Expression of Calcitonin Gene-Related Peptide Induces Ligament Degeneration Through Endochondral Ossification in Osteoarthritis

Expression of Calcitonin Gene-Related Peptide Induces Ligament Degeneration Through Endochondral Ossification in Osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of Calcitonin Gene-Related Peptide Induces Ligament Degeneration Through Endochondral Ossification in Osteoarthritis Maya Tokumoto, Tomoyuki Nakasa, Akinori Nekomoto, Masakazu Ishikawa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1264720/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Degeneration occurs in various tissues such as cartilage and subchondral bone, and ligament deficiency also plays an important role in the osteoarthritis (OA) progression, but factor which it causes is still unclear. Calcitonin gene-related peptide (CGRP), one of the neuropeptides, its expression increases in OA progression. The purpose of this study is to analyze the mechanism of ligament degeneration and the function of CGRP. Methods To examine the relationship between ligament degeneration and CGRP expression, human posterior cruciate ligament (PCL) from OA patients and SAMP8 mice were histologically analyzed. The effect of CGRP of human ligament cells on the differentiation was also examined. Results In human PCL, CGRP expression increased as degeneration progressed, and decreased in the severe degeneration. CGRP expressed in the chondrocyte like cells with SOX9. In SAMP8 mice, the expression of CGRP increased as OA progression, but it decreased in the severe OA. CGRP up regulated the gene expression of VEGF, SOX9, RUNX2, Col10a1and MMP13. CGRP also promoted chondrogenesis and osteogenesis in the human ligament cells. Conclusions During OA progression, CGRP induces the transdifferentiation from ligament cells to chondrocytes, and promotes endochondral ossification in the ligament. CGRP would be the therapeutic target to prevent the ligament degeneration. Calcitonin gene-related peptide endochondral ossification ligament degeneration osteoarthritis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Osteoarthritis (OA) is a progressive joint disorder that increases its prevalence annually. Although OA is a common disease encountered in daily clinical practice, the mechanisms of OA initiation and progression have not been completely elucidated. In the pathogenesis of OA, various tissues including articular cartilage, subchondral bone, meniscus, joint capsule and ligament exhibit progressive degenerative changes, which is one of the reasons for the difficult OA treatment [ 1 ]. Thus, elucidating the mechanism of degenerative changes in all joint structures is important. However, few studies on the degeneration mechanism of ligaments have been conducted compared with those on the cartilage, bone, and meniscus despite the importance of ligament degeneration in OA pathogenesis. Cushner et al. reported that forty-seven percent of patients with OA have ligament degeneration, which may cause joint laxity [ 2 ]. Levy et al. demonstrated that histological degenerative changes of the posterior cruciate ligament (PCL) were observed before articular cartilage degeneration [ 3 ]. The anterior cruciate ligament and PCL insufficiency frequently cause meniscal and cartilage injuries, which can accelerate the progression of OA due to lateral thrust [ 4 ]. Elucidation of the mechanism of ligament degeneration would enable to prevent OA progression. Increasing chondrogenic potential in the ligament has been reported to be a key factor in the degeneration process [ 5 ]. During the degenerative process of the ligament, chondrogenic cells increase, and ossification finally occurs in the fibroblast-based ligament [ 6 ]. This suggests that some factors may induce chondrogenesis and osteogenesis in the ligament during OA pathogenesis. Neuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, are well known to play an important role in OA-related pain in the joint [ 7 ]. Neuropeptides have various functions, including bone metabolism and angiogenesis, to maintain homeostasis [ 8 ]. In the pathogenesis of OA, excessive expression of neuropeptides in the subchondral bone causes sclerotic changes in the subchondral bone, and subsequently, cartilage degeneration progresses [ 9 , 10 ]. CGRP is a neuropeptide belonging to the calcitonin superfamily; it consists of 37 amino acids and is released from sensory nerves [ 11 ]. CGRP has various functions, including blood vessel dilation, accelerating inflammatory pain, and promotion of bone remodeling by differentiation of bone marrow mesenchymal cells into osteoblasts, stimulation of osteoblast proliferation, and acceleration of bone metabolism by inhibiting osteoclast differentiation [ 12 ]. We focused on CGRP in ligament degeneration because CGRP induces osteogenesis, and the sensory nerve extends into the affected joints during OA progression [ 13 ]. We hypothesized that the elongated CGRP-positive sensory nerves into the joint might cause increased chondrogenic potential in the ligament, subsequently progressing ligament degeneration. The aim of this study was to analyze the expression pattern and function of CGRP in the ligament cells in OA pathogenesis. Materials And Methods Between June 2016 and October 2018, 30 patients with advanced knee OA were treated with total knee arthroplasty (TKA). They consisted of 11 men and 19 women, with a mean age of 73.5 years. Knee joints were classified as Kellgren-Lawrence grades 3 and 4. Patients with a history of ligament injury, malunited fractures, intra-articular steroid injection in the previous 6 months, joint infection, or previous knee surgery were excluded. During surgery, PCL was harvested for histological analysis according to a previous report [ 14 ]. This study was approved by the institutional review board and ethics committee of our hospital and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all patients. Histological analysis of human ligament Harvested PCL was fixed in 4% paraformaldehyde and embedded in paraffin. Four-micrometer-thick sections were prepared for histological analysis. Sections were stained with hematoxylin & eosin (HE) and safranin- O/fast green, and histologically graded using a scoring system according to a previous report [ 15 ]. The following categories were examined and scored for each ligament: (1) inflammation in the ligament substance, (2) mucoid degeneration, (3) chondroid metaplasia, (4) cystic changes, and (5) orientation of collagen fibers. Five fields of the PCL were randomly selected and each slide was evaluated at 200X magnification. The histological changes were scored and graded with reference to previous report as follows [ 15 ]: 0, no changes; 0.5, minimal changes; 1, mild changes; 2, moderate changes; and 3, severe changes. The highest summed score of ligament degeneration (total score) was 15 if all five histological categories were scored as severe. Furthermore, the total score was classified into three groups: mild (0–5), moderate (6–10), severe (11–15). Animals All procedures were performed in accordance with the Guidelines for Animal Experimentation at our university, and with the approval of the Committee of Research Facilities for Laboratory Animal Sciences, Graduate School of Biomedical Sciences, Hiroshima University. Senescence-accelerated mouse-prone 8 (SAMP8) mice which develop spontaneous joint OA signs that resemble human disease at the ages of 4, 18, and 42 weeks (n = 9 at each time point) were used for the evaluation of the spontaneous OA model. Harvested knee joints fixed in 4% paraformaldehyde were decalcified for 2 weeks in 20% EDTA and, embedded in paraffin. Four-micrometer-thick sagittal sections, where the whole length of the PCL in the knee joint was observed, were prepared for histological analysis. Sections were stained with HE and safranin-O, and graded histologically using OARSI and ligament scores [ 15 , 16 ]. Immunohistochemical analysis For immunohistochemical analysis, each section was immunostained using anti-CGRP antibody (1:500 dilution; Abcam, Cambridge, MA), anti-SOX 9 (1:800 dilution; Abcam, Cambridge, MA), anti-MMP13 (1:20 dilution; Neo Markers, Fremont, CA), and anti-CRLR/CGRP1 polyclonal antibody (1:100 dilution; Bioss, Boston, MA, USA). For double immunofluorescence staining of CGRP and SOX9, the anti-CGRP antibody was labeled with FITC using Dojindo Ab-10 Rapid Fluorescein Labeling Kit (Dojindo Laboratories, Kumamoto, Japan), and anti-SOX9 antibody was labeled using the Dojindo Ab-10 Rapid HiLyte Fluor 555 Labeling Kit (Dojindo Laboratories, Kumamoto, Japan) according to the method described in a previous report [ 17 ]. DAPI (Dojindo Laboratories, Kumamoto, Japan) solution was used for nuclear staining. For immunohistochemical signals, five fields in the ligament were randomly selected among the areas where ligamentous structures were found; the total number of cells was counted, and the number of SOX9, MMP13, and CGRP positive cells was measured at 200X magnification using ImageJ software (National Institution of Health) according to the method of a previous report [ 18 , 19 ]. Cell culture Human ligament cells were obtained from the anterior cruciate ligament resected with TKA, because in TKA the anterior cruciate ligament is removed and therefore more tissue can be harvested. Ligament tissues were placed in 10-cm diameter Petri dishes under sterile conditions and washed five times with phosphate buffered saline (PBS; WAKO, Osaka, Japan) to remove red blood cells after removing synovial tissues, adipose tissue, and small blood vessels. The tissue was minced into 1–2 mm pieces, and 0.25% type 1 collagenase was added. Tissues were shaken in a water bath at 37°C for 60 min, and