J Oral Pathol Med (2014) 43: 388–394 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
doi: 10.1111/jop.12146
wileyonlinelibrary.com/journal/jop
Fibroblast growth factor 2 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint Yingjie Li, Hengxing Cai, Wei Fang, Qinggong Meng, Jian Li, Mohong Deng, Xing Long Department of Oral and Maxillofacial Surgery, The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
BACKGROUND: Synovial chondromatosis (SC) of temporomandibular joint (TMJ) is a rare proliferative disorder characterized by the formation of cartilaginous or osteocartilaginous nodules in synovium and joint space. Fibroblast growth factor 2 (FGF-2) is frequently applied in chondrogenic differentiation assays. Therefore, we hypothesized that FGF-2 might involved in the pathogenesis of SC. METHODS: SC synovium and loose bodies (LBs) specimens were observed by histological and immunohistochemical methods. Real-time PCR was conducted for comparing genes expressions in SC and normal synovium. SC synoviocytes were stimulated by FGF-2 in the presence or absence of its antagonist long pentraxin-3 (PTX3) for 6 days. Real-time PCR and alkaline phosphatase (ALP) activity were performed to examine the effects exerted by FGF-2 and PTX3. RESULTS: SC synovium, no matter facing the articular cavity or covering LB, was characterized by increased quantity of synoviocytes and blood vessels. FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs, and the wall of blood vessels. Expressions of chondrogenic genes (Sox9 and Wnt-4), osteogenic genes (Foxc2), FGF-2, and VEGF-A mRNA were significantly higher in SC synovium than that of the control group. The stimulation of FGF-2 on SC synoviocytes increased ALP activity and expressions of chondrogenic genes (Sox9, Col2a1, and Aggrecan), osteogenic genes (Foxc2, osteocalcin, and Col1a1), and VEGF-A, but PTX3 inhibited these effects. CONCLUSION: FGF-2 was responsible for the formation of cartilaginous loose bodies and involved in the pathogenesis of SC. J Oral Pathol Med (2014) 43: 388–394
Correspondence: Xing Long, Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, no. 237 Luo Yu Road, Wuhan, Hubei 430079, China. Tel: +86 27 87646312, Fax: +86 27 87873260/647443, E-mail: [email protected] Accepted for publication November 18, 2013
Keywords: fibroblast growth factor 2; Runx2; Sox9; synovial chondromatosis; temporomandibular joint
Introduction Synovial chondromatosis (SC) of temporomandibular joint (TMJ) is a monoarticular and rare proliferative disorder characterized by the formation of cartilaginous or osteocartilaginous nodules in synovium and joint space (1–3), as well as secondary calcification and ossification (4). The main symptoms of TMJ SC include joint pain, limited mouth opening, swelling, clicking, and crepitation (5, 6). It was reported that exposure of mesenchymal stem cell (MSC) to fibroblast growth factor 2 (FGF-2) improves the ability of MSC to undergo chondrogenesis (7), which is evidenced by increased glycosaminoglycan (GAG) accumulation (8–10), toluidine blue staining (11, 12), and chondrogenic genes including Sox9, Col II, and aggrecan (9, 11, 13, 14). The synoviocytes from TMJ are supposed to be MSC because they can be induced to differentiate into osteocyte, chondrocyte, and adipocyte lineages (15, 16). Therefore, we hypothesized that FGF-2 might induce TMJ synoviocytes to undergo chondrogenic differentiation and was responsible for the pathogenesis of SC. In this study, the synovium and loose bodies (LBs) of SC were obtained. The existence of FGF-2 was investigated by real-time PCR and immunohistochemical staining. The effects that FGF-2 exerted were explored.
Materials and methods Samples Written informed consents were obtained from all patients. All experiments were permitted by the Ethics Committee, School & Hospital of Stomatology, Wuhan University, on March 8, 2010. Synovium and loose bodies (LBs) were obtained from three SC patients. Synovium specimens obtained from three patients who were subjected to open reduction for condylar fracture were taken as control. SC synovium and normal synovium specimens were obtained from the area facing the articular cavity.
