Bilateral pseudo-disc changes in a minipig model for unilateral anterior disc displacement of the temporomandibular joint

Bilateral pseudo-disc changes in a minipig model for unilateral anterior disc displacement of the temporomandibular joint | 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 Bilateral pseudo-disc changes in a minipig model for unilateral anterior disc displacement of the temporomandibular joint Wei Liu, Xiaofeng Bai, Wenlin Yuan, Cunyi Wang, Yijie Hu, Jianghang Yu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5392138/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 Objective Current animal models of anterior disc displacement (ADD) of the temporomandibular joint (TMJ) have primarily been developed in small animals, using sutures or elastic bands to pull the disc anteriorly. These models often exhibit severe osteoarthritis (OA) and rapid progression to late-stage ADD, which do not accurately reflect the natural course of ADD in humans. This study aimed to establish a unilateral ADD-induced surgical protocol in a minipig model and evaluate the early changes of ADD. Methods Six Bama minipigs (12 joints) were divided into 3 groups: control group (CG), ADD experimental group (ADD-EG), and contralateral experimental group (CL-EG). Nickel-titanium coil springs were used to induce mild, sustained traction to anteriorly displace the TMJ discs. After a 5-week observation period, in-vivo magnetic resonance imaging (MRI) scans confirmed the anterior position of the TMJ discs. Results Micro-computed tomography showed minimal condylar bone changes, while histological analysis revealed mild OA-like condylar cartilage changes. MRI identified hypointense lesions posterior to the TMJ discs, indicative of pseudo-disc changes in the posterior attachments (PAs). Histological staining further confirmed chondroid metaplasia in the PAs surrounded by transitional zones in both ADD-EG and CL-EG. Conclusion This study successfully established a unilateral ADD-induced minipig model and identified pseudo-disc changes in the PAs under abnormal mechanical stress, which may offer new perspectives for ADD therapies. To our knowledge, this is the first study to observe bilateral pseudo-disc changes in a unilateral ADD-induced model. biomechanics chondrogenesis fibrocartilage temporomandibular disorder temporomandibular joint disc Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Anterior disc displacement (ADD) is the most common subtype of temporomandibular disorder (TMD), characterized by joint clicking, limited mouth opening, and pain 1 , 2 . In adolescents, it can affect mandibular growth, leading to facial asymmetry and deformity, thus necessitating prompt attention 3 . When ADD progresses to an advanced stage, over half of the patients tend to develop degenerative joint disease which is difficult to reverse 2 , 4 . However, early intervention is limited due to a lack of understanding and suitable animal models for early-stage ADD. Several animal models of ADD have been established, including mice, rats, and rabbits. Ali et al. induced ADD in rabbits within 24 hours by severing the discal attachments and pulling the discs anteriorly with sutures 5 . Nguyen et al. reported a surgical approach in rats that did not involve dissection of the discal attachments, achieving a success rate of 58.3% 6 . Gu et al. developed a minimally invasive surgery without opening the temporomandibular joint (TMJ) capsules in rabbits, with 15 out of 16 rabbits achieving ADD 7 . Despite modification to make surgical protocols minimally invasive, the use of sutures remained. Some studies identified the success of ADD induction by immediate verification during surgery, while such abrupt induction is unlikely in humans 6 , 8 – 10 . Therefore, Wu et al. used double-strand elastic rubbers and found that apoptosis-associated proteins such as Fas and caspase-8, increased after ADD within 4 weeks, implying chondrocytes loss in the condyle 11 . Almost all the ADD animal models developed severe TMJ osteoarthritis (OA) quickly, particularly the degeneration of condyle chondrocytes, inhibition of TMJ growth and mandibular deviation 5 – 9 , 11 – 13 . Clinically, however, patients with early-stage ADD do not typically present with serious OA. Meanwhile, a few studies have reported pseudo-disc changes in the posterior attachments (PAs) after ADD, characterized by chondrocytes and denser collagen, but evidence concerning a comprehensive view of the entire disc is absent 7 , 10 . Comparing the human TMJ to various species for biochemical and biomechanical properties, the minipig is often cited as the gold standard model for TMJ research 14 , 15 . No previous study has induced ADD in minipigs. The aim of this study is to surgically induce unilateral ADD (U-ADD) in minipigs and investigate the early-stage changes associated with ADD. It is hypothesized that the early stage of ADD will exhibit minimal OA-like changes or mandibular underdevelopment. Additionally, the specific aims of the study were to investigate pseudo-disc changes in the PAs and explore whether these changes were reconstructive or degenerative. Methods Study D esign Six female minipigs (6 months old, weighing 25 and 33 kilograms) were used, considering the susceptible age and gender for TMD (Fig. 1A). Two minipigs (4 joints) served as the control group (CG). Given the nature of the TMJ as a bilateral linkage joint, the left TMJs of the remaining 4 minipigs were chosen for ADD experimental group (ADD-EG), while the right TMJs were left untreated as contralateral experimental group (CL-EG). Five weeks after the initial surgery, a second surgery was performed to remove the nickel-titanium coil spring and other metal instruments used in the initial U-ADD surgery, allowing for in-vivo magnetic resonance imaging (MRI) scanning. Subsequently, all animals were humanely sacrificed, ensuring that each group had at least 3 samples available for analysis. The experimental protocols were approved by the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (IACUC-202306-17) and conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Surgical P rotocols The lateral surface of the minipig’s head was shaved and sterilized. A 4-cm curvilinear incision was made with a #10 blade, aligned with the lateral canthus of the eye. An electrosurgical knife was used to control bleeding and incise through the subcutaneous tissue and periosteum, followed by reflecting the periosteum with periosteal elevators. The soft tissue lateral to the condylar head, composed of the TMJ capsule and ligament, was identified. An incision was then made through the anterolateral capsule, below the disc, to expose the inferior compartment of the TMJ (Fig. 1B(b, b’)). At this point, the anterior and lateral attachment was partially loosened, and the PA was not exposed and left intact. An orthodontic mini-implant (Ormco, Brea, California, USA) was screwed into the zygomatic arch, about 20 mm from the posterior margin of zygomatic arch. The anterolateral attachment, tightly linked to the disc, was penetrated by a round needle with a 4/0 nylon suture (Fig. 1B(c, c’)). The suture was then knotted to a nickel-titanium coil spring (Shinye, Hangzhou, Zhejiang, China) which was stretched from 5 mm to 14 mm, and a ligature wire was pierced through to attach the