then an equal volume of high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich Corp., St. Louis, Missouri) and antibiotics (at a final concentration of 100 units/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B; Nacalai Tesque, Kyoto, Japan) was added to stop degradation. The tissue was then filtered through a 200-mesh nylon filter and centrifuged at 400 rpm for 10 min. The supernatant was discarded, and the human ligament cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics. The harvested cells were homogeneous and were further identified as ligament cells by real time polymerase chain reaction (PCR) analysis as they expressed SCX (Hs 03054634_g1). When the cell coverage reached 80%-90%, the cells were passaged. The cells from passages 2 and 3 were seeded onto 24-well plates (BD Falcon, Franklin Lakes, NJ), and incubated with or without 10 nM and 100 nM CGRP receptor agonist (Peptide Research Institute, Inc., Osaka, Japan) or 10 nM and100 nM CGRP receptor antagonist (BIBN4096, TOCRIS, Bioscience, Bristol, UK) or the same amount of PBS as a control group added to each well twice a week in a humidified 5% CO2/95% air atmosphere at 37°C (n = 8 each group). After 24 h, 48 h, and 21 days, RNA was extracted for PCR analysis. Osteogenic differentiation medium (StemPro Osteogenic Differentiation Kit, Life Technologies, Grand Island, NY) and antibiotics with a final concentration of 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Nacalai Tesque) were used. Calcified medium containing 100 nM of CGRP receptor agonist, or the same amount of PBS were added and cultured for 3 weeks. Osteogenesis capacity was evaluated by PCR and staining with 0.5% alizarin red solution (Nacalai Tesque). To induce adipogenesis, the cells were cultured in adipogenic differentiation medium (StemPro adipogenesis differentiation kit, Life Technologies) and antibiotics, to which 100 nM of CGRP receptor agonist or the same amount of PBS was added. After 3 weeks, adipogenic capacity was assessed using PCR and staining with 0.3% oil red-O (SIGMA O-0625; Sigma-Aldrich Corp.). To induce chondrogenesis, 5 × 10 5 cells were placed in 15-ml polypropylene tubes (BD Falcon) and pelleted by centrifugation at 500 × g for 5 min. The pellets were cultured in chondrogenesis medium (StemPro chondrogenesis differentiation kit, Life Technologies) and antibiotics, to which 100 nM of CGRP receptor agonist or the same amount of PBS was added for 3 weeks. Chondrogenic capacity was evaluated using PCR and histological analysis. For histological analysis, the pellets were embedded in paraffin, cut into 6-ml sections, and stained with safranin-O/fast green. Real time PCR RNA was isolated using TRIzol (Life Technologies) for real-time PCR analysis, and proteins were extracted for analysis of alkaline phosphatase activity. Complementary DNA was synthesized using 1 µg of total RNA using the Superscript VLIO kit (Invitrogen) according to the manufacturer's protocol: MMP13 (Hs 00233992_m1), RUNX2 (Hs 00231692_m1), VEGFA (Hs 0090055_m1), SOX9 (Hs 00165814_m1), COL2A1(Hs 01064869_m1), COL10A1(Hs 00166657_m1), PPARγ (Hs 01115513_m1) and GAPDH(Hs99999905_m1) using TaqMan gene expression assays probes (Life Technologies), and real-time PCR assays were performed. The expression level of each gene was evaluated relative to the expression level of GAPDH. The ΔΔCt method was used to analyze the real-time PCR data. Statistical analysis All results in this study are expressed as mean ± standard deviation (SD). Comparisons among the three or four groups were performed using the Tukey–Kramer post hoc test, and the Mann–Whitney U test was used to determine the differences between the two groups. Statistical significance was set at P < 0.05. Results Expression pattern of the CGRP in human PCL Of the 30 knees with human ligament degeneration, 7 were in the mild group, 12 were in the moderate group, and 11 were in the severe group. The mean histopathologic score of degeneration of the mild group was 3.3 ± 0.3 points (range, 2-4.5), and the ligament was composed of parallel fiber arrangement, and the cells were mostly fibroblasts. In the moderate group, the mean histopathologic score of degeneration was 6.7 ± 1.5 points (range, 5–9), and the number of chondrocyte-like cells with round nuclei increased, and the surrounding tissue was stained with safranin O. In addition, the ligament fibers become wavy, and the parallel fiber arrangement was disrupted. In advanced degeneration, the mean histopathologic score of degeneration was 12.9 ± 1.5 points (range, 10–14), and the number of chondrocyte-like cells increased and the area stained with safranin O stain also increased. In addition, ossification areas were observed in this study. The number of SOX9-positive cells was significantly higher in the moderate group than in the mild and advanced groups (p < 0.01). The number of MMP13-positive cells was significantly higher in the moderate group than in the advanced group (p < 0.01). The number of CGRP-positive cells was lower in the mild group, significantly higher in the moderate group than in the other groups (p < 0.01), and decreased again in the severe group (Fig. 1 a, b). Immunofluorescent analyses revealed that CGRP and SOX9 were co-expressed in the chondrocyte-like cells in ligament (Fig. 1 c). Expression pattern of the CGRP in the PCL of OA model mouse To investigate longitudinal ligament degeneration, PCL in spontaneous OA mice (SAMP8) was histologically analyzed. As the number of weeks increased, the OARSI and ligament scores significantly increased with articular cartilage degeneration (Fig. 2 a, b). HE and safranin-O/fast green staining revealed parallel fiber arrangement and spindle-shaped nuclei. Ligaments were rarely stained with safranin- O at 4 weeks. At 18 weeks, the increased area in the ligament stained with safranin- O stain and the parallel fiber arrangement decreased, and chondrocyte-like cells were observed. At 42 weeks, ligaments stained almost exclusively with safranin- O stain and the parallel fiber structure disappeared, and the number of chondrocyte-like cells increased; its degeneration was similar to that of human ligament degeneration. The number of CGRP-positive cells was significantly higher at 18 weeks than at 4 weeks and 42 weeks (Fig. 2 a, c). Immunohistochemical analysis of the CGRP receptor showed few positive cells at 4 weeks. However, the positive cells in both fibroblasts and chondrocyte-like cells increased at 18 weeks, and they decreased and were expressed only in chondrocyte-like cells at 42 weeks. Immunohistochemical analysis of CGRP and SOX9 showed that the co-expression of CGRP and SOX9 in the chondrocyte-like cells was highest at 18 weeks, and then decreased at 42 weeks (Fig. 3 ). Functional analysis of CGRP on ligament in vitro To investigate the chondrogenic effects of CGRP on ligament cells, the expression of SOX9, RUNX2, COL10A1, VEGF, and MMP13 was evaluated using real-time PCR. SOX9 expression increased in a CGRP dose-dependent manner at all stages and was significantly suppressed by BIBN4096 treatment. Runx2 expression increased in a CGRP dose-dependent manner at all stages and was suppressed by BIBN4096 treatment. Col10a1 expression significantly increased in a CGRP dose-dependent manner at 3 weeks post-treatment and was significantly suppressed by BIBN4096 treatment (Fig. 4 ). VEGF expression was significantly upregulated at 24 h post-treatment in the CGRP 100 nM group and at 3 weeks post-treatment in the CGRP 10 nM group. MMP13 expression increased in a dose-dependent manner at 3 weeks and was significantly suppressed by BIBN4096 treatment (Fig. 5 ). Real-time PCR analyses revealed that CGRP has an effect on ligament cells to promote endochondral ossification, which is inhibited by BIBN4096. The effect of CGRP on chondrogenesis, osteogenesis, and adipogenesis of ligament-derived cells was examined. In chondrogenesis, the diameter of the pellet with CGRP treatment was larger than that without CGRP treatment (Fig. 6 a). In addition, the expression of SOX9 and Col2a1 was significantly higher in the pellet with CGRP treatment than in the pellet without CGRP treatment (Fig. 6 a). In osteogenesis, alizarin red staining showed more staining in the ligament cells with CGRP treatment (Fig. 6 b). In addition, Runx2 expression in the ligament cells with CGRP treatment was significantly higher than that without CGRP treatment (Fig. 6 b). In adipogenic differentiation, oil red staining showed no staining of the ligament after CGRP treatment (Fig. 6 c). The expression of PPARγ was significantly lower in the ligament cells treated with CGRP than in those without CGRP treatment (Fig. 6 c). Discussion This study showed that CGRP is expressed in chondrocyte-like cells in the ligament as degeneration develops, and CGRP induces chondrogenesis and osteogenesis in the ligament during OA progression. Since ligament dysfunction due to degeneration is an important factor in the pathogenesis of OA, elucidating the factors inducing ligament degeneration will lead to the development of new therapeutic strategies. Neuropeptides, including CGRP, have been recognized as therapeutic targets for various diseases, and receptor antagonists or antibodies have been developed as drugs targeting neuropeptides [ 20 ]. In addition, CGRP is a potential target to prevent OA progression by administering a CGRP receptor antagonist through inhibition of subchondral bone sclerosis [ 9 ]. Therefore, the evidence that neuropeptides play a crucial role in OA pathogenesis could lead to a novel therapeutic strategy for OA. Ligament degeneration is histologically characterized by disorganization of collagen fiber arrangement and chondroid metaplasia [ 15 , 21 , 22 ]. Degenerating