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Histological and immunostaining observation Both synovium and LBs were fixed in solution with 4% paraformaldehyde. LBs were treated with 10% ethylene diamine tetraacetic acid (EDTA) for 3 months. After a series of traditional treatments for histology observations, the 4-lm-thick paraffin-embedded sections were obtained and treated with hematoxylin and eosin (HE) staining. Immunohistochemical staining was performed by the streptavidin–peroxidase conjugated method, as described previously (17). Antigen retrieval was performed using pepsin (DIG-3009; Maixin, Fuzhou, China) for 30 min at 37°C. Rabbit monoclonal antibodies against human FGF-2 (1:500, ZS-79; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and CD 34 (1:400, ZA-0550) were applied as primary antibodies for 18 h at 4°C. The histological sections were then washed with PBS and stained by the anti-rabbit streptavidin-peroxidase kit (SP-9001) according to the instructions of the manufacturer. Finally, the specimens were reacted with 3, 3′-diaminobenzidine (DAB-0031, Maixin). Counterstaining was performed using hematoxylin for light microscopy observation. Real-time PCR for comparing SC and normal synovium The real-time PCR assay was performed as previously described (18). Briefly, the SC synovium and normal synovium were powdered in liquid nitrogen. Total RNA was extracted according to the manufacturer’s instructions using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reverse-transcribed into cDNA using RevertAid First Strand cDNA Synthesis kit (ReverTra Ace-a, K-1622). To quantify the mRNA expressions of FGF-2, VEGF-A, Sox9, Wnt-4, Foxc2, and Runx2, semi-quantitative PCR of cDNA samples was performed with TOYOBO THUNDERBIRD SYBR qPCR Mix (QPS-201). The sequences of the primers were listed in Table 1. The assay was performed in triplicate using the following protocols: 1-min preincubation at 95°C, followed by 40 PCR cycles at 95°C for 15 s, 58°C for 20 s, and 72°C for 20 s. The expression levels of target genes were obtained using the comparative threshold cycle (DDCt) method and normalized relative to b-actin mRNA level in each sample. Cell culture As previously described (19), the obtained SC synovium and control synovium specimens were washed three times with PBS. The specimens were cut into 1-mm3 sections and cultured with the DMEM (SH30022.01B; HyCloneâ, Logan, UT, USA) containing 15% fetal bovine serum (FBS, SV30087, HyCloneâ), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Trypsin (SH30042, HyCloneâ) was applied to dissociate the synoviocytes. Effect of FGF-2 on chondrogenic and osteogenic differentiation As previously described (20), the 3rd- to 6th-passage SC synoviocytes were cultured in a 6-well plate with DMEM containing 10% FBS. After reaching 70% confluence, culture media were changed every 2 days and maintained for 6 days in DMEM media containing 4% FBS with or without 10 ng/ml recombinant human FGF-2 (rhFGF-2,
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Table 1 Primer sequence of mRNA templates mRNA template FGF-2 Sense Antisense Sox9 Sense Antisense Wnt-4 Sense Antisense Wnt-11 Sense Antisense Foxc2 Sense Antisense Runx2 Sense Antisense VEGF-A Sense Antisense Col2a1 Sense Antisense Aggrecan Sense Antisense Osteocalcin Sense Antisense Col1a1 Sense Antisense b-actin Sense Antisense
Primer sequence 5′- AGAGCGACCCTCACATCAAG-3′ 5′- TCGTTTCAGTGCCACATAC-3′ 5′- TACGACTACACCGACCACCA-3′ 5′- TCAAGGTCGAGTGAGCTGTG-3′ 5′- CTAGCCCCGACTTCTGTGAG-3′ 5′- AAGCAGCACCAGTGGAATTT-3′ 5′-TGACCTCAAGACCCGATACC-3′ 5′-CGTTGGATGTCTTGTTGCAC-3′ 5′- ATCTCAACCACAGCGGGGAC-3′ 5′- AGTTGAACATCTCCCGCACG-3′ 5′- TCAACGATCTGAGATTTGTGGG-3′ 5′- GGGGAGGATTTGTGAAGACGG-3′ 5′- AAGGAGGAGGGCAGAATCAT-3′ 5′- ATCTGCATGGTGATGTTGGA-3′ 5′-CAATCCAGCAAACGTTCCCA-3′ 5′-CAGGCGTAGGAAGGTCATCT-3′ 5′-AGGTCTCACTGCCCAACTAC-3′ 5′-AACACGATGCCTTTCACCAC-3′ 5′-CTCACACTCCTCGCCCTATT-3′ 5′-AACTCGTCACAGTCCGGATT-3′ 5′-GGCAAAGATGGACTCAACGG-3′ 5′-ATCATCAGCCCGGTAGTAGC-3′ 5′- AGCGAGCATCCCCCAAAGTT-3′ 5′- GGGCACGAAGGCTCATCATT-3′
AF-100-18B; PeproTech, Rocky Hill, NJ, USA). Total RNA was extracted from all the cultured synoviocytes as mentioned above. Real-time PCR was performed, as mentioned above, to investigate the expressions of Sox9, Col2a1, Aggrecan, VEGF-A, Runx2, Foxc2, osteocalcin, and Col1a1 mRNA. Alkaline phosphatase activity SC synoviocytes were cultured in the presence or absence of 10 ng/ml FGF-2 for 6 days. As described previously (21), the synoviocytes were washed with PBS for three times and lysed in 1% Triton X-100. The Alkaline phosphatase (ALP) activity was measured according to the manufacturer’s instructions with colorimetric Alkaline Phosphatase Assay kit (A059-2; Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) and normalized to the concentration of total protein via BCA Assay (P0012; Beyotime Institute of Biotechnology, Jiangsu, China). The co-effect of long pentraxin-3 (PTX3) and FGF-2 After reaching 70% confluence, the SC synoviocytes were cultured with DMEM containing 4% FBS and 10 ng/ml rhFGF-2 in the presence or absence of 660 nM human recombinant long PTX3 (PRO-694, Protein Specialists) for 6 days, as described previously (22). Then total RNA was extracted from the cultured cells to analyze the mRNA J Oral Pathol Med
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expressions of Sox9, Foxc2, and VEGF-A by real-time PCR. Meanwhile, the ALP activity was measured as described above. Statistical analysis The data were presented as mean standard deviation. Statistical software (SPSS for Windows, Release 11.0.0; SPSS Inc., Chicago, IL, USA) was used in this study. Twosample Student’s t-test was used to measure significance. Significance levels were set to a = 0.05.