coil spring to the mini-implant. Measured by an orthodontic spring dynamometer during the surgery, this setup created a tension force of approximately 1 N, ensuring tension in the anterior attachment and disc, without immediate visible disc displacement (Fig. 1B(d, d’)). After that, a non-absorbable membrane (INNOD, Yanan, Jilin, China), made from expanded polytetrafluoroethylene (e-PTFE) and titanium mesh, was fixed on the zygomatic arch by a single bone nail, functioning as a barrier to avoid bone osseointegration within the nickel-titanium coil spring (Fig. 1B(e, e’)). Finally, the joint capsule and other incised tissues were closed in layers. After a 5-week observation period, a brief second procedure was required to remove the metal implants for in-vivo MRI. Second S urgery and MRI After the 5-week observation period, a brief second procedure was required to remove the metal implants for in-vivo magnetic resonance imaging. The surgical wounds of all the pigs healed well, with no other symptoms. Following the initial operative incision, the soft tissue was dissected, and after detaching the periosteum of the zygomatic arch, the draw gear was exposed. All the metal components, including the nickel-titanium coil spring, mini-screw, bone nail, and e-PTFE membrane, were removed followed by wound closure. Shortly after the second surgery, all 6 pigs were maintained under anesthesia for in-vivo MRI scans. Their snouts were ferruled using rubber bands, to ensure a closed-mouth position during examination. A 7 Tesla research MR scanner (Siemens Healthcare, Erlangen, Germany) equipped with a 1Tx/28Rx QED knee coil (Mayfield Village, OH, USA) was used for the scanning experiments. A Proton Density TSE MRI sequence was performed with the following parameters: echo time of 19 ms, repetition time of 5200 ms, and a voxel size of 0.3 × 0.3 × 2.5 mm. Specimen P reparation and Micro-CT S canning Following the MRI, all the animals were sacrificed. Intact TMJs were dissected, carefully preserving the discal attachments for gross examination to evaluate the disc-condyle relationship. Then, the TMJ discs and condyles were separated and fixed in 4% paraformaldehyde for 48 hours. Next, the condyles were scanned using a micro-computed tomography (Micro-CT) scanner (Milabs, Houten, Netherlands) with specific parameters: 50 kV voltage, 0.24 μA current, 15 ms exposure time, angle speed 30 deg/s. Imalytics Preclinical 2.1 software was used for 3-dimensional reconstruction, condylar length and width were measured 16 . A virtual acid was sprayed over the surface of condyles and the acid grew into the condyles by a 1.2 mm thickness. The region covered by this acid was considered to be subchondral bone. Bone volume fraction (BV/TV, %), bone surface/bone volume (BS/BV, %), bone mineral density (BMD, g/cm 3 ), trabecular separation (Tb. Sp, mm) and trabecular thickness (Tb. Th, mm) were calculated (Supplementary material, Fig. S1). Histological E xamination After taking micro-CT images, the condyles were decalcified with 15% EDTA for approximately 2 months. All the TMJ discs and condyles were dehydrated in an ascending graded alcohol series, then paraffin-embedded in the anterior-posterior direction and consecutively cut at 5 μm. Hematoxylin-eosin (H&E) staining and Safranin O/ fast green (SO&FG) staining were performed on the condyles. The thickness of the condylar cartilage, measured using SO&FG stained sections, was assessed 3 times in the functional slopes of the condyles and the average thickness was calculated for statistical analysis. The Osteoarthritis Research Society International (OARSI) scoring system was used to assess the severity of cartilage OA, based on SO&FG stained sections 17 . In addition to H&E staining, Alcian blue (AB) staining was used on TMJ discs for chondrocyte characterization. Statistical A naly ses Results are presented as the mean ± standard deviation. Two-way analysis of variance was performed to compare the data of the 3 groups using GraphPad Prism software, paired t-test was used to compare the ADD-EG and CL-EG. Differences were considered significant at P < 0.05. Results Of the 4 minipigs in the EG, 1 minipig exhibited a loosened suture with no obvious disc displacement, indicating a failed model. This resulted in final sample sizes of ADD-EG (n=3), CL-EG (n=3), and CG (n=4). Gross E valuation All the groups followed the same diet during the observation period and weight gains were observed (Fig. 2A). During the second surgery, gross examination showed that the coil springs had recovered from elastic deformation, confirming that the anterior attachments were displaced and slackened (Fig. 2B(a)). Osseointegration on the surface of the zygomatic arch was observed above the membrane, but no bone had formed within the springs (Fig. 2B(b)). After sacrifice, gross examination revealed that TMJ discs in the ADD-EG were positioned anteriorly compared to CL-EG, with the intermediate portion of the TMJ discs anterior to the transverse ridges of the condyles (Fig. 2C(d)). No morphological differences were observed among the 3 groups by visual inspection. Magnetic R esonance I maging Figure 2D shows the TMJ disc-condyle relationships by MRI in the 3 groups. Anterior disc displacement was confirmed in the ADD-EG, while the other groups maintained a normal disc-condyle relationship. TMJ discs in all groups retained double-concave shape but were elongated in the ADD-EG. In addition, all PAs in the EG (n = 6) presented lower signal intensity than the CG, suggesting the occurrence of pseudo-disc changes. Histological Results of TMJ Discs and PAs In the CG, collagen fibers oriented strictly in anterior-posterior direction in the intermediate portion of discs but were less organized in the anterior and posterior portions (Fig. 3). The anterior and posterior attachments exhibited a higher degree of multidirectional orientation with blood vessels and sparse pockets of fat vacuoles. A few chondrocyte-like cells were distributed in the TMJ discs and PAs, with lightly-staining intracellular matrix, and they were more frequent on the upper or lower surfaces of the intermediate portion of discs (Fig. 4C). Compared to the CG, with an increased number of chondrocyte-like cells in the PAs, TMJ discs from the EG were less stained with AB staining broadly, but focal condensations were observed in the PAs (Fig. 4). In the center of the condensed mass, chondrocyte clusters were observed with hyperchromatism of the surrounding matrix. There was an apparent transitional zone around the condensed mass, showing evolution from hyperchromatic fibroblasts at the outermost edge to immature chondrocytes to mature cartilaginous tissue at the core (Fig. 4D). Typically, fat vacuoles and blood vessels were distributed in the periphery, but they could also exist within the mass occasionally (Fig. 4E). In conclusion, well-developed heterotopic chondrogenesis was observed in all PAs from EG, corresponding to the low intensity seen in MRI. Micro-CT S can ning The average condylar length of ADD-EG was 13.82 ± 1.55 mm, lower than that of the CL-EG (14.60 ± 2.09 mm) and CG (14.72 ± 0.29 mm). The condylar width among the 3 groups showed basically no difference (Fig. 5A(b)). Micro-CT analysis showed generally similar values across the groups, indicating relatively normal subchondral