: 35697779. PMCID: PMC9191761.

39. Liu Z, Suh JS, Deng P, et al. 2022; Epigenetic regulation of NGF-mediated osteogenic differentiation in human dental mesenchymal stem cells. Stem Cells. 40:818–830. DOI: 10.1093/stmcls/sxac042. PMID: 35728620. PMCID: PMC9512103.

40. Lee J, Jung H, Park N, Park SH, Ju JH. 2019; Induced osteogenesis in plants decellularized scaffolds. Sci Rep. 9:20194. DOI: 10.1038/s41598-019-56651-0. PMID: 31882858. PMCID: PMC6934596.