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Introduction
Cartilage can be roughly categorized into three types; hyaline cartilage, fibrotic cartilage and hypertrophic cartilage. Among them, articular cartilage is mainly known as hyaline cartilage, which is smooth and translucent cartilage composed of proteoglycan, Collagen Type II, and chondrocytes with in lacunae. The chondrocytes embedded in the dense extracellular matrix (ECM) is supplied with nutrients by tissue fluid without blood vessels or lymphatic vessels. Additionally, as it covers the surface of the bone, it absorbs external pressure or stimulation, allowing joint movement without pain and with less friction.
Osteoarthritis (OA) is a degenerative disease that commonly affects the cartilages of the hands, hips, and knee (1). It is characterized by pain, swelling, and loss of mobility due to mild inflammation, which reduces the patient’s quality of life (2). Cartilage damage in the joint can also induce bone spur, also known as osteophyte formation, which are bony growths affecting the subchondral bone remodeling in OA. Although OA is mostly treated using nonsteroidal anti-inflammatory drugs, no current treatment can stop osteophyte formation in OA joints and the exact mechanism underlying OA progression and osteophyte formation remain unknown (3).
Nerve growth factor (NGF) belongs to a family of neurotrophic factors and functions in sensory neuron growth, as well as in nociceptive function (4). NGF also function in cell survival, differentiation, and has been recently suggested as an important factor in OA that can induce inflammation-associated hyperalgesia and joint damage (5, 6). Inflamed tissues resulting from joint pain, increase the expression of NGF, which leads to increased pain sensation (7). NGF targeting drugs has been developed and Tanezumab, a monoclonal anti-NGF antibody that blocks NGF activity by selectively targeting the NGF receptors tropomyosin receptor kinase A (TrkA) and p75NTR was treated to OA patients (8). Tanezumab showed therapeutic effectiveness in reducing pain-related behaviors in OA antimal models (9). Nevertheless, side effects such as rapidly progressive OA and allodynia due to nerve damage have been reported in few patients (10).
OA pathogenesis was usually focused on low state inflammation and cartilage degeneration by generated defect or the increased production of matrix-degrading enzymes (11). However, the hypertrophic and fibrotic changes that occurs in the OA chondrocytes and cartilage are now suggested as one of the major hallmarks of OA, gaining more focus in research (12). When the cartilage tissue is injured or degenerated, cartilage changes toward fibrotic or hypertrophic chondrocytes. Cartilage hypertrophy can be characterized by the shift from hyaline-like Collagen Type II proteins to Collagen Type X and increased activity of degradative enzymes such as Matrix metalloproteinase 13 (MMP13). Also, markers that increase during bone formation such as RUNX2, osteocalcin, vascular endothelial growth factor A (VEGFA), as well as inflammatory cytokines are increased as well (13). On the other hand, fibrosis is evinced by the deposition of Collagen Type I and III. Recently, a study by Jiang et al. (14). confirmed the involvement of NGF signaling in in vitro calcification of human articular chondrocytes (hACs). Using a small peptide of NGF, increased mineralization and hypertrophic marker expression was confirmed in hACs, suggesting its role in early development of OA. However, the effect of NGF in chondrocytes, chondrogenesis, and OA pathogenesis still remains poorly understood.
In this study, we attempted to further investigate the effects of NGF on normal cartilage tissue using human induced pluripotent stem cell (hiPSC)-derived chondrogenic pellets. We first determined NGF expression in cartilage tissue from OA patients and in chondrogenic pellets derived from hiPSCs treated with NGF. Chondrogenic pellets were analyzed based on expression levels of pro-chondrogenic markers along with OA-specific markers, particularly hypertrophic and fibrosis markers. Through this study, we hope to contribute to confirming the effect of NGF on phenotypic changes in cartilage tissue.
Materials and Methods
hiPSCs culture
hiPSCs used in this study were generated using umbilical cord blood mononuclear cells and cells were cultured as described previously (15, 16). Cells were maintained in Essential 8 medium (Thermo Fisher Scientific) on vitronectin-coated dishes (Thermo Fisher Scientific), and attached cells were maintained at 37℃ in 5% CO2.