bone. Histological Results of Condyles Figure 5B shows an intact and continuous surface of the condyle in all 3 groups. Each condyle exhibited a clear hierarchy of cell layers in the anterior and middle regions, but the distribution became disordered in posterior regions including CG (Supplementary material, Fig. S2). Compared to the CG, condyles from the EG showed more hyperchromatic chondrocytes and more stained hypertrophic chondrocyte layers, suggesting a compensatory increase in cartilage matrix proteoglycan and an early-stage OA feature. Additionally, condyles from the EG showed increased cartilage thickness compared to the CG, but with no significant difference. SO&FG staining revealed a significant increase in OARSI grades in the EG, in accordance with the safranin O + area. Discussion In this study, we used a nickel-titanium coil spring to provide lasting traction to displace the TMJ disc anteriorly, as the elasticity of rubber might decay quickly in a complex body fluid environment. This traction simulated the hyperactivity of lateral pterygoid muscle and no visible disc displacement was detected during the U-ADD surgery, allowing us to mimic the natural course of human ADD to some degree. According to the pilot study, the unabsorbable e-PTFE membrane was necessary to prevent osseointegration within the nickel-titanium coil spring, which would otherwise prevent the spring from returning to its original shape, leading to failure of U-ADD (Supplementary material, Fig. S3). Considering the observation time of previous studies in small animals, a 5-week observation period was adopted. MRI assessment confirmed the U-ADD, and subsequent analysis showed no significant OA-like condylar bony changes and compensatory cartilage changes. Notably, bilateral pseudo-disc changes were observed in a U-ADD model for the first time. Pseudo-disc changes were first identified in human cadaveric histological analysis by Scalpino in 1983, who reported that the fibers formed a compact mass with reduced vessels in the thickened PA 18 . Leonardi et al. further added that islands of fibro chondrocytes also existed in the remodeled PA 19 . With the development of ADD animal models, most studies observed degenerative changes in the PAs, such as disordered fibers, hyalinization and loss of adipose tissue, while a few studies reported the pseudo-disc changes, which might be related to variations in model establishment 5 , 6 , 10 , 20 , 21 . Gu et al. found chondrocyte-like cells in both TMJ discs and PAs, and observed immature chondroid metaplasia in 1 of 16 experimental rabbits 8 weeks after U-ADD surgery, but the contralateral side was not mentioned 7 , 10 . Partially aligned with this study, we observed that the chondrocytes in the PA gathered into a mass with a clear transition zone around it. The transition zone showed apparent transformation from fibroblasts to chondrocytes, suggesting mature chondroid metaplasia in the PA. In addition, the degenerative tendency of TMJ discs and PAs was not apparent in this study. With the abnormal stress on the contralateral side as indicated by finite element analyses, bilateral pseudo-disc changes occurred 22 , 23 . Considering the clinical outcomes of ADD, some patients develop severe degenerative changes leading to disc perforation over time, while others gradually attain a stable TMJ structure. We believe pseudo-disc change belongs to the latter category. While widespread ossification or chondrification within the joint is typically considered degenerative due to its tendency to restrict mandibular movement and generation of abnormal stress, our results suggest that heterotopic chondrogenesis confined to the PA, which might represent a new treatment avenue 24 , 25 . The PA is one of the most vulnerable sites of perforation, often associated with degenerative bony changes like osteophytes and flattening 26 . Radiological follow-up studies suggest that bony changes in the condyle tend to become steady when pseudo-disc changes occur 27 . Together with this study, it can be inferred that pseudo-changes are related to stabilized bony changes, although the cause-and-effect relationship is not yet fully understood. MRI seems to be the only method to detect pseudo-disc changes in the clinic, showing a hypointense lesion posterior to the disc in the sagittal plane, consistent with our results. Though no evidence links pseudo-disc changes to pain relief, it has been reported that high signal intensity of the PA is closely correlated with TMJ pain, possibly indicating increased vascularity or inflammation 28 , 29 . In our study, without the observation of significant inflammatory cells or vascularization, heterotopic chondrogenesis was found in the PAs, which expressed increased collagen II and aggrecan, according to the study by Gu et al 10 . These results suggest that this change might optimize the mechanical properties of the original fibrocyte-composed PA, allowing it to better endure compressive stress. Considering the above, we hypothesis that inducing pseudo-disc changes could reshape the PA into fibrocartilage, allowing it to endure compressive stress during ADD. This may stabilize condylar cartilage, contribute to condyle repair, prevent disc perforation, and ultimately alleviate OA. However, there were a few conflicts with conventional opinions in this study. Several radiological follow-up studies reported the occurrence rate of pseudo-disc change to be 8.9–45% among TMD patients 27 , 29 , 31 . In this study, however, all the ADD-induced TMJs presented pseudo-disc changes, possibly due to the small sample size (n = 3). With the disturbance in the direction of stress distribution through U-ADD, pseudo-disc changes in the contralateral sides (n = 3) were observed as well, a finding not previously reported in humans. Furthermore, it is generally accepted that the reconstruction of the PA can take years, and pseudo-disc changes are considered a symbol of late-stage ADD 27 . Yet, in this study, these changes occurred within 5 weeks. Little research has been done on the pseudo-disc changes, and these questions need further study. The chondrogenic potential of fibroblasts or synoviocytes to spontaneously generate heterotopic chondrogenesis is reflected in diseases such as chondromatosis. With continuous overexpression of transforming growth factor-beta 1, Watson et al. observed aggressive fibrocartilaginous metaplasia in the knee joints of rats 32 . Our studies observed heterotopic chondrogenesis in TMJs of minipigs and revealed the key role of mechanical stress in this change. This study had some limitations. According to the outcomes of pilot study and due to economic considerations, there was no sham surgery group set up in this study. A longer-term observation period with multiple observation timepoints is needed. The mechanical properties of the TMJ disc complex after pseudo-change have not been tested, and further studies are required. Conclusion Our study developed a U-ADD minipig model with bilateral pseudo-disc changes in the PAs, suggesting the key role of mechanical stress in these changes. These findings could provide potential insights for future ADD therapies. Abbreviations ADD: Anterior disc displacement TMJ: Temporomandibular joint OA: Osteoarthritis CG: Control group ADD-EG: Anterior disc displacement