ligaments exhibit fibrocartilaginous regions with collagen type Ⅱ and Ⅲ and an increasing distribution of chondrocytes in addition to disorganization of fiber arrangements [ 2 , 23 , 24 ]. Experimental tendon degeneration showed that approximately 5% of the degenerating area has calcific foci with chondrocytes expressing SOX9 [ 25 ]. Kumagai et al. demonstrated that Sclelaxis- positive cells decreased and SOX9-positive cells increased as ligament degeneration progressed [ 5 ]. Thus, chondrogenic differentiation plays a crucial role in ligament degeneration. However, the factors that induce or promote chondrogenic differentiation of the ligament during OA progression have not been elucidated. We focused on CGRP regarding chondrogenic differentiation in ligament degeneration. CGRP has various functions, including pain perception, osteogenesis, and angiogenesis [ 26 ]. The sensory nerve around the joint has been reported to elongate into the joint during OA progression, and the neuropeptides released from the sensory nerve then causes pain, inflammation, and subchondral bone sclerosis [ 27 ]. CGRP is distributed in the synovium, subchondral bone, cartilage, and meniscus in the joints with OA [ 28 ]. In particular, CGRP expression in the subchondral bone increases in destabilization of the medial meniscus (DMM) mice and induces subchondral bone sclerosis [ 9 ]. CGRP may also induces ossification of the ligament during the degeneration process. Moreover, CGRP has been reported to be involved in heterotopic ossification [ 29 ], and administration of CGRP to the tibialis anterior muscle of mice promotes heterotopic ossification [ 30 ]. We examined the effects of CGRP on the differentiation of ligament cells. Furumatsu et al. demonstrated that ligament cells exhibit chondrogenic properties with SOX9 expression in chondrogenic induction medium [ 31 ]. Takimoto et al. showed the direct conversion of tenocytes to chondrocytes by SOX9 overexpression [ 32 ]. In our study, CGRP upregulated SOX9 expression in pellet cultures during chondrogenesis, and the size of the pellet was enlarged. Furthermore, CGRP promoted the osteogenesis in the ligament cells while it inhibiting adipogenesis. In fact, with the addition of CGRP to the ligament cells, the expression of SOX9 and Runx2 was increased up to 3 weeks, suggesting that CGRP induces chondrogenesis and osteogenesis. At 3 weeks of culture, the expression of Col10a1 and MMP13, which are important factors in endochondral ossification, increased in a dose-dependent manner, indicating that CGRP induced endochondral ossification [ 33 ]. VEGF, important factor in endochondral ossification, is also upregulated by CGRP. Hypervascularization in various tissues in the joint, including the ligament, has been reported in the OA joint [ 35 ]. Our study revealed that CGRP might play a role in inducing endochondral ossification and angiogenesis in the ligament through VEGF expression. In our study, CGRP expression increased as the degeneration of the ligament progressed, but severe degeneration ligaments did not exhibit a decrease in CGRP expression accompanied by MMP13 expression. It is suspected that CGRP expression by the sensory nerve that elongates from the surrounding joint induces transdifferentiation of the ligament cells to chondrocyte-like cells to promote endochondral ossification [ 13 ]. In the severe degeneration ligament, fibroblastic cells were sparsely present, and CGRP was no longer required. CGRP induces VEGF at 24 h after CGRP addition to the ligament cells. CGRP has been reported to induce angiogenesis [ 26 ]. CGRP may also induce angiogenesis in the early phase of OA, which may lead to further increased expression of CGRP [ 35 ]. This study had several limitations. First, the natural course of ligament degeneration during OA progression could not be observed in human samples. This is an unsolvable problem for histological evaluation, but we compensated for it by stratifying the degree of ligament degeneration. Moreover, SAMP8, which has been established as a spontaneous OA model, was used to evaluate the time course of ligament degeneration in OA progression [ 34 ]. Second, the factors that induce the expression of CGRP in the ligament have not been elucidated, although this study explored important role of CGRP in ligament degeneration. However, the factors that induce OA have not been clarified, despite many studies on OA pathogenesis. Finally, the loss of function of CGRP was not examined in vivo in this study. Knockdown of the CGRP prevents ligament degeneration. Nakasa et al. reported that DMM mice which were administered the CGRP receptor antagonist reduced the OARSI score until 8 weeks, which suggested that inhibition of the CGRP expression may lead to prevent ligament degeneration [ 9 ]. However, DMM mice are recognized as a traumatic OA model, and whether DMM affects ligament degeneration is unclear. Therefore, it is desirable to examine CGRP knockdown in a spontaneous OA mouse model. Conclusion The present study shows that with regard to ligament degeneration, angiogenesis occurs in the early stages, followed by chondrogenesis and ossification via endochondral ossification, and CGRP is deeply involved in this process. Furthermore, CGRP receptor antagonists were shown to have the potential to effectively attenuate OA progression. Abbreviations OA Osteoarthritis CGRP Calcitonin gene-related peptide PCL posterior cruciate ligament TKA Total knee arthroplasty HE Hematoxylin & eosin SAMP8 Senescence-accelerated mouse-prone 8 PBS Phosphate buffered saline DMEM Dulbecco’s modified Eagle’s medium FBS Fetal bovine serum PCR Polymerase chain reaction SD Standard deviation DMM Destabilization of the medial meniscus Declarations Ethics approval and consent to participate This study was approved by the institutional review board and ethics committee of our hospital and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all patients. The study was approved by the Ethics Committee of Hiroshima University Hospital, and written consent was obtained from all patients. All procedures were performed in accordance with the Guidelines for Animal Experimentation at our university, and with the approval of the Committee of Research Facilities for Laboratory Animal Sciences, Graduate School of Biomedical Sciences, Hiroshima University. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was partly supported by MEXT KAKENHI Grant-in-Aid for Scientific Research (C); Grant Number 18K09066 (T.N.). Research grant from the Nakatomi Foundation (T.N.). Authors’ contributions Dr. MT and TN designed this study, analyzed and interpreted the data, and drafted the manuscript. TN provided the study materials and funding. AN, MI, YI and SM obtained the data, analyzed and interpreted the data, drafting the article. TN and NA contributed to the critical revision of the article. All authors have read and approved the final manuscript. Acknowledgements The authors would like to thank Editage (www.editage.com) for English language editing. References Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: A disease of the joint as an organ. Arthritis Rheum. 2012;64: 1697–707. Cushner FD, La Rosa DF, Vigorita VJ, Scuderi GR, Scott WN, Insall NS. A quantitative histologic comparison: ACL degeneration in the osteoarthritic knee. J Arthroplasty. 2003;18: 687–92. Levy YD, Hasegawa A, Patil S, Koziol JA, Lotz MK, D’Lima DD. Histopathological changes in the human posterior cruciate ligament during aging and osteoarthritis: correlations with anterior cruciate ligament and cartilage changes. Ann Rheum Dis. 2013; 72: 271–7. Yoshimura I, Naito M, Zhang J. Lateral thrust of anterior cruciate ligament-insufficient knees and posterior cruciate ligament-insufficient knees. International Orthop. 2002; 26: 303–305. Kumagai K, Sakai K, Kusayama Y, Akamatsu Y, Sakamaki K, Morita T, et al. The extent of degeneration of cruciate ligament is associated with chondrogenic differentiation in patients with osteoarthritis of the knee. Osteoarthritis Cartilage. 2012;20: 1258–67. Hasegawa A, Nakahara H, Kinoshita M, Asahara H, Koziol J, Lotz MK et al. Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis. Arthritis Res Therapy. 2013;15: R29. Saxler G, Loer F, Skumavc M, Pfortner J, Hanesch U. Localization of SP- and CGRP-immunopositive nerve fibers in the hip joint of patients with painful osteoarthritis and of patients with painless failed total hip arthroplasties. Eur J Pain. 2007; 11: 67–74. Irie K, Hara-Irie F, Ozawa H, Yajima T. Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling. Microsc Res Tech. 2002; 15;58: 85–90. Nakasa T, Ishikawa M, Takada T, Miyaki S, Mitsuo Ochi. Attenuation of cartilage degeneration by calcitonin gene-related paptide receptor antagonist via inhibition of subchondral bone sclerosis in osteoarthritis mice. J Orthop Res. 2016; 34: 1177–84. Kanemitsu M, Nakasa T, Shirakawa Y, Ishikawa M, Miyaki S, Adachi N. Role of vasoactive intestinal peptide in the progression of osteoarthritis through bone sclerosis and angiogenesis in subchondral bone. J Orthop Sci. 2020; 25: 897–906. Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, et al. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature. 1983; 14–20;304(5922): 129–35. Brain SD, Williams TJ, Tippins JR, Morris HR, Maclntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature. 1985; 313: 54–6. Grässel S, Muschter D. Peripheral nerve fibers and their neurotransmitters in osteoarthritis pathology. Int J Mol Sci. 2017; 18: 931. Hayashi S, Nakasa T, Matsuoka Y, Akiyama Y, Ishikawa M, Nakamae A, et al. Evaluation of the degenerative pattern of PCL in osteoarthritis patients using UTE-T2 mapping. Asia Pac J Sports Med Arthrosc Rehabil Technol. 