Human OA synovial fluid preparation
This study was approved by the Institutional Review Board of the Catholic University of Korea (approval number: No. MC22SISI0085). written informed consents were obtained from all participants. Synovial fluid (SF) was obtained from the knee joints of patients with OA (n=3) by Seoul St. Mary’s Hospital and transferred to a 15 mL conical tube. The samples were centrifuged at 1,800 g for 10 minutes. The supernatant was collected, aliquoted, and stored at −80℃. After random selection, Chondrogenic pellets in the samples were treated with 5% SF in medium once on day 3 of differentiation (17).
NGF treatment after chondrogenic differentiation using hiPSCs
hiPSCs were expanded and 4.5×106 cells were seeded into Aggrewell plates (STEMCELL Technologies) and cultured in T75 cell culture flasks to generate embryoid bodies (EBs). EBs were harvested and placed in a 0.1% gelatin-coated dish to induce cell outgrowth. Next, 3×105 outgrowth cells (OGCs) were resuspended in chondrogenic differentiation medium (DMEM containing 20% knockout serum replacement, 1× non-essential amino acids, 1 mM L-glutamine, 1% sodium pyruvate, 1% insulin-transferrin-selenium＋, 10−7 M dexamethasone, 50 mM ascorbic acid, and 40 μg/mL L-proline supplemented with 10 ng/mL recombinant human bone morphogenetic protein 2 and transforming growth factor beta-3 and transferred to a 15 mL conical tube. The cells were then centrifuged at 1,800 g for 5 minutes. The generated pellets were maintained for 14 days, and medium was replaced every other day (18). Recombinant human NGF protein (10 ng/mL; Abcam) (14, 19) was added to the differentiated chondrogenic pellets on day 3 and its expression level was analyzed on days 7 and 14 of differentiation.
Polymerase chain reaction assays
Samples of total RNA were extracted using TRIzol reagent (Thermo Fisher Scientific). cDNA synthesis was performed using a RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific). Real-time polymerase chain reaction (RT-PCR) was performed using the StepOneTM Real-Time PCR System (Thermo Fisher Scientific). Gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase and relative gene expression was calculated using the 2−ΔΔCt method. The PCR primer sequences used are listed in Table 1.
Western blotting
Proteins were extracted from the chondrogenic pellets using radioimmunoprecipitation assay buffer (Sigma-Aldrich). Protein concentration was measured using the Bradford assay (Bio-Rad). Protein samples were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred using nitrocellulose membrane (Cytiva). The membranes were blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.1% TweenⓇ 20 detergent (TBST) for 1 hour. The primary antibodies used were: anti-NGF (1：1,000, ab52918; Abcam), anti-TrkA (1：1,000, 06-574; Sigma- Aldrich), anti-Collagen Type III (1：1,000, ab7778; Abcam),anti-Alpha-smooth muscle actin (4A4) (1：1,000, MA5-15871; Invitrogen), anti-ALP (1：1,000, ab67228; Abcam), anti-RUNX2 (1：1,000, ab23981; Abcam), anti-GSK3β (1：1,000, ab32391; Abcam), anti-phospho-GSK3β (Ser9) antibody (serine 9) (1：500, ab75814; Abcam) were incubated at 4℃ overnight. The membranes were washed with 0.1% TBST and incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies. After three washes with 0.1% TBST, proteins were detected using a Bio-Image Analysis System (Amersham Imager 600; Fuji Photo Film Co., Ltd.). The all images are shown in Supplementary Fig. S1 and band intensity was analyzed using ImageJ software (National Institutes of Health).