experimental group CL-EG: Contralateral experimental group MRI: Magnetic resonance imaging PA: Posterior attachment TMD: Temporomandibular disorder U-ADD: Unilateral anterior disc displacement E-PTFE: Expanded polytetrafluoroethylene Micro-CT: Micro-computed tomography BV/TV: Bone volume fraction BS/BV: Bone surface/bone volume BMD: Bone mineral density Tb. Sp: Trabecular separation Tb. Th: Trabecular thickness H&E: Hematoxylin-eosin SO&FG: Safranin O/ fast green AB: Alcian blue Declarations Ethics approval and consent to participate The experimental protocols were approved by the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (IACUC-202306-17). Clinical trial number: not applicable. Consent to Participate declaration: not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 82170984), Research and development Foundation of Stomatology Hospital in Zhejiang University (RD2023YYYB01). Authors’ contributions W.L. contributed to conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; X.B. contributed to design, data acquisition and interpretation, critically revised the manuscript; W.Y. contributed to data acquisition, analysis and interpretation, critically revised the manuscript; C.W., Y.H., J.Y. and Y.Z. all contributed to data acquisition, critically revised the manuscript; J.S. contributed to conception and design, critically revised the manuscript. 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Decreased signal from the retrodiskal tissue. Oral Surg Oral Med Oral Pathol 1993; 76 (5):631–5. https://doi.org/10.1016/0030-4220(93)90074-e Bristela M, Schmid-Schwap M, Eder J, Reichenberg G, Kundi M, Piehslinger E, Robinson S. Magnetic resonance imaging of temporomandibular joint with anterior disk dislocation without reposition - long-term results. Clin Oral Investig 2017; 21 (1):237–45. https://doi.org/10.1007/s00784-016-1800-9 Watson RS, Gouze E, Levings PP, Bush ML, Kay JD, Jorgensen MS, Dacanay EA, Reith JW, Wright TW, Ghivizzani SC. Gene delivery of TGF-β1 induces arthrofibrosis and chondrometaplasia of synovium in vivo. Lab Invest 2010; 90 (11):1615–27. https://doi.org/10.1038/labinvest.2010.145 Additional Declarations No competing interests reported. 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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-5392138","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":378851257,"identity":"8f16d8ef-14ac-4799-a6d4-c7beb5f5048e","order_by":0,"name":"Wei Liu","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Liu","suffix":""},{"id":378851258,"identity":"eb00370c-79e3-4333-bc49-8e765721c2c1","order_by":1,"name":"Xiaofeng Bai","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Bai","suffix":""},{"id":378851259,"identity":"3f9d4f08-aa04-40ad-83e7-5c6f403a0a0d","order_by":2,"name":"Wenlin Yuan","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenlin","middleName":"","lastName":"Yuan","suffix":""},{"id":378851260,"identity":"418cc856-9520-4220-be0d-23b8914c0664","order_by":3,"name":"Cunyi Wang","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cunyi","middleName":"","lastName":"Wang","suffix":""},{"id":378851261,"identity":"76de27f7-ba9c-4e64-a713-a553db19b4c5","order_by":4,"name":"Yijie Hu","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yijie","middleName":"","lastName":"Hu","suffix":""},{"id":378851262,"identity":"edc7cf50-5d63-4019-9349-eb511bc5b17e","order_by":5,"name":"Jianghang Yu","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jianghang","middleName":"","lastName":"Yu","suffix":""},{"id":378851263,"identity":"c316016b-8d81-431e-ac8b-b4c85466ff66","order_by":6,"name":"Yi Zhang","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":378851264,"identity":"f55ffb9c-f1a7-47b7-8563-78a52c27949d","order_by":7,"name":"Jiejun Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIie3PMQrCMBTG8a8ITpWuz6W9QsRJvIxOnQSnzilCXDxAwMEzeIOEgC6ls0OHujgXXFwEU3UUUzeH/JeXQH7wAvh8f5nmICAGlL30fyDjX8irOX+OLiTJTV5PRJXu8oKhyQyiLf9OmNIrNhSXRc4LFsjSgCrlINCChsIsVihYbyAMGM0ci3G9vlmS9lty70KgtIAls7AlQRfS/oWoNCOJw1JvyjSkk2sxeTxfKTNJIs2+vmXTOJKuxUihR++DsiN0vLdFHEHzPvh8Pp/vYw9rk0kv+HsQ9gAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jiejun","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2024-11-05 04:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5392138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5392138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70477889,"identity":"075a5772-c31c-4c54-8d2c-5511e9b5b847","added_by":"auto","created_at":"2024-12-03 14:30:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19494080,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of unilateral ADD-induced minipig models.\u003c/p\u003e\n\u003cp\u003e(A) Scheme of the animal experimental design. (B) Intraoperative images and illustrations of the ADD-induced surgical procedures. (a, a’) Brief cranial anatomy of minipigs and the surgical site (red square). (b, b’) The inferior compartment of the TMJ was opened with a periosteal elevator burrowing into it. (c, c’) Part of the anterior and lateral attachment was loosened and penetrated with a 4-0 nylon suture. (d, d’) The anchorage system was implanted, and the nickel-titanium coil spring was tensioned to about 14 mm, generating 1 N force measured by an orthodontic spring dynamometer intraoperatively. (e, e’) The e-PTFE membrane was fixed with a bone nail, to \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cbr\u003e\n segregate the anchorage system and zygomatic arch.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/f95cabc2cc9209bfbd72edc3.png"},{"id":70477890,"identity":"327e3c14-b2e6-4e7c-9365-d964da1ded46","added_by":"auto","created_at":"2024-12-03 14:30:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24194505,"visible":true,"origin":"","legend":"\u003cp\u003eSurgical outcomes of ADD 5 weeks postoperatively.\u003c/p\u003e\n\u003cp\u003e(A) Weight gain during the 5-week observation period. (B) The intraoperative examination during the second surgery in the main study. The coil spring recovered from the elastic deformation (a, green arrow) to 5 mm with osseointegration forming over the e-PTFE membrane and not inside the nickel-titanium coil spring (b, blue arrow). (C) Gross examination from the side view and top view of the left-side TMJ in the ADD-EG (c, d), right-side TMJ in the CL-EG (e, f), and left-side TMJ in the CG (g, h). To maintain consistent orientation for better comparison, images (c) and (e) were horizontally flipped. (D) MRI scans of TMJs in the 3 groups. The red-shaded areas represent TMJ discs, and the yellow arrows point to the PAs, which were comparatively low-intensity in the EG (a, b). A, anterior; C, condyle; D, TMJ disc; L, lateral; M, medial; P, posterior.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/6f0348aa4a089fb564ff29fe.png"},{"id":70477888,"identity":"6bddad4a-ffa2-438f-8c37-891347a8c26d","added_by":"auto","created_at":"2024-12-03 14:30:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20696659,"visible":true,"origin":"","legend":"\u003cp\u003eHistological features of the TMJ disc complex from the CG.\u003c/p\u003e\n\u003cp\u003eBlood vessels are distributed in every portion of the TMJ disc complex (blue arrows). A, anterior; AA: anterior attachment; AP: anterior portion; I, inferior; IP: intermediate portion; P, posterior; PA: posterior attachment; PP: posterior portion; S, superior.