2021; 24: 35–40. Mullaji AB, Marawar SV, Simha M, Jindal G. Cruciate Ligaments in Arthritic Knees. A Histologic Study With Radiologic Correlation. J Arthroplasty. 2008; 23: 567–72. Glasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010; 18: S17-23. Miyazaki K, Oyanagi J, Hoshino D, Togo S, Kumagai H, Miyagi Y. Cancer cell migration on elongate protrusions of fibroblasts in collagen matrix. Sci Rep. 2019; 9: 292. Zemmyo M, Meharra EJ, Kühn K, Creighton-Achermann L, Lotz M. Accelerated, Aging-Dependent Development of Osteoarthritis in α1 Integrin-Deficient Mice. Arthritis Rheum. 2003; 48: 2873–80. Yan D, Liu X, Guo SW. Neuropeptides Substance P and Calcitonin Gene Related Peptide Accelerate the Development and Fibrogenesis of Endometriosis. Sci Rep. 2019; 9: 2698. Tepper SJ. History and Review of anti-Calcitonin Gene-Related Peptide (CGRP) Therapies: From Translational Research to Treatment. Headache. 2018; 58: 238–275. Hasegawa A, Otsuki S, Pauli C, Miyaki S, Patil S, Steklov N, et al. Anterior cruciate ligament changes in human joint in aging and osteoarthritis. Arthritis Rheum. 2012; 64: 696–704. Tallon C, Maffulli N, Ewen SWB. Ruptured Achilles tendons are significantly more degenerated than tendinopathic tendons. Med Sci Sports Exerc. 2001; 33: 1983–90. Riley GP, Harrall RL, Constant CR, Chard MD, Cawston T, Hazleman B. Tendon degeneration and chronic shoulder pain: changes in the collagen composition of the human rotator cuff tendons in rotator cuff tendinitis. Ann Rheum Dis. 1994; 53: 359–66. Samiric T, Parkinson J, Ilic MZ, Cook J, Feller JA, Handley CJ. Changes in the composition of the extracellular matrix in patellar tendinopathy. Matrix Biol. 2009; 28: 230–6. Lui PP, Fu SC, Chan LS, Hung LK, Chan KM. Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration. J Histochem Cytochem. 2009; 57: 91–100. Zheng S, Li W, Xu M, Bai X, Han J, Shyy JY, Wang X. Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase. Am J Physiol Cell Physiol. 2010; 299: 1485–92. Grässel, S. The role of peripheral nerve fibers and their neurotransmitters in cartilage and bone physiology and pathophysiology. Arthritis Res Ther. 2014; 16: 485. Suri S, Gill SE, De Camin SM, Wilson D, McWiliams DF, Walsh DA. Neurovascular invasion at the osteochondral junction and in osteophytes in osteoarthritis. Ann Rheum Dis. 2007; 66: 1423–8. Tuzmen C, Verdelis K, Weiss L, Campbell P. Crosstalk between substance P and calcitonin gene-related peptide during heterotopic ossification in murine Achilles tendon. J Orthop Res. 2018; 36: 1444–1455. Sang X, Wang Z, Shi P, Li Y, Cheng L. CGRP accelerates the pathogenesis of neurological heterotopic ossification following spinal cord injury. Artif Cells Nanomed Biotechnol. 2019; 47: 2569–2574. Furumatsu T, Hachioji M, Saiga K, Takata N, YokoyamaY. Anterior cruciate ligament-derived cells have high chondrogenic potential. Biochem Biophys Res Commun. 2010; 391: 1142–7. Takimoto A, Oro M, Hiraki Y, Shukunami C. Direct conversion of tenocytes into chondrocytes by Sox9. Exp Cell Res. 2012; 318: 1492–507. Kim JS, Ali MH, Wydra F, Li X, Hamilton JL, An HS, et al. Characterization of degenerative human facet joints and facet joint capsular tissues. Osteoarthritis Cartilage. 2015; 23: 2242–2251. Korchi AM, Cengarle-Samak A, Okuno Y, Martel-Pelletier J, Pelletier JP, Boesen M, et al. Inflammation and Hypervascularization in a Large Animal Model of Knee Osteoarthritis: Imaging with Pathohistologic Correlation. J Vasc Interv Radiol. 2019; 30: 1116–1127. Ashraf S, Wibberley H, Mapp PI, Hill R, Wilson D, Walsh DA. Increased vascular penetration and nerve growth in the meniscus: A potential source of pain in osteoarthritis. Ann Rheum Dis. 2011; 70: 523–9. Nagira K, Ikuta Y, Shinohara M, Sanada Y, Omoto T, Kanaya H, et al. Histological scoring system for subchondral bone changes in murine models of joint aging and osteoarthritis. Sci Rep. 2020; 10: 1–14. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1264720","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":84567249,"identity":"6b11f840-c243-4e36-a4c8-3b22c79ee25a","order_by":0,"name":"Maya Tokumoto","email":"","orcid":"","institution":"Hiroshima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Tokumoto","suffix":""},{"id":84567250,"identity":"d2620edd-7328-4b9f-937c-5f02acdb1289","order_by":1,"name":"Tomoyuki Nakasa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYHACZhDBww8iEwqIUM8D1SIn2QDSYkCCFmODAyCKGC320s2PDX78sUvcfH514ocHBgzy/GIHCNgic8w4sbctOXHbjbebJYAOM5w5O4GAFokE4wO8DcxALWc3gLQkGNwmqCX988E/f+oTN884u/kHkVpyjJN52A4bG/D3biPSlhs5xcaybcflJG7wbrNIMJAg7Bf2GembJd/8qebh7z+7+eaPCht5fmkCWhBAAqxSgljlIMB/gBTVo2AUjIJRMJIAADdyQrAHFeT9AAAAAElFTkSuQmCC","orcid":"","institution":"Hiroshima University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tomoyuki","middleName":"","lastName":"Nakasa","suffix":""},{"id":84567251,"identity":"c77ce14e-2b0f-458d-92e2-0c8bee8c2d2b","order_by":2,"name":"Akinori Nekomoto","email":"","orcid":"","institution":"Hiroshima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akinori","middleName":"","lastName":"Nekomoto","suffix":""},{"id":84567252,"identity":"f5cd8cbf-e7ad-4dad-a3be-59db3727f5ce","order_by":3,"name":"Masakazu Ishikawa","email":"","orcid":"","institution":"Hiroshima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Masakazu","middleName":"","lastName":"Ishikawa","suffix":""},{"id":84567253,"identity":"6a06e521-60a6-4326-8276-2a93b03f3ebb","order_by":4,"name":"Yasunari Ikuta","email":"","orcid":"","institution":"Hiroshima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yasunari","middleName":"","lastName":"Ikuta","suffix":""},{"id":84567254,"identity":"a9c4c1ea-434e-4175-9135-f55a23e0c3b5","order_by":5,"name":"Shigeru Miyaki","email":"","orcid":"","institution":"Hiroshima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shigeru","middleName":"","lastName":"Miyaki","suffix":""},{"id":84567255,"identity":"a0bc77f5-4dce-4fed-8134-455f3d1a6880","order_by":6,"name":"Nobuo Adachi","email":"","orcid":"","institution":"Hiroshima University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nobuo","middleName":"","lastName":"Adachi","suffix":""}],"badges":[],"createdAt":"2022-01-16 04:29:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1264720/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1264720/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18438502,"identity":"75cfcc66-6e3c-4ee8-a958-1ed2e90087de","added_by":"auto","created_at":"2022-02-21 15:05:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146202,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of the calcitonin gene-related peptide (CGRP) in human posterior cruciate ligament (PCL). (a) Expression pattern of SOX9, MMP13 and CGRP with mild, moderate, and severe degeneration in safranin O staining and immunohistochemistry. (b) Percentage of SOX9-, MMP13-, and CGRP-positive cells. *;p\u0026lt;0.05, **;p\u0026lt;0.01. (c) Safranin O staining and immunohistochemistry of CGRP and SOX9\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/afdd12734964d120683b62d4.jpg"},{"id":18439218,"identity":"a3e18adf-564f-4fef-bbf7-853e07a61f4a","added_by":"auto","created_at":"2022-02-21 15:08:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":161696,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of the CGRP in the SAMP8 mice at 4, 18 and 42 weeks. (a) Safranin O staining and immunohistochemistry of the CGRP and CGRP receptor (CGRPR). Dashed line; PCL. Arrows; CGRP in the chondrocyte-like cells. Bar; 100 μm. (b) OARSI and ligament scores. *; p\u0026lt;0.05, **; p\u0026lt;0.01. (c) Number of CGRP positive cells. **; p\u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/8354c9002937524177613c80.jpg"},{"id":18438498,"identity":"ab1bfc14-9430-4155-bf47-491e0164054f","added_by":"auto","created_at":"2022-02-21 15:05:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119205,"visible":true,"origin":"","legend":"\u003cp\u003eDouble staining of the CGRP and Sox9 in the PCL of SAMP8 mice. Bar; 100 μm.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/87215268a6b98be743218ede.jpg"},{"id":18438503,"identity":"e00c8885-d5c0-4879-a878-693ccbdb9041","added_by":"auto","created_at":"2022-02-21 15:05:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125269,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analyses of SOX9, Runx2, and Col10a1 using polymerase chain reaction (PCR). *; p\u0026lt;0.05, **; p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/9f61576b4efa4f06a66e67f2.jpg"},{"id":18439812,"identity":"c04e08ff-ad62-4ae3-9f71-55a9837b5571","added_by":"auto","created_at":"2022-02-21 15:11:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":122217,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analyses of VEGF and MMP13 using polymerase chain reaction (PCR). *; p\u0026lt;0.05, **; p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/b636cf2510f32e1919ab6858.jpg"},{"id":18438499,"identity":"224291b5-09ba-4500-bd35-7eeced081269","added_by":"auto","created_at":"2022-02-21 15:05:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":139213,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentiation analyses of the human ligament cells by the CGRP. (a) Chondrogenesis capacity using pellet culture.\u0026nbsp;(b) Osteogenesis evaluated using alizarin red staining and real-time PCR of Runx2. (c) Adipogenesis evaluated using oil red O staining and real-time PCR of PPARγ.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/829f1e100285a0f805fae17c.jpg"},{"id":22486134,"identity":"53056177-c80b-4f71-a69c-033decac4f28","added_by":"auto","created_at":"2022-06-10 04:29:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":843441,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1264720/v1/8dfb1966-d292-4a9e-9cc2-f7e2f53eb4ce.