Histological analysis of chondrogenesis pellets
Three slides containing OA cartilage samples were provided by Dr. Chan Kwon Jung. All slide samples were deparaffinized using two cycles of xylene treatment and then rehydrated. Samples were stained with toluidine blue for 10 minutes using 0.05% toluidine staining reagent and then washed with tap water. Alcian blue staining was performed by incubating the slides in hematoxylin for 5 minutes, followed by washing with tap water for 5 minutes. The slides were then treated with 1% Alcian blue solution for 30 minutes and washed with tap water. The slides were incubated with nuclear fast red dye for 1 minute. Safranin O staining was performed by incubating the slides with 0.001% fast green solution (Sigma-Aldrich) for 5 minutes. After dipping in 1% acetic acid for 5 minutes, the slides were incubated with 0.1% Safranin O (Sigma-Aldrich) for 5 minutes. Finally, after all staining procedures were performed, the slides were dehydrated and samples were mounted. The stained slides were observed under a light microscope.
Immunohistological staining
For immunohistological (IHC) staining, sample slides were heated at 60℃ in an oven for 10 minutes and deparaffinized using xylene following rehydration. Endogenous peroxidase activity was blocked via treatment with 3% hydrogen peroxide (Sigma-Aldrich) for 10 minutes. The slides were washed and blocked with 1× tris-buffered saline (TBS) containing 10% normal goat serum for 20 minutes at room temperature (RT). Primary antibodies were diluted in the blocking solution at the following ratios: NGF (1：250), TrkA (1：200), Collagen Type II (1：200, ab34712; Abcam), Collagen Type X (1：250, ab58632; Abcam), Osteocalcin (1：100, sc-30044; Santa Cruz Biotechnology), and Collagen Type I (1：400, ab138492; Abcam). The samples were incubated with the diluted primary antibodies at 4℃ overnight. The next day, the slides were washed in 1× TBS and incubated with a biotinylated secondary antibody for 40 minutes. The slides were washed and treated with avidin-biotin complex reagent (Vector Laboratories) for 30 minutes. The washed slides were subsequently incubated with DAB solution (Vector Laboratories) for 1 minute. Finally, the slides were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) for 1 minute followed by dehydration and mounting.
For immunofluorescence staining, the slides were washed with 1× TBS and treated with Alexa Fluor-594 and -488-conjugated secondary antibodies diluted in blocking buffer after primary antibody treatment. Secondary antibodies were then incubated for 40 minutes at RT. The slides were stained with 4’,6-diamidino-2-phenylindole (10236276001; Roche) for 10 minutes. Subsequently, the slides were washed with 1× phosphate-buffered saline (PBS) and mounted using antifade. All immunostained samples were analyzed using bright-field or fluorescence microscopy.
Human inflammatory cytokine array
The Human XL Cytokine Array Kit (ARY022B; R&D Systems) for cytokine analysis in culture medium was used according to the protocol. The experiment began with the membranes being blocked using blocking buffer for 1 hour at RT. and then incubated with cell culture supernatant overnight at 4℃. The next day, washed with the 1× wash buffer contained in the kit for 10 minutes. After, detection antibody cocktail diluted in 1x array buffer was added and incubated for 1 hour. Finally, 1× streptavidin-HRP was added to the wells and incubate for 30 minutes at RT. Images were detected by a bio-image analysis system (Amersham Imager 600).
Alizarin Red S staining
Paraffin sections were deparaffinized. The slides were washed with 1× PBS. Alizarin Red S solution (#8678; ScienCell Research Laboratories) was added to the slides at RT for 30 minutes. The stained slides were then washed with distilled water. Calcium deposition on the samples was confirmed using light microscopy analysis.
Von Kossa staining of chondrogenic pellets
Von Kossa staining was performed on paraffin slides. The slides were washed with tap water after deparaffinization and incubated with 5% silver nitrate solution under a UV lamp for 40 minutes. The slides were washed with tap water and then treated with 5% sodium thiosulfate for 20 minutes. Stained slides were viewed under a light microscope.
Statistical analysis
All experiments were performed identically in triplicate, and graphs of the experimental data were generated using GraphPad Prism 9 (GraphPad Software). To analyze non-parametric quantitative data sets, we used t-tests and calculated one-sided p-values. Values indicate significance using t-test analyzed (*p<0.05, **p<0.01, and ***p<0.001). Otherwise, the difference values between the two groups were analyzed to indicate significance using the Mann– Whitney U-test (#p<0.05, ##p<0.01, and ###p<0.001).