Results Histological and immunohistochemical observation Normal synovium facing the TMJ articular cavity was characterized by 3–4 layers of synoviocytes in the lining layer and a few sporadic fibroblasts in the sublining layer (Fig. 1a). Few blood vessels were observed in control synovium. The SC synovium facing the TMJ articular cavity was characterized by an increase in the number of sublining cells and blood vessels containing blood cells (Fig. 1b). The wall of blood vessels and some of the sublining synoviocytes were positively stained by antibodies against FGF-2 (Fig. 1c) and CD 34 (Fig. 1d). The LBs were observed in articular cavity and synovium of TMJ, showing a smooth texture (Fig. 2a). Most of them were calcified too completely to be cut into pieces by scissors. Through histological observation, two kinds of LBs were discovered. One kind was the single cartilaginous nodule (Fig. 2b–d), and the other kind was numerous small nodules forming a large nodule (Fig. 2e–h). In the case of single nodule, LB was covered by the thick synovium, in which increased synoviocytes and bloods vessels containing red blood cells were found (Fig. 2b and c). In the case of LB formed by numerous small nodules, nodules were separated by a connective tissue containing fibroblast-like cells and blood vessels (Fig. 2e and g). The surface of the LB was covered by a connective tissue containing numerous rounded cells and blood vessels (Fig. 2e and f). FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs (Fig. 2d and h) and in the wall of blood vessels (Fig. 2d). Real-time PCR for comparing SC and normal synovium The expressions of FGF-2 and VEGF-A mRNA in SC synovium were 4.12 and 15.9 times that of the control synovium (Fig. 3a and b), respectively. As for chondrogenic genes, the expressions of Sox9 and Wnt-4 mRNA were 4.26
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and 9.69 times that of the control group (Fig. 3c and d), respectively. As for osteogenic genes, the expression of Foxc2 mRNA was 4.74 times that of the control group (Fig. 3e), but the expression of Runx2 mRNA was slightly increased without statistical significance (Fig. 3f). The effect of FGF-2 stimulation on mRNA expressions Compared with the SC synoviocytes without FGF-2 stimulation, the FGF-2 treatment increased the chondrogenic expressions of Sox9, Col2a1, Aggrecan mRNA over 7.42fold, 2.81-fold, and 1.54-fold (Fig. 4a–c), respectively. Also, the osteogenic expressions of Foxc2, osteocalcin, and Col1a1 mRNA were increased over 5.20-fold, 2.33fold, and 2.89-fold (Fig. 4f, g, and h), respectively. However, the expression of Runx2 mRNA was significantly down-regulated by FGF-2 treatment (Fig. 4e). The angiogenic expression of VEGF-A mRNA was increased over 2.13-fold (Fig. 4d). The restraining effect of long pentraxin-3 (PTX3) Through real-time PCR measurement, the expressions of Sox9, Foxc2, and VEGF-A mRNA were significantly downregulated by the addition of PTX3 (Fig. 5a–c). The quantity of expressions decreased to a level that earned no statistical difference with the control (absence of PTX3 and FGF-2). The ALP activity in the group with the addition of FGF-2 only was 22.93 U/gprot which was 2.64 times that of the control group (Fig. 5d). However, the ALP activity in the group with the addition PTX3 decreased dramatically to 10.36 U/gprot, a data without statistical difference with the control group (Fig. 5d).
Discussion As the SC in TMJ is a rare disease, most studies are case report. Basic researches concerning SC focus mainly on cytokines or protein in synovium and LBs (1, 6, 23–25), using immunohistochemical staining methods. Comprehensive and basic studies on this disease are rarely seen. This study focused on the involvement of FGF-2 in the pathogenesis of SC, because FGF-2 was frequently applied in the chondrogenic differentiation assays (7, 8, 10, 12). Besides, the LBs were found in both synovium and articular cavity (4, 5, 26), made of cartilaginous extracelluar matrix and chondrocytes which are absent in normal synovium (27, 28). Therefore, we hypothesized that FGF-2 might exist in
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Figure 1 Normal synovium facing the articular cavity stained by HE (a). SC synovium facing the articular cavity stained by HE (b), antibodies against FGF-2 (c), and CD34 (d), respectively. SC synovium was characterized by increased quantity of blood vessels and sublining synoviocytes (b). FGF-2 (c) and CD34 (d) were expressed widespread in extracelluar matrix, wall of blood vessels, and most synoviocytes within the synovium. HE: hematoxylin and eosin. SC: synovial chondromatosis. *represents for articular cavity. Scale bars: a–d: 50 lm. J Oral Pathol Med
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Figure 2 Gross view of loose bodies (LBs) (a). LBs showed a smooth texture. The LBs were observed in articular cavity and synovium of TMJ, showing a smooth texture (a). Two forms of LBs were discovered. One kind was the single nodule (b–d), and the other kind was many nodules composing a large nodule (e–h). In the former form, LB was covered by a thick synovium, in which increased synoviocytes and bloods vessels containing red blood cells were observed (b and c). In the latter form, nodules were separated by a connective tissue containing fibroblast-like cells and blood vessels (e and g). The surface of the LB was covered by a connective tissue containing numerous round-shaped cells and blood vessels (e and f). FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs (d and h), and in the wall of blood vessels (d). c is the amplification of the rectangle in b. f and g are the amplification of the left and right rectangle in e, respectively. Scale bars: b and e: 200 lm; c and d, f–h: 50 lm.