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/0782f4b0596b0fbb557abfe2.png"},{"id":70478741,"identity":"4eeac6fa-b36b-47c0-90a3-fde86f4ae2f5","added_by":"auto","created_at":"2024-12-03 14:38:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18270445,"visible":true,"origin":"","legend":"\u003cp\u003ePseudo-disc changes of PAs in the EG with H\u0026amp;E (A) and AB (B) staining.\u003c/p\u003e\n\u003cp\u003e(C) Chondrocyte-like cells in the IP of the TMJ disc (green arrows). (D) The transition zone presented the transformation from fibroblasts to mature chondrocytes. (E) Fat cells distributed in or around the chondrocyte mass (red arrows). Deeply stained arterioles around the chondrocyte mass (yellow arrow). A, anterior; AA: anterior attachment; I, inferior; IP: intermediate portion; P, posterior; PA: posterior attachment; S, superior.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/a29c921890f52e5e6b043fa6.png"},{"id":70477885,"identity":"45f0f88a-7280-47fb-9a1d-a2f02cb2b4b4","added_by":"auto","created_at":"2024-12-03 14:30:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15903232,"visible":true,"origin":"","legend":"\u003cp\u003eThe ADD-induced surgery caused mild OA in the condyle cartilage.\u003c/p\u003e\n\u003cp\u003e(A) Minimal bone changes of the condyles measured by Micro-CT. (a) Representative image of the TMJ from the CL-EG. The orange region within 3D reconstruction represents the measurement range of subchondral bone depicting by the acid spray tool. (b) The condylar length and width at 5 weeks after the ADD-induced surgery. (c) Micro-CT assessment of the subchondral bone of TMJ condyles. (B) H\u0026amp;E staining and SO\u0026amp;FG staining of the condyles in the 3 groups. (j) The semi-quantitative analysis of cartilage thickness, safranin O + area, and OARSI grade in the 3 groups. A, anterior; P, posterior. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/6c953a48280b9b4052942973.png"},{"id":91105754,"identity":"99a90d12-149c-4ac1-bbf4-dab9684cc720","added_by":"auto","created_at":"2025-09-11 15:32:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":88446866,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/71a65903-d94d-4629-8b22-9f8ce2a3e35d.pdf"},{"id":70477886,"identity":"1ba307d5-ecfd-4a28-bd0c-0821847342e5","added_by":"auto","created_at":"2024-12-03 14:30:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14244794,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5392138/v1/48d5e92bb3d7a1355816434d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bilateral pseudo-disc changes in a minipig model for unilateral anterior disc displacement of the temporomandibular joint","fulltext":[{"header":"Background","content":"\u003cp\u003eAnterior disc displacement (ADD) is the most common subtype of temporomandibular disorder (TMD), characterized by joint clicking, limited mouth opening, and pain\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In adolescents, it can affect mandibular growth, leading to facial asymmetry and deformity, thus necessitating prompt attention\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. When ADD progresses to an advanced stage, over half of the patients tend to develop degenerative joint disease which is difficult to reverse\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, early intervention is limited due to a lack of understanding and suitable animal models for early-stage ADD.\u003c/p\u003e \u003cp\u003eSeveral animal models of ADD have been established, including mice, rats, and rabbits. Ali et al. induced ADD in rabbits within 24 hours by severing the discal attachments and pulling the discs anteriorly with sutures\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Nguyen et al. reported a surgical approach in rats that did not involve dissection of the discal attachments, achieving a success rate of 58.3%\u003csup\u003e6\u003c/sup\u003e. Gu et al. developed a minimally invasive surgery without opening the temporomandibular joint (TMJ) capsules in rabbits, with 15 out of 16 rabbits achieving ADD\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Despite modification to make surgical protocols minimally invasive, the use of sutures remained. Some studies identified the success of ADD induction by immediate verification during surgery, while such abrupt induction is unlikely in humans\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, Wu et al. used double-strand elastic rubbers and found that apoptosis-associated proteins such as Fas and caspase-8, increased after ADD within 4 weeks, implying chondrocytes loss in the condyle\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Almost all the ADD animal models developed severe TMJ osteoarthritis (OA) quickly, particularly the degeneration of condyle chondrocytes, inhibition of TMJ growth and mandibular deviation\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Clinically, however, patients with early-stage ADD do not typically present with serious OA. Meanwhile, a few studies have reported pseudo-disc changes in the posterior attachments (PAs) after ADD, characterized by chondrocytes and denser collagen, but evidence concerning a comprehensive view of the entire disc is absent\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eComparing the human TMJ to various species for biochemical and biomechanical properties, the minipig is often cited as the gold standard model for TMJ research\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. No previous study has induced ADD in minipigs. The aim of this study is to surgically induce unilateral ADD (U-ADD) in minipigs and investigate the early-stage changes associated with ADD. It is hypothesized that the early stage of ADD will exhibit minimal OA-like changes or mandibular underdevelopment. Additionally, the specific aims of the study were to investigate pseudo-disc changes in the PAs and explore whether these changes were reconstructive or degenerative.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eStudy\u0026nbsp;\u003c/em\u003e\u003cem\u003eD\u003c/em\u003e\u003cem\u003eesign\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSix female minipigs (6 months old, weighing 25 and 33 kilograms) were used, considering the susceptible age and gender for TMD (Fig. 1A). Two minipigs (4 joints) served as the control group (CG). Given the nature of the TMJ as a bilateral linkage joint, the left TMJs of the remaining 4 minipigs were chosen for ADD experimental group (ADD-EG), while the right TMJs were left untreated as contralateral experimental group (CL-EG). Five weeks after the initial surgery, a second surgery was performed to remove the nickel-titanium coil spring and other metal instruments used in the initial\u0026nbsp;U-ADD\u0026nbsp;surgery, allowing for in-vivo magnetic resonance imaging (MRI) scanning. Subsequently, all animals were humanely sacrificed, ensuring that each group had at least 3 samples available for analysis.