Results
Human OA-SF treatment increased NGF and TrkA expression in hiPSCs-derived chondrogenic pellets
We first evaluated NGF expression in the OA articular cartilage tissue. NGF expression was the highest in the damaged regions, suggesting that NGF may be involved in OA progression (Fig. 1A). Next, we attempted to confirm if treating OA patient-derived SF can induce NGF expression in hiPSCs-derived chondrogenic pellets and mimic the OA microenvironment (Fig. 1B). OA-SF was treated once on day 3 differentiated chondrogenic pellets. OA-SF treatment did not induce any morphological changes in the EBs and mesenchymal-like OGCs while differentiating chondrogenic pellets (Fig. 1C). Also, we expected that the aggregates in SF-treated cartilaginous pellets would be larger, not perfectly spherical, and show morphological changes such as vacuoles, but we did not observe any specific morphological changes in pellet images after treating OA patient’s SF in the cartilaginous pellets compared to the normal control and SF-treated pellets. (Fig. 1D). The change in NGF and its receptor, NTRK1 gene expression was evaluated in the SF-treated pellets. NGF gene expression did show any difference, however, NTRK1 was significantly increased (p<0.001, t-test and Mann–Whitney U-test) on day 7 chondrogenic pellets treated with OA-SF (Fig. 1E). A similar trend was seen in protein expression of NGF and TrkA (Fig. 1F). Quantitative evaluation also showed that NGF and TrkA protein expression was significantly increased (p<0.001, t-test and Mann–Whitney U-test) in SF-treated pellets at day 7 (Fig. 1G). In addition, the expression of NGF and TrkA was confirmed through immunofluorescence staining of chondrogenic pellets (Fig. 1H). Quantitative evaluation showed that NGF showed similar expression levels in both groups, whereas TrkA expression was increased by OA-SF treatment (Fig. 1I). Taken together, we confirmed that NGF was induced in chondrogenic pellets treated with OA-SF. as well as its receptor TrkA.
NGF treatment reduced the hyaline cartilage-like characteristics in hiPSCs-derived chondrogenic pellets
We confirmed that OA-SF treatment induced NGF and TrkA expression in iPSC-derived chondrogenic pellets. To confirm the direct effect of NGF during in vitro chondrogenesis, we treated the cells with human recombinant NGF protein on day 3 of chondrogenic differentiation and analyzed the pellets on days 7 and 14 (Fig. 2A). There was no significant difference in pellet morphology or size between normal and NGF-treated pellets (Fig. 2B). Interestingly, NGF treatment temporarily increased the NGF gene expression on day 7, as well as NTRK1 (Fig. 2C). Protein expression of NGF and TrkA treatment increased NGF expression in the chondrogenic pellets on day 7 (Fig. 2D). Also, the expression of TrkA was simultaneously detected (Fig. 2E).
We confirmed the expression of pro-chondrogenic markers, including SOX9, ACAN, and COL2A1, as well as the expression of hypertrophic markers, COL10A1, VEGFA, and MMP13. The expression of SOX9, ACAN, and COL2A1 greatly decreased on day 14 in the NGF-treated pellets (Fig. 2F). However, the expression of hypertrophic markers appeared to increase in NGF-treated pellets on day 14. Additionally, Toluidine blue, Safranin O, and Alcian blue staining were further performed to confirm ECM accumulation in differentiated chondrogenic pellets. The morphology of the chondrogenic pellets and ECM accumulation levels did not show any significant difference between the two groups (Fig. 2G). Increased ECM accumulation was confirmed during chondrogenesis in both groups on day 14 of differentiation. The expression of the hyaline cartilage marker Collagen Type II and the hypertrophy marker Collagen Type X was confirmed in the chondrogenic pellets (Fig. 2H). Notably, Collagen Type II levels decreased in the NGF treatment group at both time points. In contrast, Collagen Type X expression was significantly higher in the NGF treatment group (Fig. 2H). Quantification of the Collagen Type II and type X positive area showed increased hypertrophic markers in pellets treated with NGF (Fig. 2I). To confirm, if there is any iPSC clone heterogeneity, we confirmed whether the changes induced by NGF in chondrogenic pellets were consistently observed in two additional hiPSC clones. As a result, chondrogenic markers tended to be decreased and hypertrophic markers increased in the NGF-treated group by IHC staining, and osteogenesis markers were increased used IFA staining (Supplementary Fig. S2). Given the heterogeneity of hiPSCs, hiPSC clones are receiving constant attention and should therefore always be analyzed together in future additional experiments (Supplementary Fig. S2). Collectively, we confirmed that NGF treatment during in vitro chondrogenesis can induce a hypertrophic cartilage-like conditions.