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eExpression of Calcitonin Gene-Related Peptide Induces Ligament Degeneration Through Endochondral Ossification in Osteoarthritis\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a progressive joint disorder that increases its prevalence annually. Although OA is a common disease encountered in daily clinical practice, the mechanisms of OA initiation and progression have not been completely elucidated. In the pathogenesis of OA, various tissues including articular cartilage, subchondral bone, meniscus, joint capsule and ligament exhibit progressive degenerative changes, which is one of the reasons for the difficult OA treatment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Thus, elucidating the mechanism of degenerative changes in all joint structures is important. However, few studies on the degeneration mechanism of ligaments have been conducted compared with those on the cartilage, bone, and meniscus despite the importance of ligament degeneration in OA pathogenesis. Cushner et al. reported that forty-seven percent of patients with OA have ligament degeneration, which may cause joint laxity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Levy et al. demonstrated that histological degenerative changes of the posterior cruciate ligament (PCL) were observed before articular cartilage degeneration [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The anterior cruciate ligament and PCL insufficiency frequently cause meniscal and cartilage injuries, which can accelerate the progression of OA due to lateral thrust [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Elucidation of the mechanism of ligament degeneration would enable to prevent OA progression. Increasing chondrogenic potential in the ligament has been reported to be a key factor in the degeneration process [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. During the degenerative process of the ligament, chondrogenic cells increase, and ossification finally occurs in the fibroblast-based ligament [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This suggests that some factors may induce chondrogenesis and osteogenesis in the ligament during OA pathogenesis.\u003c/p\u003e \u003cp\u003eNeuropeptides, such as calcitonin gene-related peptide (CGRP) and substance P, are well known to play an important role in OA-related pain in the joint [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Neuropeptides have various functions, including bone metabolism and angiogenesis, to maintain homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the pathogenesis of OA, excessive expression of neuropeptides in the subchondral bone causes sclerotic changes in the subchondral bone, and subsequently, cartilage degeneration progresses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CGRP is a neuropeptide belonging to the calcitonin superfamily; it consists of 37 amino acids and is released from sensory nerves [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CGRP has various functions, including blood vessel dilation, accelerating inflammatory pain, and promotion of bone remodeling by differentiation of bone marrow mesenchymal cells into osteoblasts, stimulation of osteoblast proliferation, and acceleration of bone metabolism by inhibiting osteoclast differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We focused on CGRP in ligament degeneration because CGRP induces osteogenesis, and the sensory nerve extends into the affected joints during OA progression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We hypothesized that the elongated CGRP-positive sensory nerves into the joint might cause increased chondrogenic potential in the ligament, subsequently progressing ligament degeneration. The aim of this study was to analyze the expression pattern and function of CGRP in the ligament cells in OA pathogenesis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eBetween June 2016 and October 2018, 30 patients with advanced knee OA were treated with total knee arthroplasty (TKA). They consisted of 11 men and 19 women, with a mean age of 73.5 years. Knee joints were classified as Kellgren-Lawrence grades 3 and 4. Patients with a history of ligament injury, malunited fractures, intra-articular steroid injection in the previous 6 months, joint infection, or previous knee surgery were excluded. During surgery, PCL was harvested for histological analysis according to a previous report [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This study was approved by the institutional review board and ethics committee of our hospital and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all patients.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis of human ligament\u003c/h2\u003e \u003cp\u003eHarvested PCL was fixed in 4% paraformaldehyde and embedded in paraffin. Four-micrometer-thick sections were prepared for histological analysis. Sections were stained with hematoxylin \u0026amp; eosin (HE) and safranin- O/fast green, and histologically graded using a scoring system according to a previous report [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The following categories were examined and scored for each ligament: (1) inflammation in the ligament substance, (2) mucoid degeneration, (3) chondroid metaplasia, (4) cystic changes, and (5) orientation of collagen fibers. Five fields of the PCL were randomly selected and each slide was evaluated at 200X magnification. The histological changes were scored and graded with reference to previous report as follows [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]: 0, no changes; 0.5, minimal changes; 1, mild changes; 2, moderate changes; and 3, severe changes. The highest summed score of ligament degeneration (total score) was 15 if all five histological categories were scored as severe. Furthermore, the total score was classified into three groups: mild (0\u0026ndash;5), moderate (6\u0026ndash;10), severe (11\u0026ndash;15).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All procedures were performed in accordance with the Guidelines for Animal Experimentation at our university, and with the approval of the Committee of Research Facilities for Laboratory Animal Sciences, Graduate School of Biomedical Sciences, Hiroshima University.\u003c/p\u003e \u003cp\u003eSenescence-accelerated mouse-prone 8 (SAMP8) mice which develop spontaneous joint OA signs that resemble human disease at the ages of 4, 18, and 42 weeks (n\u0026thinsp;=\u0026thinsp;9 at each time point) were used for the evaluation of the spontaneous OA model. Harvested knee joints fixed in 4% paraformaldehyde were decalcified for 2 weeks in 20% EDTA and, embedded in paraffin. Four-micrometer-thick sagittal sections, where the whole length of the PCL in the knee joint was observed, were prepared for histological analysis. Sections were stained with HE and safranin-O, and graded histologically using OARSI and ligament scores [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical analysis\u003c/h2\u003e \u003cp\u003eFor immunohistochemical analysis, each section was immunostained using anti-CGRP antibody (1:500 dilution; Abcam, Cambridge, MA), anti-SOX 9 (1:800 dilution; Abcam, Cambridge, MA), anti-MMP13 (1:20 dilution; Neo Markers, Fremont, CA), and anti-CRLR/CGRP1 polyclonal antibody (1:100 dilution; Bioss, Boston, MA, USA). For double immunofluorescence staining of CGRP and SOX9, the anti-CGRP antibody was labeled with FITC using Dojindo Ab-10 Rapid Fluorescein Labeling Kit (Dojindo Laboratories, Kumamoto, Japan), and anti-SOX9 antibody was labeled using the Dojindo Ab-10 Rapid HiLyte Fluor 555 Labeling Kit (Dojindo Laboratories, Kumamoto, Japan) according to the method described in a previous report [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. DAPI (Dojindo Laboratories, Kumamoto, Japan) solution was used for nuclear staining. For immunohistochemical signals, five fields in the ligament were randomly selected among the areas where ligamentous structures were found; the total number of cells was counted, and the number of SOX9, MMP13, and CGRP positive cells was measured at 200X magnification using ImageJ software (National Institution of Health) according to the method of a previous report [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman ligament cells were obtained from the anterior cruciate ligament resected with TKA, because in TKA the anterior cruciate ligament is removed and therefore more tissue can be harvested. Ligament tissues were placed in 10-cm diameter Petri dishes under sterile conditions and washed five times with phosphate buffered saline (PBS; WAKO, Osaka, Japan) to remove red blood cells after removing synovial tissues, adipose tissue, and small blood vessels. The tissue was minced into 1\u0026ndash;2 mm pieces, and 0.25% type 1 collagenase was added. Tissues were shaken in a water bath at 37\u0026deg;C for 60 min, and then an equal volume of high-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich Corp., St. Louis, Missouri) and antibiotics (at a final concentration of 100 units/ml penicillin, 100 \u0026micro;g/ml streptomycin and 0.25 \u0026micro;g/ml amphotericin B; Nacalai Tesque, Kyoto, Japan) was added to stop degradation. The tissue was then filtered through a 200-mesh nylon filter and centrifuged at 400 rpm for 10 min. The supernatant was discarded, and the human ligament cells were suspended in DMEM medium containing 10% FBS and 1% antibiotics. The harvested cells were homogeneous and were further identified as ligament cells by real time polymerase chain reaction (PCR) analysis as they expressed SCX (Hs 03054634_g1). When the cell coverage reached 80%-90%, the cells were passaged. The cells from passages 2 and 3 were seeded onto 24-well plates (BD Falcon, Franklin Lakes, NJ), and incubated with or without 10 nM and 100 nM CGRP receptor agonist (Peptide Research Institute, Inc., Osaka, Japan) or 10 nM and100 nM CGRP receptor antagonist (BIBN4096, TOCRIS, Bioscience, Bristol, UK) or the same amount of PBS as a control group added to each well twice a week in a humidified 5% CO2/95% air atmosphere at 37\u0026deg;C (n\u0026thinsp;=\u0026thinsp;8 each group). After 24 h, 48 h, and 21 days, RNA was extracted for PCR analysis.