\u003c/p\u003e\n\u003cp\u003eThe experimental protocols were approved by the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (IACUC-202306-17) and conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurgical\u0026nbsp;\u003c/em\u003e\u003cem\u003eP\u003c/em\u003e\u003cem\u003erotocols\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe lateral surface of the minipig\u0026rsquo;s head was shaved and sterilized. A 4-cm curvilinear incision was made with a #10 blade, aligned with the lateral canthus of the eye. An electrosurgical knife was used to control bleeding and incise through the subcutaneous tissue and periosteum, followed by reflecting the periosteum with periosteal elevators. The soft tissue lateral to the condylar head, composed of the TMJ capsule and ligament, was identified. An incision was then made through the anterolateral capsule, below the disc, to expose the inferior compartment of the TMJ (Fig. 1B(b, b\u0026rsquo;)). At this point, the anterior and lateral attachment was partially loosened, and the PA was not exposed and left intact.\u003c/p\u003e\n\u003cp\u003eAn orthodontic mini-implant (Ormco, Brea, California, USA) was screwed into the zygomatic arch, about 20 mm from the posterior margin of zygomatic arch. The anterolateral attachment, tightly linked to the disc, was penetrated by a round needle with a 4/0 nylon suture (Fig. 1B(c, c\u0026rsquo;)).\u0026nbsp;The suture was then knotted to a nickel-titanium coil spring (Shinye, Hangzhou, Zhejiang, China) which was stretched from 5 mm to 14 mm, and a ligature wire was pierced through to attach the coil spring to the mini-implant. Measured by an orthodontic spring dynamometer during the surgery, this setup created a tension force of approximately 1 N, ensuring tension in the anterior attachment and disc, without immediate visible disc displacement (Fig. 1B(d, d\u0026rsquo;)). After that, a non-absorbable membrane (INNOD, Yanan, Jilin, China), made from expanded polytetrafluoroethylene (e-PTFE) and titanium mesh,\u0026nbsp;was fixed on the zygomatic arch by a single bone nail, functioning as a barrier to avoid bone osseointegration within the nickel-titanium coil spring (Fig. 1B(e, e\u0026rsquo;)). Finally, the joint capsule and other incised tissues were closed in layers.\u003c/p\u003e\n\u003cp\u003eAfter a 5-week observation period, a brief second procedure was required to remove the metal implants for in-vivo MRI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSecond\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eurgery and MRI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the 5-week observation period, a brief second procedure was required to remove the metal implants for \u003cem\u003ein-vivo\u003c/em\u003e magnetic resonance imaging. The surgical wounds of all the pigs healed well, with no other symptoms. Following the initial operative incision, the soft tissue was dissected, and after detaching the periosteum of the zygomatic arch, the draw gear was exposed. All the metal components, including the nickel-titanium coil spring, mini-screw, bone nail, and e-PTFE membrane, were removed followed by wound closure.\u003c/p\u003e\n\u003cp\u003eShortly after the second surgery, all 6 pigs were maintained under anesthesia for\u003cem\u003e\u0026nbsp;in-vivo\u0026nbsp;\u003c/em\u003eMRI scans. Their snouts were ferruled using rubber bands, to ensure a closed-mouth position during examination. A 7 Tesla research MR scanner (Siemens Healthcare, Erlangen, Germany) equipped with a 1Tx/28Rx QED knee coil (Mayfield Village, OH, USA) was used for the scanning experiments. A Proton Density TSE MRI sequence was performed with the following parameters: echo time of 19 ms, repetition time of 5200 ms, and a voxel size of 0.3 \u0026times; 0.3 \u0026times; 2.5 mm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSpecimen\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ereparation and Micro-CT\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ecanning\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the MRI, all the animals were sacrificed. Intact TMJs were dissected, carefully preserving the discal attachments for gross examination to evaluate the disc-condyle relationship. Then, the TMJ discs and condyles were separated and fixed in 4% paraformaldehyde for 48 hours.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, the condyles were scanned using a micro-computed tomography (Micro-CT) scanner (Milabs, Houten, Netherlands) with specific parameters: 50 kV voltage, 0.24 \u0026mu;A current, 15 ms exposure time, angle speed 30 deg/s. Imalytics Preclinical 2.1 software was used for 3-dimensional reconstruction, condylar length and width were measured\u003csup\u003e16\u003c/sup\u003e.\u0026nbsp;A virtual acid was sprayed over the surface of condyles and the acid grew into the condyles by a 1.2 mm thickness. The region covered by this acid was considered to be subchondral bone. Bone volume fraction (BV/TV, %), bone surface/bone volume (BS/BV, %), bone mineral density (BMD, g/cm\u003csup\u003e3\u003c/sup\u003e), trabecular separation (Tb. Sp, mm) and trabecular thickness (Tb. Th, mm) were calculated (Supplementary material, Fig. S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistological\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eE\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003examination\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter taking micro-CT images, the condyles were decalcified with 15% EDTA for approximately 2 months. All the TMJ discs and condyles were dehydrated in an ascending graded alcohol series, then paraffin-embedded in the anterior-posterior direction and consecutively cut at 5 \u0026mu;m.\u003c/p\u003e\n\u003cp\u003eHematoxylin-eosin (H\u0026amp;E) staining and Safranin O/ fast green (SO\u0026amp;FG) staining were performed on the condyles. The thickness of the condylar cartilage, measured using SO\u0026amp;FG stained sections, was assessed 3 times in the functional slopes of the condyles and the average thickness was calculated for statistical analysis. The Osteoarthritis Research Society International (OARSI) scoring system was used to assess the severity of cartilage OA, based on SO\u0026amp;FG stained sections\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to H\u0026amp;E staining, Alcian blue (AB) staining was used on TMJ discs for chondrocyte characterization.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical\u0026nbsp;\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e\u003cem\u003enaly\u003c/em\u003e\u003cem\u003eses\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eResults are presented as the mean \u0026plusmn; standard deviation. Two-way analysis of variance was performed to compare the data of the 3 groups using GraphPad Prism software, paired t-test was used to compare the ADD-EG and CL-EG. Differences were considered significant at \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOf the 4 minipigs in the EG, 1 minipig exhibited a loosened suture with no obvious disc displacement, indicating a failed model. This resulted in final sample sizes of ADD-EG (n=3), CL-EG (n=3), and CG (n=4).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGross\u0026nbsp;\u003c/em\u003e\u003cem\u003eE\u003c/em\u003e\u003cem\u003evaluation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll the groups followed the same diet during the observation period and weight gains were observed (Fig. 2A).\u003c/p\u003e\n\u003cp\u003eDuring the second surgery, gross examination showed that the coil springs had recovered from elastic deformation, confirming that the anterior attachments were displaced and slackened (Fig. 2B(a)). Osseointegration on the surface of the zygomatic arch was observed above the membrane, but no bone had formed within the springs (Fig. 2B(b)).