NGF treatment induce pro-osteogenic and angiogenic cytokine expression
To confirm additional changes induced by NGF treatment, inflammatory cytokine expression was analyzed in the cultured media of chondrogenic pellets (Fig. 3A, Supplementary Fig. S3A). In the NGF-treated group, increased expression of angiogenesis related markers, angiogenin, RBP-4, and VCAM-1 was confirmed (Fig. 3B). RBP-4 was significantly increased by more than 5-fold and VCAM-1 (CD106) also increased by 4-fold relative to control. Increased levels of pro-osteogenic cytokines, IGFBP-2, IGFBP-3, IP-10, and HGF were confirmed (Fig. 3C). The levels of IGFBP-2, -3, and IP-10 (CXCL10) increased approximately 2-fold, and HGF was higher expressed more than 3-fold, showing a significant difference. Additionally, MIF and EMMPRIN are known to induce MMPs expression during inflammation. MIF and EMMPRIN (CD147) were found to increase approximately 2-fold in NGF-treated media (Fig. 3D). However, Osteopontin (OPN), which its role is controversial in chondrogenesis, increased noticeably in the normal group and decreased in the NGF-treated group (Fig. 3E). Other markers related to inflammation showed a slight change in expression (Supplementary Fig. S3A). Inflammatory markers such as chitinase 3-like 1, DPPIV (CD26), and MCP-1 (CCL2) showed a slight decrease in the NGF-treated group. Although some of the markers showed no significant difference, Cystatin C, Dkk-1, GDF-15, and Upar were increased, among which IL-17A increased approximately 3-fold (Supplementary Fig. S3B).
Increased fibrotic and osteogenic marker expression in NGF-treated pellets
During OA progression, abnormal proliferative chondrocytes inevitably induce cartilage fibrosis and hypertrophy. We further determined whether osteogenic characteristics were induced in chondrogenic pellets treated with NGF. Interestingly, the gene level of osteogenic markers, including ALP, OCN, and RUNX2, was highly expressed in day 14 chondrogenic pellets treated with NGF (Fig. 4A). Likewise, the gene expression of fibrotic markers such as COL1A1, COL3A1, and α-SMA showed a similar tendency to that of the osteogenic markers (Fig. 4B). Increased protein levels of alkaline phosphatase (ALP) and RUNX2 were confirmed in day 7 NGF-treated pellets. Also the protein levels of Collagen Type III and α-SMA were also increased in day 7 chondrogenic pellets treated with NGF (Fig. 4C, 4D). Calcium deposition was confirmed via Alizarin Red S staining and Von Kossa staining and increased staining intensity was confirmed in both staining methods (Fig.s 4E). Further examination confirmed that NGF treated day 7 pellets had increased levels of Collagen Type I and osteocalcin were expressed in the NGF-treated pellets (Fig. 4F). When a quantitative evaluation was performed, it showed the same results (Fig. 4G). Increased canonical Wnt signaling expression inhibit chondrogenesis in both in vitro and in vivo, reducing Collagen Type II expression and activate RUNX2 expression, which leads to osteogenic stimulation. Additionally, the protein levels of glycogen synthase kinase-3 beta (GSK3β) and p-GSK3β were confirmed in the chondrogenic pellets (Fig. 4H). As a result, there was no significant difference in GSK3β between the two groups, but interestingly, p-GSK3β was highly expressed in the NGF-treated group (Fig. 4I). This may suggest that the Wnt signaling pathway may be involved in the fibrotic and hypertrophical changes induced by NGF treatment. Taken all together, we confirmed that NGF affect the expression of hypertrophic and fibrotic markers in the chondrogenic pellets and possibly through the Wnt signaling pathway.