\u003c/p\u003e \u003cp\u003eOsteogenic differentiation medium (StemPro Osteogenic Differentiation Kit, Life Technologies, Grand Island, NY) and antibiotics with a final concentration of 100 units/ml penicillin, 100 \u0026micro;g/ml streptomycin, and 0.25 \u0026micro;g/ml amphotericin B (Nacalai Tesque) were used. Calcified medium containing 100 nM of CGRP receptor agonist, or the same amount of PBS were added and cultured for 3 weeks. Osteogenesis capacity was evaluated by PCR and staining with 0.5% alizarin red solution (Nacalai Tesque). To induce adipogenesis, the cells were cultured in adipogenic differentiation medium (StemPro adipogenesis differentiation kit, Life Technologies) and antibiotics, to which 100 nM of CGRP receptor agonist or the same amount of PBS was added. After 3 weeks, adipogenic capacity was assessed using PCR and staining with 0.3% oil red-O (SIGMA O-0625; Sigma-Aldrich Corp.). To induce chondrogenesis, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were placed in 15-ml polypropylene tubes (BD Falcon) and pelleted by centrifugation at 500 \u0026times; g for 5 min. The pellets were cultured in chondrogenesis medium (StemPro chondrogenesis differentiation kit, Life Technologies) and antibiotics, to which 100 nM of CGRP receptor agonist or the same amount of PBS was added for 3 weeks. Chondrogenic capacity was evaluated using PCR and histological analysis. For histological analysis, the pellets were embedded in paraffin, cut into 6-ml sections, and stained with safranin-O/fast green.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eReal time PCR\u003c/h2\u003e \u003cp\u003eRNA was isolated using TRIzol (Life Technologies) for real-time PCR analysis, and proteins were extracted for analysis of alkaline phosphatase activity. Complementary DNA was synthesized using 1 \u0026micro;g of total RNA using the Superscript VLIO kit (Invitrogen) according to the manufacturer's protocol: MMP13 (Hs 00233992_m1), RUNX2 (Hs 00231692_m1), VEGFA (Hs 0090055_m1), SOX9 (Hs 00165814_m1), COL2A1(Hs 01064869_m1), COL10A1(Hs 00166657_m1), PPARγ (Hs 01115513_m1) and GAPDH(Hs99999905_m1) using TaqMan gene expression assays probes (Life Technologies), and real-time PCR assays were performed. The expression level of each gene was evaluated relative to the expression level of GAPDH. The ΔΔCt method was used to analyze the real-time PCR data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results in this study are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons among the three or four groups were performed using the Tukey\u0026ndash;Kramer post hoc test, and the Mann\u0026ndash;Whitney U test was used to determine the differences between the two groups. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExpression pattern of the CGRP in human PCL\u003c/h2\u003e \u003cp\u003eOf the 30 knees with human ligament degeneration, 7 were in the mild group, 12 were in the moderate group, and 11 were in the severe group. The mean histopathologic score of degeneration of the mild group was 3.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 points (range, 2-4.5), and the ligament was composed of parallel fiber arrangement, and the cells were mostly fibroblasts. In the moderate group, the mean histopathologic score of degeneration was 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 points (range, 5\u0026ndash;9), and the number of chondrocyte-like cells with round nuclei increased, and the surrounding tissue was stained with safranin O. In addition, the ligament fibers become wavy, and the parallel fiber arrangement was disrupted. In advanced degeneration, the mean histopathologic score of degeneration was 12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 points (range, 10\u0026ndash;14), and the number of chondrocyte-like cells increased and the area stained with safranin O stain also increased. In addition, ossification areas were observed in this study.\u003c/p\u003e \u003cp\u003eThe number of SOX9-positive cells was significantly higher in the moderate group than in the mild and advanced groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The number of MMP13-positive cells was significantly higher in the moderate group than in the advanced group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The number of CGRP-positive cells was lower in the mild group, significantly higher in the moderate group than in the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and decreased again in the severe group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Immunofluorescent analyses revealed that CGRP and SOX9 were co-expressed in the chondrocyte-like cells in ligament (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExpression pattern of the CGRP in the PCL of OA model mouse\u003c/h2\u003e \u003cp\u003eTo investigate longitudinal ligament degeneration, PCL in spontaneous OA mice (SAMP8) was histologically analyzed. As the number of weeks increased, the OARSI and ligament scores significantly increased with articular cartilage degeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). HE and safranin-O/fast green staining revealed parallel fiber arrangement and spindle-shaped nuclei. Ligaments were rarely stained with safranin- O at 4 weeks. At 18 weeks, the increased area in the ligament stained with safranin- O stain and the parallel fiber arrangement decreased, and chondrocyte-like cells were observed. At 42 weeks, ligaments stained almost exclusively with safranin- O stain and the parallel fiber structure disappeared, and the number of chondrocyte-like cells increased; its degeneration was similar to that of human ligament degeneration. The number of CGRP-positive cells was significantly higher at 18 weeks than at 4 weeks and 42 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, c). Immunohistochemical analysis of the CGRP receptor showed few positive cells at 4 weeks. However, the positive cells in both fibroblasts and chondrocyte-like cells increased at 18 weeks, and they decreased and were expressed only in chondrocyte-like cells at 42 weeks. Immunohistochemical analysis of CGRP and SOX9 showed that the co-expression of CGRP and SOX9 in the chondrocyte-like cells was highest at 18 weeks, and then decreased at 42 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFunctional analysis of CGRP on ligament in vitro\u003c/h2\u003e \u003cp\u003eTo investigate the chondrogenic effects of CGRP on ligament cells, the expression of SOX9, RUNX2, COL10A1, VEGF, and MMP13 was evaluated using real-time PCR. SOX9 expression increased in a CGRP dose-dependent manner at all stages and was significantly suppressed by BIBN4096 treatment. Runx2 expression increased in a CGRP dose-dependent manner at all stages and was suppressed by BIBN4096 treatment. Col10a1 expression significantly increased in a CGRP dose-dependent manner at 3 weeks post-treatment and was significantly suppressed by BIBN4096 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). VEGF expression was significantly upregulated at 24 h post-treatment in the CGRP 100 nM group and at 3 weeks post-treatment in the CGRP 10 nM group. MMP13 expression increased in a dose-dependent manner at 3 weeks and was significantly suppressed by BIBN4096 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Real-time PCR analyses revealed that CGRP has an effect on ligament cells to promote endochondral ossification, which is inhibited by BIBN4096.\u003c/p\u003e\u003cp\u003eThe effect of CGRP on chondrogenesis, osteogenesis, and adipogenesis of ligament-derived cells was examined. In chondrogenesis, the diameter of the pellet with CGRP treatment was larger than that without CGRP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In addition, the expression of SOX9 and Col2a1 was significantly higher in the pellet with CGRP treatment than in the pellet without CGRP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In osteogenesis, alizarin red staining showed more staining in the ligament cells with CGRP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In addition, Runx2 expression in the ligament cells with CGRP treatment was significantly higher than that without CGRP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). In adipogenic differentiation, oil red staining showed no staining of the ligament after CGRP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The expression of PPARγ was significantly lower in the ligament cells treated with CGRP than in those without CGRP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study showed that CGRP is expressed in chondrocyte-like cells in the ligament as degeneration develops, and CGRP induces chondrogenesis and osteogenesis in the ligament during OA progression. Since ligament dysfunction due to degeneration is an important factor in the pathogenesis of OA, elucidating the factors inducing ligament degeneration will lead to the development of new therapeutic strategies. Neuropeptides, including CGRP, have been recognized as therapeutic targets for various diseases, and receptor antagonists or antibodies have been developed as drugs targeting neuropeptides [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, CGRP is a potential target to prevent OA progression by administering a CGRP receptor antagonist through inhibition of subchondral bone sclerosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, the evidence that neuropeptides play a crucial role in OA pathogenesis could lead to a novel therapeutic strategy for OA.