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Figure 3 Real-time PCR for determining the mRNA expressions in SC synovium and normal synovium. SC: synovial chondromatosis. *P < 0.05, **P < 0.01 with respect to the control.
the synovium and LBs. The existence of FGF-2 was confirmed by the results of the positive immunostaining observation (Fig. 1c and d, Fig. 2d and h) and the real-time PCR for SC synovium (Fig. 3a), in accordance with the previous study (24).
Then, this study focused on the effects that FGF-2 exerted. In accord with the previous findings (7, 10), FGF-2 could induce SC synoviocytes to undergo chondrogenic differentiation, evidenced by the increased levels of mRNA expressions of Sox9, Col2a1, and aggrecan in this study J Oral Pathol Med
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Figure 4 Real-time PCR for determining the mRNA expressions in SC synoviocytes stimulated by FGF-2 for 6 days. Except for Runx2 mRNA, other gene expressions were up-regulated with statistical significance due to the addition of FGF-2. SC: synovial chondromatosis. *P < 0.05, **P < 0.01 with respect to the control.
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Figure 5 Real-time PCR for determining the mRNA expressions in SC synoviocytes cultured in medium with or without FGF-2 or PTX3 (a, b, and c). ALP activity assessment for SC synoviocytes cultured in medium with or without FGF-2 or PTX3 (d). SC: synovial chondromatosis. **P < 0.01 with respect to the control. J Oral Pathol Med
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(Fig. 4a–c). Sox9 is a potent regulator of the chondrocyte phenotype and modulates the expressions of chondrogenic key genes including Col2a1, Col9a1, Col11a2, and aggrecan (29, 30). Besides, FGF-2 increased expressions of osteogenic differentiation genes, such as Foxc2, osteocalcin, and Col1a1 in this study (Fig. 4f–h). Foxc2 was reported to be responsible for regulating both osteogenesis and angiogenesis (31). The effect of Foxc2 on angiogenesis might account partly for the phenomenon of increased quantity of blood vessels in synovium of SC (Figs 1b and 2b, c, e–g). Moreover, in this study, FGF-2 increased the expression level of VEGF-A mRNA (Fig. 4d), a finding that accorded with the previous observations that FGF-2 was a potent angiogenic growth factor (32). It was reported that FGF-2 exerts the angiogenic activities on ECs through binding with high-affinity FGF receptors (FGFRs) (33) and low-affinity heparan sulfate proteoglycans (HSPGs) (34, 35), forming the HSPG/FGF2/FGFR ternary complex (36). Then, we applied an FGF-2 antagonist named PTX3 which inhibits the establishment of the complex (37). It was shown that PTX3 binds FGF-2 with high specificity and affinity and therefore inhibits FGF2-dependent proliferation of ECs in vitro and angiogenesis in vivo (38). In this study, PTX3 not only exerted anti-angiogenic effects, but also down-regulated the gene expressions of chondrogenic and osteogenic differentiation (Fig. 5). Consequently, from this angle, we proved that in SC pathogenesis, FGF-2 exerted three effects including promoting angiogenesis, chondrogenic, and osteogenic differentiation through the FGFRs. The catabolic or anabolic effects of FGF-2 on chondrocytes remain controversial (39, 40). Some researchers found that, by interacting with FGFR3 (41) or FGFR1 (40), FGF-2 reduces differentiation of chondrocytes, causing the decrease in the production of aggrecan and collagen type II (39, 40, 42) and the increase in the production of matrix metalloproteinase 13 (MMP13) (40). Mitogen-activated protein kinase (MAPK), nuclear factor jB (NF-jB), and Elk-1 signaling pathways are found involved in the process of the effects that FGF-2 exerts (43). However, some researchers showed the opposite phenomenon that FGF-2 reduces dedifferentiation of chondrocytes (44) and regenerates the joint cartilage (45–47). Although the influences of FGF-2 on chondrocytes remain controversial, MSCs, such as adipose derived stem cells (ADSCs) (48) and bone marrow derived mesenchymal stem cells (BMSCs) (8, 49), are confirmed to undergo chondrogenic differentiation with the exposure of FGF-2. FGF-2 improves the morphological appearance of MSCs, the accumulation of extracellular matrix, and transcription of cartilage-related genes, including Sox9, Col II, and aggrecan (7, 10, 12). The synoviocytes of TMJ are proved to resemble the characters of MSCs because they can be induced to differentiate into osteocyte, chondrocyte, and adipocyte lineages (15, 16). In this study, the real-time PCR results (Fig. 4) accorded with the previous findings, suggesting the chondrogenic differentiation of synoviocytes. Therefore, we concluded that the formation of cartilage nodules in synovium is attributed to the effect of FGF-2 to induce chondrogenic differentiation of synoviocytes.
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Conclusion The expression of FGF-2 was observed in the synovium and loose bodies (LBs) of synovial chondromatosis (SC). FGF-2 promoted chondrogenic, osteogenic, and angiogenic differentiation of SC synoviocytes. FGF-2 antagonist long pentraxin-3 inhibited these effects. Therefore, FGF-2 was responsible for the formation of cartilaginous LBs and involved in the pathogenesis of SC.
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Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant Nos. 81071266 and 30901683) and the Independent Research Project for Postgraduates of Wuhan University (Grant No. 201130402020010).