Discussion
OA is a degenerative disease that can affect any joint. It is characterized by mild inflammation and destruction of cartilage structures by osteophytes in which bone growth appears (20). This results in swelling, stiffness, pain, and loss of mobility. The use of tanezumab has received attention as a promising treatment for reducing pain in OA patients after blocking NGF; however, its use is currently being discontinued owing to various side effects (21). NGF is involved in nerve growth and survival, and reported to be associated with OA pain, however, the mechanisms underlying the effects of NGF on OA are yet to be elucidated (22).
To determine the effect of NGF on cartilage, we generated chondrogenic pellets from hiPSCs and treated them with human NGF protein. There were no significant changes in the size or shape of the pellets during chondrogenic differentiation with or without NGF treatment. Gene expression of chondrogenic markers SOX9, ACAN, and COL2A1, however, was decreased in the NGF-treated group and the expression of hypertrophic markers COL10A1, VEGFA, and MMP13 increased significantly, especially at day 14 of differentiation (Fig. 2F). Additionally, this was once again proven through histological analysis (Fig. 2G, 2H).
Based on the hypothesis that NGF levels are increased in OA joint cartilage, there may be also an imbalance between the pro-inflammatory cytokine components and expression levels (23). We performed an inflammatory cytokine array to determine which factors were altered by NGF treatment. Angiogenin, a key protein involved in angiogenesis, was increased in the NGF treatment group. BRP-4, a specific transporter of retinol (vitamin A) in the blood, showed high expression in the NGF-treated group, and is known to induce the expression of pro-inflammatory cytokines in macrophages involved in angiogenesis (24, 25) It has recently been reported to be present in the plasma and SF of OA patients. In addition, RBP-4 is known to induce VCAM-1 (CD106), a vascular cell adhesion molecule 1, and VCAM-1 (CD106) is expressed in large and small blood vessels after endothelial cells are stimulated by cytokines. These results suggest that increased levels of NGF induce angiogenin, RBP-4, and VCAM-1, which may be involve in angiogenesis in the OA cartilage (Fig. 3B). Next, it has been reported that IGFBP-2, -3 of insulin-like growth factor binding protein (IGFBP) increases in OA cartilage, inducing chondrocyte death and at the same time interfering with chondrocyte proliferation and proteoglycan secretion. This suggests that it may be an important factor in the development of OA (26). Also, IP-10 (CXCL10) involves in cartilage degeneration (27), and HGF is usually produced in subchondral bone osteoblasts and at a higher level in OA (28). The increase of these factors in the NGF treated chondrogenic pellet cultured media, suggest a possible role in chondrocyte hypertrophic and fibrotic changes. In addition, the release of pro-inflammatory cytokines such as MIF, which upregulates the expression of MMP13 (29) and EMMPRIM, also showed high expression (30). These results demonstrated that increased NGF might possibly induce increased levels of MMPs and inflammatory cytokine expression (Fig. 3C, 3D). Nevertheless, unlike previous results, OPN was decreased in the NGF-treated group and increased in the normal group (Fig. 3E). Recent studies have shown that it can be a promising therapeutic agent by promoting ECM synthesis in cartilage and up-regulation of CD44 and HA (31). Among the other cytokines that were altered by NGF treatment, IL-17A is usually expressed in synovial fibroblasts, macrophages, osteocytes, and osteocytes. It has been reported to promote bone formation by promote the production of inflammatory cytokines such as IL-6 and MMPs by interfering the ECM homeostasis that can lead to serious joint damage and pain (32). Taken all together, we suggest that increased NGF expression in the articular cartilage is related to all the hallmark symptoms of OA, which is (1) induced inflammation, (2) ECM degradation, (3) MMP induction, and (4) promoted angiogenesis. All these symptoms can possibly lead to pro-fibrotic and hypertrophic conditions in the articular cartilage.