\u003c/p\u003e \u003cp\u003eLigament degeneration is histologically characterized by disorganization of collagen fiber arrangement and chondroid metaplasia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Degenerating ligaments exhibit fibrocartilaginous regions with collagen type Ⅱ and Ⅲ and an increasing distribution of chondrocytes in addition to disorganization of fiber arrangements [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Experimental tendon degeneration showed that approximately 5% of the degenerating area has calcific foci with chondrocytes expressing SOX9 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Kumagai et al. demonstrated that Sclelaxis- positive cells decreased and SOX9-positive cells increased as ligament degeneration progressed [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, chondrogenic differentiation plays a crucial role in ligament degeneration. However, the factors that induce or promote chondrogenic differentiation of the ligament during OA progression have not been elucidated. We focused on CGRP regarding chondrogenic differentiation in ligament degeneration. CGRP has various functions, including pain perception, osteogenesis, and angiogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The sensory nerve around the joint has been reported to elongate into the joint during OA progression, and the neuropeptides released from the sensory nerve then causes pain, inflammation, and subchondral bone sclerosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. CGRP is distributed in the synovium, subchondral bone, cartilage, and meniscus in the joints with OA [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In particular, CGRP expression in the subchondral bone increases in destabilization of the medial meniscus (DMM) mice and induces subchondral bone sclerosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CGRP may also induces ossification of the ligament during the degeneration process. Moreover, CGRP has been reported to be involved in heterotopic ossification [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and administration of CGRP to the tibialis anterior muscle of mice promotes heterotopic ossification [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe examined the effects of CGRP on the differentiation of ligament cells. Furumatsu et al. demonstrated that ligament cells exhibit chondrogenic properties with SOX9 expression in chondrogenic induction medium [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Takimoto et al. showed the direct conversion of tenocytes to chondrocytes by SOX9 overexpression [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, CGRP upregulated SOX9 expression in pellet cultures during chondrogenesis, and the size of the pellet was enlarged. Furthermore, CGRP promoted the osteogenesis in the ligament cells while it inhibiting adipogenesis. In fact, with the addition of CGRP to the ligament cells, the expression of SOX9 and Runx2 was increased up to 3 weeks, suggesting that CGRP induces chondrogenesis and osteogenesis. At 3 weeks of culture, the expression of Col10a1 and MMP13, which are important factors in endochondral ossification, increased in a dose-dependent manner, indicating that CGRP induced endochondral ossification [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. VEGF, important factor in endochondral ossification, is also upregulated by CGRP. Hypervascularization in various tissues in the joint, including the ligament, has been reported in the OA joint [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our study revealed that CGRP might play a role in inducing endochondral ossification and angiogenesis in the ligament through VEGF expression. In our study, CGRP expression increased as the degeneration of the ligament progressed, but severe degeneration ligaments did not exhibit a decrease in CGRP expression accompanied by MMP13 expression. It is suspected that CGRP expression by the sensory nerve that elongates from the surrounding joint induces transdifferentiation of the ligament cells to chondrocyte-like cells to promote endochondral ossification [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the severe degeneration ligament, fibroblastic cells were sparsely present, and CGRP was no longer required. CGRP induces VEGF at 24 h after CGRP addition to the ligament cells. CGRP has been reported to induce angiogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. CGRP may also induce angiogenesis in the early phase of OA, which may lead to further increased expression of CGRP [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study had several limitations. First, the natural course of ligament degeneration during OA progression could not be observed in human samples. This is an unsolvable problem for histological evaluation, but we compensated for it by stratifying the degree of ligament degeneration. Moreover, SAMP8, which has been established as a spontaneous OA model, was used to evaluate the time course of ligament degeneration in OA progression [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Second, the factors that induce the expression of CGRP in the ligament have not been elucidated, although this study explored important role of CGRP in ligament degeneration. However, the factors that induce OA have not been clarified, despite many studies on OA pathogenesis. Finally, the loss of function of CGRP was not examined in vivo in this study. Knockdown of the CGRP prevents ligament degeneration. Nakasa et al. reported that DMM mice which were administered the CGRP receptor antagonist reduced the OARSI score until 8 weeks, which suggested that inhibition of the CGRP expression may lead to prevent ligament degeneration [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, DMM mice are recognized as a traumatic OA model, and whether DMM affects ligament degeneration is unclear. Therefore, it is desirable to examine CGRP knockdown in a spontaneous OA mouse model.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study shows that with regard to ligament degeneration, angiogenesis occurs in the early stages, followed by chondrogenesis and ossification via endochondral ossification, and CGRP is deeply involved in this process. Furthermore, CGRP receptor antagonists were shown to have the potential to effectively attenuate OA progression.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteoarthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCGRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCalcitonin gene-related peptide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eposterior cruciate ligament\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTKA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal knee arthroplasty\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin \u0026amp; eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSAMP8\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSenescence-accelerated mouse-prone 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDestabilization of the medial meniscus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the institutional review board and ethics committee of our hospital and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all patients.\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Hiroshima University Hospital, and written consent was obtained from all patients. All procedures were performed in accordance with the Guidelines for Animal Experimentation at our university, and with the approval of the Committee of Research Facilities for Laboratory Animal Sciences, Graduate School of Biomedical Sciences, Hiroshima University. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research was partly supported by MEXT KAKENHI Grant-in-Aid for Scientific Research (C); Grant Number 18K09066 (T.N.). Research grant from the Nakatomi Foundation (T.N.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. MT and TN designed this study, analyzed and interpreted the data, and drafted the manuscript. TN provided the study materials and funding. AN, MI, YI and SM obtained the data, analyzed and interpreted the data, drafting the article. TN and NA contributed to the critical revision of the article. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Editage (www.editage.com) for English language editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLoeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: A disease of the joint as an organ. Arthritis Rheum. 2012;64: 1697\u0026ndash;707.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCushner FD, La Rosa DF, Vigorita VJ, Scuderi GR, Scott WN, Insall NS. A quantitative histologic comparison: ACL degeneration in the osteoarthritic knee. J Arthroplasty. 2003;18: 687\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevy YD, Hasegawa A, Patil S, Koziol JA, Lotz MK, D\u0026rsquo;Lima DD. Histopathological changes in the human posterior cruciate ligament during aging and osteoarthritis: correlations with anterior cruciate ligament and cartilage changes. Ann Rheum Dis. 2013; 72: 271\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimura I, Naito M, Zhang J. Lateral thrust of anterior cruciate ligament-insufficient knees and posterior cruciate ligament-insufficient knees. International Orthop. 2002; 26: 303\u0026ndash;305.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumagai K, Sakai K, Kusayama Y, Akamatsu Y, Sakamaki K, Morita T, et al. The extent of degeneration of cruciate ligament is associated with chondrogenic differentiation in patients with osteoarthritis of the knee. Osteoarthritis Cartilage. 