Conflict of interest We declare no conflict of interest on this manuscript.
doi: 10.1111/jop.12146
wileyonlinelibrary.com/journal/jop
Fibroblast growth factor 2 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint Yingjie Li, Hengxing Cai, Wei Fang, Qinggong Meng, Jian Li, Mohong Deng, Xing Long Department of Oral and Maxillofacial Surgery, The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
BACKGROUND: Synovial chondromatosis (SC) of temporomandibular joint (TMJ) is a rare proliferative disorder characterized by the formation of cartilaginous or osteocartilaginous nodules in synovium and joint space. Fibroblast growth factor 2 (FGF-2) is frequently applied in chondrogenic differentiation assays. Therefore, we hypothesized that FGF-2 might involved in the pathogenesis of SC. METHODS: SC synovium and loose bodies (LBs) specimens were observed by histological and immunohistochemical methods. Real-time PCR was conducted for comparing genes expressions in SC and normal synovium. SC synoviocytes were stimulated by FGF-2 in the presence or absence of its antagonist long pentraxin-3 (PTX3) for 6 days. Real-time PCR and alkaline phosphatase (ALP) activity were performed to examine the effects exerted by FGF-2 and PTX3. RESULTS: SC synovium, no matter facing the articular cavity or covering LB, was characterized by increased quantity of synoviocytes and blood vessels. FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs, and the wall of blood vessels. Expressions of chondrogenic genes (Sox9 and Wnt-4), osteogenic genes (Foxc2), FGF-2, and VEGF-A mRNA were significantly higher in SC synovium than that of the control group. The stimulation of FGF-2 on SC synoviocytes increased ALP activity and expressions of chondrogenic genes (Sox9, Col2a1, and Aggrecan), osteogenic genes (Foxc2, osteocalcin, and Col1a1), and VEGF-A, but PTX3 inhibited these effects. CONCLUSION: FGF-2 was responsible for the formation of cartilaginous loose bodies and involved in the pathogenesis of SC. J Oral Pathol Med (2014) 43: 388–394
Correspondence: Xing Long, Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, no. 237 Luo Yu Road, Wuhan, Hubei 430079, China. Tel: +86 27 87646312, Fax: +86 27 87873260/647443, E-mail: [email protected] Accepted for publication November 18, 2013
Keywords: fibroblast growth factor 2; Runx2; Sox9; synovial chondromatosis; temporomandibular joint
Introduction Synovial chondromatosis (SC) of temporomandibular joint (TMJ) is a monoarticular and rare proliferative disorder characterized by the formation of cartilaginous or osteocartilaginous nodules in synovium and joint space (1–3), as well as secondary calcification and ossification (4). The main symptoms of TMJ SC include joint pain, limited mouth opening, swelling, clicking, and crepitation (5, 6). It was reported that exposure of mesenchymal stem cell (MSC) to fibroblast growth factor 2 (FGF-2) improves the ability of MSC to undergo chondrogenesis (7), which is evidenced by increased glycosaminoglycan (GAG) accumulation (8–10), toluidine blue staining (11, 12), and chondrogenic genes including Sox9, Col II, and aggrecan (9, 11, 13, 14). The synoviocytes from TMJ are supposed to be MSC because they can be induced to differentiate into osteocyte, chondrocyte, and adipocyte lineages (15, 16). Therefore, we hypothesized that FGF-2 might induce TMJ synoviocytes to undergo chondrogenic differentiation and was responsible for the pathogenesis of SC. In this study, the synovium and loose bodies (LBs) of SC were obtained. The existence of FGF-2 was investigated by real-time PCR and immunohistochemical staining. The effects that FGF-2 exerted were explored.
Materials and methods Samples Written informed consents were obtained from all patients. All experiments were permitted by the Ethics Committee, School & Hospital of Stomatology, Wuhan University, on March 8, 2010. Synovium and loose bodies (LBs) were obtained from three SC patients. Synovium specimens obtained from three patients who were subjected to open reduction for condylar fracture were taken as control. SC synovium and normal synovium specimens were obtained from the area facing the articular cavity.