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter sacrifice, gross examination revealed that TMJ discs in the ADD-EG were positioned anteriorly compared to CL-EG, with the intermediate portion of the TMJ discs anterior to the transverse ridges of the condyles (Fig. 2C(d)). No morphological differences were observed among the 3 groups by visual inspection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMagnetic\u0026nbsp;\u003c/em\u003e\u003cem\u003eR\u003c/em\u003e\u003cem\u003eesonance\u0026nbsp;\u003c/em\u003e\u003cem\u003eI\u003c/em\u003e\u003cem\u003emaging\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2D shows the TMJ disc-condyle relationships by MRI in the 3 groups. Anterior disc displacement was confirmed in the ADD-EG, while the other groups maintained a normal disc-condyle relationship. TMJ discs in all groups retained double-concave shape but were elongated in the ADD-EG. In addition, all PAs in the EG (n = 6) presented lower signal intensity than the CG, suggesting the occurrence of pseudo-disc changes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHistological Results of TMJ Discs and PAs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the CG, collagen fibers oriented strictly in anterior-posterior direction in the intermediate portion of discs but were less organized in the anterior and posterior portions (Fig. 3). The anterior and posterior attachments exhibited a higher degree of multidirectional orientation with blood vessels and sparse pockets of fat vacuoles. A few chondrocyte-like cells were distributed in the TMJ discs and PAs, with lightly-staining intracellular matrix, and they were more frequent on the upper or lower surfaces of the intermediate portion of discs (Fig. 4C).\u003c/p\u003e\n\u003cp\u003eCompared to the CG, with an increased number of chondrocyte-like cells in the PAs, TMJ discs from the EG were less stained with AB staining broadly, but focal condensations were observed in the PAs (Fig. 4). In the center of the condensed mass, chondrocyte clusters were observed with hyperchromatism of the surrounding matrix. There was an apparent transitional zone around the condensed mass, showing evolution from hyperchromatic fibroblasts at the outermost edge to immature chondrocytes to mature cartilaginous tissue at the core (Fig. 4D). Typically, fat vacuoles and blood vessels were distributed in the periphery, but they could also exist within the mass occasionally (Fig. 4E). In conclusion, well-developed heterotopic chondrogenesis was observed in all PAs from EG, corresponding to the low intensity seen in MRI.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicro-CT\u0026nbsp;\u003c/em\u003e\u003cem\u003eS\u003c/em\u003e\u003cem\u003ecan\u003c/em\u003e\u003cem\u003ening\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe average condylar length of ADD-EG was 13.82 \u0026plusmn; 1.55 mm, lower than that of the CL-EG (14.60 \u0026plusmn; 2.09 mm) and CG (14.72 \u0026plusmn; 0.29 mm). The condylar width among the 3 groups showed basically no difference (Fig. 5A(b)). Micro-CT analysis showed generally similar values across the groups, indicating relatively normal subchondral bone.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHistological Results of Condyles\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5B shows an intact and continuous surface of the condyle in all 3 groups. Each condyle exhibited a clear hierarchy of cell layers in the anterior and middle regions, but the distribution became disordered in posterior regions including CG (Supplementary material, Fig. S2). Compared to the CG, condyles from the EG showed more hyperchromatic chondrocytes and more stained hypertrophic chondrocyte layers, suggesting a compensatory increase in cartilage matrix proteoglycan and an early-stage OA feature. Additionally, condyles from the EG showed increased cartilage thickness compared to the CG, but with no significant difference. SO\u0026amp;FG staining revealed a significant increase in OARSI grades in the EG, in accordance with the safranin O + area.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used a nickel-titanium coil spring to provide lasting traction to displace the TMJ disc anteriorly, as the elasticity of rubber might decay quickly in a complex body fluid environment. This traction simulated the hyperactivity of lateral pterygoid muscle and no visible disc displacement was detected during the U-ADD surgery, allowing us to mimic the natural course of human ADD to some degree. According to the pilot study, the unabsorbable e-PTFE membrane was necessary to prevent osseointegration within the nickel-titanium coil spring, which would otherwise prevent the spring from returning to its original shape, leading to failure of U-ADD (Supplementary material, Fig. S3). Considering the observation time of previous studies in small animals, a 5-week observation period was adopted. MRI assessment confirmed the U-ADD, and subsequent analysis showed no significant OA-like condylar bony changes and compensatory cartilage changes. Notably, bilateral pseudo-disc changes were observed in a U-ADD model for the first time.\u003c/p\u003e \u003cp\u003ePseudo-disc changes were first identified in human cadaveric histological analysis by Scalpino in 1983, who reported that the fibers formed a compact mass with reduced vessels in the thickened PA\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Leonardi et al. further added that islands of fibro chondrocytes also existed in the remodeled PA\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. With the development of ADD animal models, most studies observed degenerative changes in the PAs, such as disordered fibers, hyalinization and loss of adipose tissue, while a few studies reported the pseudo-disc changes, which might be related to variations in model establishment\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Gu et al. found chondrocyte-like cells in both TMJ discs and PAs, and observed immature chondroid metaplasia in 1 of 16 experimental rabbits 8 weeks after U-ADD surgery, but the contralateral side was not mentioned\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Partially aligned with this study, we observed that the chondrocytes in the PA gathered into a mass with a clear transition zone around it. The transition zone showed apparent transformation from fibroblasts to chondrocytes, suggesting mature chondroid metaplasia in the PA. In addition, the degenerative tendency of TMJ discs and PAs was not apparent in this study. With the abnormal stress on the contralateral side as indicated by finite element analyses, bilateral pseudo-disc changes occurred\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsidering the clinical outcomes of ADD, some patients develop severe degenerative changes leading to disc perforation over time, while others gradually attain a stable TMJ structure. We believe pseudo-disc change belongs to the latter category. While widespread ossification or chondrification within the joint is typically considered degenerative due to its tendency to restrict mandibular movement and generation of abnormal stress, our results suggest that heterotopic chondrogenesis confined to the