2012;20: 1258\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasegawa A, Nakahara H, Kinoshita M, Asahara H, Koziol J, Lotz MK et al. Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis. Arthritis Res Therapy. 2013;15: R29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxler G, Loer F, Skumavc M, Pfortner J, Hanesch U. Localization of SP- and CGRP-immunopositive nerve fibers in the hip joint of patients with painful osteoarthritis and of patients with painless failed total hip arthroplasties. Eur J Pain. 2007; 11: 67\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIrie K, Hara-Irie F, Ozawa H, Yajima T. Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling. Microsc Res Tech. 2002; 15;58: 85\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakasa T, Ishikawa M, Takada T, Miyaki S, Mitsuo Ochi. Attenuation of cartilage degeneration by calcitonin gene-related paptide receptor antagonist via inhibition of subchondral bone sclerosis in osteoarthritis mice. J Orthop Res. 2016; 34: 1177\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanemitsu M, Nakasa T, Shirakawa Y, Ishikawa M, Miyaki S, Adachi N. Role of vasoactive intestinal peptide in the progression of osteoarthritis through bone sclerosis and angiogenesis in subchondral bone. J Orthop Sci. 2020; 25: 897\u0026ndash;906.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, et al. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature. 1983; 14\u0026ndash;20;304(5922): 129\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrain SD, Williams TJ, Tippins JR, Morris HR, Maclntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature. 1985; 313: 54\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGr\u0026auml;ssel S, Muschter D. Peripheral nerve fibers and their neurotransmitters in osteoarthritis pathology. Int J Mol Sci. 2017; 18: 931.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayashi S, Nakasa T, Matsuoka Y, Akiyama Y, Ishikawa M, Nakamae A, et al. Evaluation of the degenerative pattern of PCL in osteoarthritis patients using UTE-T2 mapping. Asia Pac J Sports Med Arthrosc Rehabil Technol. 2021; 24: 35\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMullaji AB, Marawar SV, Simha M, Jindal G. Cruciate Ligaments in Arthritic Knees. A Histologic Study With Radiologic Correlation. J Arthroplasty. 2008; 23: 567\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage. 2010; 18: S17-23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyazaki K, Oyanagi J, Hoshino D, Togo S, Kumagai H, Miyagi Y. Cancer cell migration on elongate protrusions of fibroblasts in collagen matrix. Sci Rep. 2019; 9: 292.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZemmyo M, Meharra EJ, K\u0026uuml;hn K, Creighton-Achermann L, Lotz M. Accelerated, Aging-Dependent Development of Osteoarthritis in α1 Integrin-Deficient Mice. Arthritis Rheum. 2003; 48: 2873\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan D, Liu X, Guo SW. Neuropeptides Substance P and Calcitonin Gene Related Peptide Accelerate the Development and Fibrogenesis of Endometriosis. Sci Rep. 2019; 9: 2698.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTepper SJ. History and Review of anti-Calcitonin Gene-Related Peptide (CGRP) Therapies: From Translational Research to Treatment. Headache. 2018; 58: 238\u0026ndash;275.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasegawa A, Otsuki S, Pauli C, Miyaki S, Patil S, Steklov N, et al. Anterior cruciate ligament changes in human joint in aging and osteoarthritis. Arthritis Rheum. 2012; 64: 696\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTallon C, Maffulli N, Ewen SWB. Ruptured Achilles tendons are significantly more degenerated than tendinopathic tendons. Med Sci Sports Exerc. 2001; 33: 1983\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiley GP, Harrall RL, Constant CR, Chard MD, Cawston T, Hazleman B. Tendon degeneration and chronic shoulder pain: changes in the collagen composition of the human rotator cuff tendons in rotator cuff tendinitis. Ann Rheum Dis. 1994; 53: 359\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamiric T, Parkinson J, Ilic MZ, Cook J, Feller JA, Handley CJ. Changes in the composition of the extracellular matrix in patellar tendinopathy. Matrix Biol. 2009; 28: 230\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLui PP, Fu SC, Chan LS, Hung LK, Chan KM. Chondrocyte phenotype and ectopic ossification in collagenase-induced tendon degeneration. J Histochem Cytochem. 2009; 57: 91\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng S, Li W, Xu M, Bai X, Han J, Shyy JY, Wang X. Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase. Am J Physiol Cell Physiol. 2010; 299: 1485\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGr\u0026auml;ssel, S. The role of peripheral nerve fibers and their neurotransmitters in cartilage and bone physiology and pathophysiology. Arthritis Res Ther. 2014; 16: 485.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuri S, Gill SE, De Camin SM, Wilson D, McWiliams DF, Walsh DA. Neurovascular invasion at the osteochondral junction and in osteophytes in osteoarthritis. Ann Rheum Dis. 2007; 66: 1423\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuzmen C, Verdelis K, Weiss L, Campbell P. Crosstalk between substance P and calcitonin gene-related peptide during heterotopic ossification in murine Achilles tendon. J Orthop Res. 2018; 36: 1444\u0026ndash;1455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSang X, Wang Z, Shi P, Li Y, Cheng L. CGRP accelerates the pathogenesis of neurological heterotopic ossification following spinal cord injury. Artif Cells Nanomed Biotechnol. 2019; 47: 2569\u0026ndash;2574.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurumatsu T, Hachioji M, Saiga K, Takata N, YokoyamaY. Anterior cruciate ligament-derived cells have high chondrogenic potential. Biochem Biophys Res Commun. 2010; 391: 1142\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakimoto A, Oro M, Hiraki Y, Shukunami C. Direct conversion of tenocytes into chondrocytes by Sox9. Exp Cell Res. 2012; 318: 1492\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JS, Ali MH, Wydra F, Li X, Hamilton JL, An HS, et al. Characterization of degenerative human facet joints and facet joint capsular tissues. Osteoarthritis Cartilage. 2015; 23: 2242\u0026ndash;2251.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorchi AM, Cengarle-Samak A, Okuno Y, Martel-Pelletier J, Pelletier JP, Boesen M, et al. Inflammation and Hypervascularization in a Large Animal Model of Knee Osteoarthritis: Imaging with Pathohistologic Correlation. J Vasc Interv Radiol. 2019; 30: 1116\u0026ndash;1127.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshraf S, Wibberley H, Mapp PI, Hill R, Wilson D, Walsh DA. Increased vascular penetration and nerve growth in the meniscus: A potential source of pain in osteoarthritis. Ann Rheum Dis. 2011; 70: 523\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagira K, Ikuta Y, Shinohara M, Sanada Y, Omoto T, Kanaya H, et al. Histological scoring system for subchondral bone changes in murine models of joint aging and osteoarthritis. Sci Rep. 2020; 10: 1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Calcitonin gene-related peptide, endochondral ossification, ligament degeneration, osteoarthritis","lastPublishedDoi":"10.21203/rs.3.rs-1264720/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1264720/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eDegeneration occurs in various tissues such as cartilage and subchondral bone, and ligament deficiency also plays an important role in the osteoarthritis (OA) progression, but factor which it causes is still unclear. Calcitonin gene-related peptide (CGRP), one of the neuropeptides, its expression increases in OA progression. The purpose of this study is to analyze the mechanism of ligament degeneration and the function of CGRP.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTo examine the relationship between ligament degeneration and CGRP expression, human posterior cruciate ligament (PCL) from OA patients and SAMP8 mice were histologically analyzed. The effect of CGRP of human ligament cells on the differentiation was also examined.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn human PCL, CGRP expression increased as degeneration progressed, and decreased in the severe degeneration. CGRP expressed in the chondrocyte like cells with SOX9. In SAMP8 mice, the expression of CGRP increased as OA progression, but it decreased in the severe OA. CGRP up regulated the gene expression of VEGF, SOX9, RUNX2, Col10a1and MMP13. CGRP also promoted chondrogenesis and osteogenesis in the human ligament cells.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eDuring OA progression, CGRP induces the transdifferentiation from ligament cells to chondrocytes, and promotes endochondral ossification in the ligament. CGRP would be the therapeutic target to prevent the ligament degeneration.\u003c/p\u003e","manuscriptTitle":"Expression of Calcitonin Gene-Related Peptide Induces Ligament Degeneration Through Endochondral Ossification in Osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-21 15:05:16","doi":"10.21203/rs.3.rs-1264720/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17726875-5176-44b6-a0ee-d9bcb40b33fc","owner":[],"postedDate":"February 21st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-10T04:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-21 15:05:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1264720","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1264720","identity":"rs-1264720","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}