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Histological and immunostaining observation Both synovium and LBs were fixed in solution with 4% paraformaldehyde. LBs were treated with 10% ethylene diamine tetraacetic acid (EDTA) for 3 months. After a series of traditional treatments for histology observations, the 4-lm-thick paraffin-embedded sections were obtained and treated with hematoxylin and eosin (HE) staining. Immunohistochemical staining was performed by the streptavidin–peroxidase conjugated method, as described previously (17). Antigen retrieval was performed using pepsin (DIG-3009; Maixin, Fuzhou, China) for 30 min at 37°C. Rabbit monoclonal antibodies against human FGF-2 (1:500, ZS-79; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) and CD 34 (1:400, ZA-0550) were applied as primary antibodies for 18 h at 4°C. The histological sections were then washed with PBS and stained by the anti-rabbit streptavidin-peroxidase kit (SP-9001) according to the instructions of the manufacturer. Finally, the specimens were reacted with 3, 3′-diaminobenzidine (DAB-0031, Maixin). Counterstaining was performed using hematoxylin for light microscopy observation. Real-time PCR for comparing SC and normal synovium The real-time PCR assay was performed as previously described (18). Briefly, the SC synovium and normal synovium were powdered in liquid nitrogen. Total RNA was extracted according to the manufacturer’s instructions using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA was reverse-transcribed into cDNA using RevertAid First Strand cDNA Synthesis kit (ReverTra Ace-a, K-1622). To quantify the mRNA expressions of FGF-2, VEGF-A, Sox9, Wnt-4, Foxc2, and Runx2, semi-quantitative PCR of cDNA samples was performed with TOYOBO THUNDERBIRD SYBR qPCR Mix (QPS-201). The sequences of the primers were listed in Table 1. The assay was performed in triplicate using the following protocols: 1-min preincubation at 95°C, followed by 40 PCR cycles at 95°C for 15 s, 58°C for 20 s, and 72°C for 20 s. The expression levels of target genes were obtained using the comparative threshold cycle (DDCt) method and normalized relative to b-actin mRNA level in each sample. Cell culture As previously described (19), the obtained SC synovium and control synovium specimens were washed three times with PBS. The specimens were cut into 1-mm3 sections and cultured with the DMEM (SH30022.01B; HyCloneâ, Logan, UT, USA) containing 15% fetal bovine serum (FBS, SV30087, HyCloneâ), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Trypsin (SH30042, HyCloneâ) was applied to dissociate the synoviocytes. Effect of FGF-2 on chondrogenic and osteogenic differentiation As previously described (20), the 3rd- to 6th-passage SC synoviocytes were cultured in a 6-well plate with DMEM containing 10% FBS. After reaching 70% confluence, culture media were changed every 2 days and maintained for 6 days in DMEM media containing 4% FBS with or without 10 ng/ml recombinant human FGF-2 (rhFGF-2,
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Table 1 Primer sequence of mRNA templates mRNA template FGF-2 Sense Antisense Sox9 Sense Antisense Wnt-4 Sense Antisense Wnt-11 Sense Antisense Foxc2 Sense Antisense Runx2 Sense Antisense VEGF-A Sense Antisense Col2a1 Sense Antisense Aggrecan Sense Antisense Osteocalcin Sense Antisense Col1a1 Sense Antisense b-actin Sense Antisense
Primer sequence 5′- AGAGCGACCCTCACATCAAG-3′ 5′- TCGTTTCAGTGCCACATAC-3′ 5′- TACGACTACACCGACCACCA-3′ 5′- TCAAGGTCGAGTGAGCTGTG-3′ 5′- CTAGCCCCGACTTCTGTGAG-3′ 5′- AAGCAGCACCAGTGGAATTT-3′ 5′-TGACCTCAAGACCCGATACC-3′ 5′-CGTTGGATGTCTTGTTGCAC-3′ 5′- ATCTCAACCACAGCGGGGAC-3′ 5′- AGTTGAACATCTCCCGCACG-3′ 5′- TCAACGATCTGAGATTTGTGGG-3′ 5′- GGGGAGGATTTGTGAAGACGG-3′ 5′- AAGGAGGAGGGCAGAATCAT-3′ 5′- ATCTGCATGGTGATGTTGGA-3′ 5′-CAATCCAGCAAACGTTCCCA-3′ 5′-CAGGCGTAGGAAGGTCATCT-3′ 5′-AGGTCTCACTGCCCAACTAC-3′ 5′-AACACGATGCCTTTCACCAC-3′ 5′-CTCACACTCCTCGCCCTATT-3′ 5′-AACTCGTCACAGTCCGGATT-3′ 5′-GGCAAAGATGGACTCAACGG-3′ 5′-ATCATCAGCCCGGTAGTAGC-3′ 5′- AGCGAGCATCCCCCAAAGTT-3′ 5′- GGGCACGAAGGCTCATCATT-3′
AF-100-18B; PeproTech, Rocky Hill, NJ, USA). Total RNA was extracted from all the cultured synoviocytes as mentioned above. Real-time PCR was performed, as mentioned above, to investigate the expressions of Sox9, Col2a1, Aggrecan, VEGF-A, Runx2, Foxc2, osteocalcin, and Col1a1 mRNA. Alkaline phosphatase activity SC synoviocytes were cultured in the presence or absence of 10 ng/ml FGF-2 for 6 days. As described previously (21), the synoviocytes were washed with PBS for three times and lysed in 1% Triton X-100. The Alkaline phosphatase (ALP) activity was measured according to the manufacturer’s instructions with colorimetric Alkaline Phosphatase Assay kit (A059-2; Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) and normalized to the concentration of total protein via BCA Assay (P0012; Beyotime Institute of Biotechnology, Jiangsu, China). The co-effect of long pentraxin-3 (PTX3) and FGF-2 After reaching 70% confluence, the SC synoviocytes were cultured with DMEM containing 4% FBS and 10 ng/ml rhFGF-2 in the presence or absence of 660 nM human recombinant long PTX3 (PRO-694, Protein Specialists) for 6 days, as described previously (22). Then total RNA was extracted from the cultured cells to analyze the mRNA J Oral Pathol Med
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expressions of Sox9, Foxc2, and VEGF-A by real-time PCR. Meanwhile, the ALP activity was measured as described above. Statistical analysis The data were presented as mean standard deviation. Statistical software (SPSS for Windows, Release 11.0.0; SPSS Inc., Chicago, IL, USA) was used in this study. Twosample Student’s t-test was used to measure significance. Significance levels were set to a = 0.05.