The expression of GSK3β in the NGF treatment group was confirmed using Western blot (Fig. 4H). On day 7, the expression of phosphorylated GSK3β was increased in the NGF-treated group (Fig. 4I). The GSK3β signaling pathway consists of GSK3α/β and is known to affect a variety of cells (33) and involved in several signaling pathways (34). Especially, the Wnt signaling pathway is involved in diverse roles in cell survival, differentiation, and apoptosis (35). According to previous research, abnormal Wnt signaling pathway promotes osteogenic differentiation and causes fibrosis by inducing markers such as RUNX2 and Dkk-1 (36, 37). Interestingly, Wnt signaling may also be involved in vascular development and homeostasis. It was found that when an abnormal Wnt ligand binds to the receptor in endothelial cells, increased expression of VCAM-1 is upregulated, as confirmed in the cytokine analysis results (Fig. 3A) (38).
OA further induces ECM differentiation due to the imbalance of cartilage ECM degrading enzymes during the disease progression, and ECM degradation induces inflammation from the synovium, while simultaneously inducing the release of cytokines and chemokines that induce chondrocytes and cartilage destruction. This process promotes phenotypic transformation of chondrocytes and dedifferentiation into fibrotic chondrocytes (e.g., fibroblasts) by abnormal proliferation and secretion of ECM proteins such as Collagen Type I. In addition, osteoclasts in the subchondral bone are activated in response to inflammatory mediators, which increase bone resorption and bone formation of osteoclast-derived mediators that regulate sensory innervation and vascular invasion, and abnormal bone remodeling that induces subchondral osteosclerosis, which is a pathological process of OA. Recently, it has been reported that osteogenic conditions created by increased NGF levels in OA result in the formation of calcified cartilage (39, 40). We confirmed that calcification was initiated in the NGF-treated chondrogenic pellets using Alizarin Red S and Von Kossa staining. In addition, the analysis confirmed that osteogenic markers were expressed in NGF-treated pellets. These findings suggest that when NGF expression is increased in articular cartilage, an OA environment develops accompanied by an increase in fibrosis and hypertrophy rather than cartilage differentiation, resulting in osteogenic differentiation and chondrocalcinosis. This suggests that the progression of osteogenesis by NGF in chondrogenic pellets differentiated from hiPSCs may represent an important cause of osteophyte formation. limitations of the study, we only treated the chondrogenic pellets with NGF once on day 3. It was interesting to find that a single NGF treatment during chondrogenesis, can affect and alter the gene expression of pro-chondrogenic, fibrosis, hypertrophic markers, and MMPs on late stages of differentiation. However, continuous treatment for a longer period would be more interesting to confirm the role of NGF on OA progression and osteophyte formation. Previously, our laboratory successfully generated human iPSCs from fibroblast-like synoviocytes of patients with OA and confirmed the expression of OA-related markers by differentiating human iPSCs from patients with early-onset finger OA into chondrogenic pellets. However, we could not confirm whether there was a difference in NGF-induced hypertrophy and fibrosis after differentiation of normal and patient-derived hiPSCs into chondrocyte pellets. Therefore, additional studies are needed in the future, and we plan to further analyze its role in vivo by confirming the effect of NGF in an OA animal model.
Conclusions
Taken together, we found that NGF treatment in chondrogenic pellets decreased expression of pro-chondrogenic markers and increased expression of hypertrophic and fibrotic markers. Furthermore, activation of GSK3β by NGF treatment was confirmed through the results. This study suggests a possible role for NGF in OA progression through inflammation-induced cartilage fibrosis and hypertrophy.
Supplementary Materials
Supplementary data including three figures can be found with this article online at https://doi.org/10.15283/ijsc24097
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