Results Histological and immunohistochemical observation Normal synovium facing the TMJ articular cavity was characterized by 3–4 layers of synoviocytes in the lining layer and a few sporadic fibroblasts in the sublining layer (Fig. 1a). Few blood vessels were observed in control synovium. The SC synovium facing the TMJ articular cavity was characterized by an increase in the number of sublining cells and blood vessels containing blood cells (Fig. 1b). The wall of blood vessels and some of the sublining synoviocytes were positively stained by antibodies against FGF-2 (Fig. 1c) and CD 34 (Fig. 1d). The LBs were observed in articular cavity and synovium of TMJ, showing a smooth texture (Fig. 2a). Most of them were calcified too completely to be cut into pieces by scissors. Through histological observation, two kinds of LBs were discovered. One kind was the single cartilaginous nodule (Fig. 2b–d), and the other kind was numerous small nodules forming a large nodule (Fig. 2e–h). In the case of single nodule, LB was covered by the thick synovium, in which increased synoviocytes and bloods vessels containing red blood cells were found (Fig. 2b and c). In the case of LB formed by numerous small nodules, nodules were separated by a connective tissue containing fibroblast-like cells and blood vessels (Fig. 2e and g). The surface of the LB was covered by a connective tissue containing numerous rounded cells and blood vessels (Fig. 2e and f). FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs (Fig. 2d and h) and in the wall of blood vessels (Fig. 2d). Real-time PCR for comparing SC and normal synovium The expressions of FGF-2 and VEGF-A mRNA in SC synovium were 4.12 and 15.9 times that of the control synovium (Fig. 3a and b), respectively. As for chondrogenic genes, the expressions of Sox9 and Wnt-4 mRNA were 4.26
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and 9.69 times that of the control group (Fig. 3c and d), respectively. As for osteogenic genes, the expression of Foxc2 mRNA was 4.74 times that of the control group (Fig. 3e), but the expression of Runx2 mRNA was slightly increased without statistical significance (Fig. 3f). The effect of FGF-2 stimulation on mRNA expressions Compared with the SC synoviocytes without FGF-2 stimulation, the FGF-2 treatment increased the chondrogenic expressions of Sox9, Col2a1, Aggrecan mRNA over 7.42fold, 2.81-fold, and 1.54-fold (Fig. 4a–c), respectively. Also, the osteogenic expressions of Foxc2, osteocalcin, and Col1a1 mRNA were increased over 5.20-fold, 2.33fold, and 2.89-fold (Fig. 4f, g, and h), respectively. However, the expression of Runx2 mRNA was significantly down-regulated by FGF-2 treatment (Fig. 4e). The angiogenic expression of VEGF-A mRNA was increased over 2.13-fold (Fig. 4d). The restraining effect of long pentraxin-3 (PTX3) Through real-time PCR measurement, the expressions of Sox9, Foxc2, and VEGF-A mRNA were significantly downregulated by the addition of PTX3 (Fig. 5a–c). The quantity of expressions decreased to a level that earned no statistical difference with the control (absence of PTX3 and FGF-2). The ALP activity in the group with the addition of FGF-2 only was 22.93 U/gprot which was 2.64 times that of the control group (Fig. 5d). However, the ALP activity in the group with the addition PTX3 decreased dramatically to 10.36 U/gprot, a data without statistical difference with the control group (Fig. 5d).
Discussion As the SC in TMJ is a rare disease, most studies are case report. Basic researches concerning SC focus mainly on cytokines or protein in synovium and LBs (1, 6, 23–25), using immunohistochemical staining methods. Comprehensive and basic studies on this disease are rarely seen. This study focused on the involvement of FGF-2 in the pathogenesis of SC, because FGF-2 was frequently applied in the chondrogenic differentiation assays (7, 8, 10, 12). Besides, the LBs were found in both synovium and articular cavity (4, 5, 26), made of cartilaginous extracelluar matrix and chondrocytes which are absent in normal synovium (27, 28). Therefore, we hypothesized that FGF-2 might exist in
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Figure 1 Normal synovium facing the articular cavity stained by HE (a). SC synovium facing the articular cavity stained by HE (b), antibodies against FGF-2 (c), and CD34 (d), respectively. SC synovium was characterized by increased quantity of blood vessels and sublining synoviocytes (b). FGF-2 (c) and CD34 (d) were expressed widespread in extracelluar matrix, wall of blood vessels, and most synoviocytes within the synovium. HE: hematoxylin and eosin. SC: synovial chondromatosis. *represents for articular cavity. Scale bars: a–d: 50 lm. J Oral Pathol Med
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Figure 2 Gross view of loose bodies (LBs) (a). LBs showed a smooth texture. The LBs were observed in articular cavity and synovium of TMJ, showing a smooth texture (a). Two forms of LBs were discovered. One kind was the single nodule (b–d), and the other kind was many nodules composing a large nodule (e–h). In the former form, LB was covered by a thick synovium, in which increased synoviocytes and bloods vessels containing red blood cells were observed (b and c). In the latter form, nodules were separated by a connective tissue containing fibroblast-like cells and blood vessels (e and g). The surface of the LB was covered by a connective tissue containing numerous round-shaped cells and blood vessels (e and f). FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs (d and h), and in the wall of blood vessels (d). c is the amplification of the rectangle in b. f and g are the amplification of the left and right rectangle in e, respectively. Scale bars: b and e: 200 lm; c and d, f–h: 50 lm.
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Figure 3 Real-time PCR for determining the mRNA expressions in SC synovium and normal synovium. SC: synovial chondromatosis. *P < 0.05, **P < 0.01 with respect to the control.
the synovium and LBs. The existence of FGF-2 was confirmed by the results of the positive immunostaining observation (Fig. 1c and d, Fig. 2d and h) and the real-time PCR for SC synovium (Fig. 3a), in accordance with the previous study (24).
Then, this study focused on the effects that FGF-2 exerted. In accord with the previous findings (7, 10), FGF-2 could induce SC synoviocytes to undergo chondrogenic differentiation, evidenced by the increased levels of mRNA expressions of Sox9, Col2a1, and aggrecan in this study J Oral Pathol Med
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Figure 4 Real-time PCR for determining the mRNA expressions in SC synoviocytes stimulated by FGF-2 for 6 days. Except for Runx2 mRNA, other gene expressions were up-regulated with statistical significance due to the addition of FGF-2. SC: synovial chondromatosis. *P < 0.05, **P < 0.01 with respect to the control.
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Figure 5 Real-time PCR for determining the mRNA expressions in SC synoviocytes cultured in medium with or without FGF-2 or PTX3 (a, b, and c). ALP activity assessment for SC synoviocytes cultured in medium with or without FGF-2 or PTX3 (d). SC: synovial chondromatosis. **P < 0.01 with respect to the control. J Oral Pathol Med
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(Fig. 4a–c). Sox9 is a potent regulator of the chondrocyte phenotype and modulates the expressions of chondrogenic key genes including Col2a1, Col9a1, Col11a2, and aggrecan (29, 30). Besides, FGF-2 increased expressions of osteogenic differentiation genes, such as Foxc2, osteocalcin, and Col1a1 in this study (Fig. 4f–h). Foxc2 was reported to be responsible for regulating both osteogenesis and angiogenesis (31). The effect of Foxc2 on angiogenesis might account partly for the phenomenon of increased quantity of blood vessels in synovium of SC (Figs 1b and 2b, c, e–g). Moreover, in this study, FGF-2 increased the expression level of VEGF-A mRNA (Fig. 4d), a finding that accorded with the previous observations that FGF-2 was a potent angiogenic growth factor (32). It was reported that FGF-2 exerts the angiogenic activities on ECs through binding with high-affinity FGF receptors (FGFRs) (33) and low-affinity heparan sulfate proteoglycans (HSPGs) (34, 35), forming the HSPG/FGF2/FGFR ternary complex (36). Then, we applied an FGF-2 antagonist named PTX3 which inhibits the establishment of the complex (37). It was shown that PTX3 binds FGF-2 with high specificity and affinity and therefore inhibits FGF2-dependent proliferation of ECs in vitro and angiogenesis in vivo (38). In this study, PTX3 not only exerted anti-angiogenic effects, but also down-regulated the gene expressions of chondrogenic and osteogenic differentiation (Fig. 5). Consequently, from this angle, we proved that in SC pathogenesis, FGF-2 exerted three effects including promoting angiogenesis, chondrogenic, and osteogenic differentiation through the FGFRs. The catabolic or anabolic effects of FGF-2 on chondrocytes remain controversial (39, 40). Some researchers found that, by interacting with FGFR3 (41) or FGFR1 (40), FGF-2 reduces differentiation of chondrocytes, causing the decrease in the production of aggrecan and collagen type II (39, 40, 42) and the increase in the production of matrix metalloproteinase 13 (MMP13) (40). Mitogen-activated protein kinase (MAPK), nuclear factor jB (NF-jB), and Elk-1 signaling pathways are found involved in the process of the effects that FGF-2 exerts (43). However, some researchers showed the opposite phenomenon that FGF-2 reduces dedifferentiation of chondrocytes (44) and regenerates the joint cartilage (45–47). Although the influences of FGF-2 on chondrocytes remain controversial, MSCs, such as adipose derived stem cells (ADSCs) (48) and bone marrow derived mesenchymal stem cells (BMSCs) (8, 49), are confirmed to undergo chondrogenic differentiation with the exposure of FGF-2. FGF-2 improves the morphological appearance of MSCs, the accumulation of extracellular matrix, and transcription of cartilage-related genes, including Sox9, Col II, and aggrecan (7, 10, 12). The synoviocytes of TMJ are proved to resemble the characters of MSCs because they can be induced to differentiate into osteocyte, chondrocyte, and adipocyte lineages (15, 16). In this study, the real-time PCR results (Fig. 4) accorded with the previous findings, suggesting the chondrogenic differentiation of synoviocytes. Therefore, we concluded that the formation of cartilage nodules in synovium is attributed to the effect of FGF-2 to induce chondrogenic differentiation of synoviocytes.
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Conclusion The expression of FGF-2 was observed in the synovium and loose bodies (LBs) of synovial chondromatosis (SC). FGF-2 promoted chondrogenic, osteogenic, and angiogenic differentiation of SC synoviocytes. FGF-2 antagonist long pentraxin-3 inhibited these effects. Therefore, FGF-2 was responsible for the formation of cartilaginous LBs and involved in the pathogenesis of SC.
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Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant Nos. 81071266 and 30901683) and the Independent Research Project for Postgraduates of Wuhan University (Grant No. 201130402020010).
Conflict of interest We declare no conflict of interest on this manuscript.