PA, which might represent a new treatment avenue\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The PA is one of the most vulnerable sites of perforation, often associated with degenerative bony changes like osteophytes and flattening\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Radiological follow-up studies suggest that bony changes in the condyle tend to become steady when pseudo-disc changes occur\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Together with this study, it can be inferred that pseudo-changes are related to stabilized bony changes, although the cause-and-effect relationship is not yet fully understood. MRI seems to be the only method to detect pseudo-disc changes in the clinic, showing a hypointense lesion posterior to the disc in the sagittal plane, consistent with our results. Though no evidence links pseudo-disc changes to pain relief, it has been reported that high signal intensity of the PA is closely correlated with TMJ pain, possibly indicating increased vascularity or inflammation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In our study, without the observation of significant inflammatory cells or vascularization, heterotopic chondrogenesis was found in the PAs, which expressed increased collagen II and aggrecan, according to the study by Gu et al\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These results suggest that this change might optimize the mechanical properties of the original fibrocyte-composed PA, allowing it to better endure compressive stress. Considering the above, we hypothesis that inducing pseudo-disc changes could reshape the PA into fibrocartilage, allowing it to endure compressive stress during ADD. This may stabilize condylar cartilage, contribute to condyle repair, prevent disc perforation, and ultimately alleviate OA.\u003c/p\u003e \u003cp\u003eHowever, there were a few conflicts with conventional opinions in this study. Several radiological follow-up studies reported the occurrence rate of pseudo-disc change to be 8.9\u0026ndash;45% among TMD patients\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In this study, however, all the ADD-induced TMJs presented pseudo-disc changes, possibly due to the small sample size (n\u0026thinsp;=\u0026thinsp;3). With the disturbance in the direction of stress distribution through U-ADD, pseudo-disc changes in the contralateral sides (n\u0026thinsp;=\u0026thinsp;3) were observed as well, a finding not previously reported in humans. Furthermore, it is generally accepted that the reconstruction of the PA can take years, and pseudo-disc changes are considered a symbol of late-stage ADD\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Yet, in this study, these changes occurred within 5 weeks. Little research has been done on the pseudo-disc changes, and these questions need further study.\u003c/p\u003e \u003cp\u003eThe chondrogenic potential of fibroblasts or synoviocytes to spontaneously generate heterotopic chondrogenesis is reflected in diseases such as chondromatosis. With continuous overexpression of transforming growth factor-beta 1, Watson et al. observed aggressive fibrocartilaginous metaplasia in the knee joints of rats\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our studies observed heterotopic chondrogenesis in TMJs of minipigs and revealed the key role of mechanical stress in this change.\u003c/p\u003e \u003cp\u003eThis study had some limitations. According to the outcomes of pilot study and due to economic considerations, there was no sham surgery group set up in this study. A longer-term observation period with multiple observation timepoints is needed. The mechanical properties of the TMJ disc complex after pseudo-change have not been tested, and further studies are required.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study developed a U-ADD minipig model with bilateral pseudo-disc changes in the PAs, suggesting the key role of mechanical stress in these changes. These findings could provide potential insights for future ADD therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eADD: Anterior disc displacement\u003c/p\u003e\n\u003cp\u003eTMJ: Temporomandibular joint\u003c/p\u003e\n\u003cp\u003eOA: Osteoarthritis\u003c/p\u003e\n\u003cp\u003eCG: Control group\u003c/p\u003e\n\u003cp\u003eADD-EG: Anterior disc displacement experimental group\u003c/p\u003e\n\u003cp\u003eCL-EG: Contralateral experimental group\u003c/p\u003e\n\u003cp\u003eMRI: Magnetic resonance imaging\u003c/p\u003e\n\u003cp\u003ePA: Posterior attachment\u003c/p\u003e\n\u003cp\u003eTMD: Temporomandibular disorder\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eU-ADD: Unilateral anterior disc displacement\u003c/p\u003e\n\u003cp\u003eE-PTFE: Expanded polytetrafluoroethylene\u003c/p\u003e\n\u003cp\u003eMicro-CT: Micro-computed tomography\u003c/p\u003e\n\u003cp\u003eBV/TV: Bone volume fraction\u003c/p\u003e\n\u003cp\u003eBS/BV: Bone surface/bone volume\u003c/p\u003e\n\u003cp\u003eBMD: Bone mineral density\u003c/p\u003e\n\u003cp\u003eTb. Sp: Trabecular separation\u003c/p\u003e\n\u003cp\u003eTb. Th: Trabecular thickness\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E: Hematoxylin-eosin\u003c/p\u003e\n\u003cp\u003eSO\u0026amp;FG: Safranin O/ fast green\u003c/p\u003e\n\u003cp\u003eAB: Alcian blue\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocols were approved by the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (IACUC-202306-17). Clinical\u0026nbsp;trial\u0026nbsp;number: not applicable. Consent to Participate declaration: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 82170984), Research and development Foundation of Stomatology Hospital in Zhejiang University (RD2023YYYB01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eW.L. contributed to conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; X.B. contributed to design, data acquisition and interpretation, critically revised the manuscript; W.Y. contributed to data acquisition, analysis and interpretation, critically revised the manuscript; C.W., Y.H., J.Y. and Y.Z. all contributed to data acquisition, critically revised the manuscript; J.S. contributed to conception and design, critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the Zhejiang University 7T Brain Imaging Research Center and technician Bin Xu for their assistance with the in-vivo MRI in minipigs.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePoluha RL, Canales GD la T, Costa YM, Grossmann E, Bonjardim LR, Conti PCR. Temporomandibular joint disc displacement with reduction: a review of mechanisms and clinical presentation. \u003cem\u003eJ Appl Oral Sci\u003c/em\u003e 2019;\u003cstrong\u003e27\u003c/strong\u003e:e20180433. https://doi.org/10.1590/1678-7757-2018-0433\u003c/li\u003e\n\u003cli\u003eSilva MAG, Pantoja LLQ, Dutra-Horstmann KL, Valladares-Neto J, Wolff FL, Porporatti AL, Guerra ENS, De Luca Canto G. 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Gene delivery of TGF-\u0026beta;1 induces arthrofibrosis and chondrometaplasia of synovium in vivo. \u003cem\u003eLab Invest\u003c/em\u003e 2010;\u003cstrong\u003e90\u003c/strong\u003e(11):1615\u0026ndash;27. https://doi.org/10.1038/labinvest.2010.145\u003c/li\u003e\n\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":"biomechanics, chondrogenesis, fibrocartilage, temporomandibular disorder, temporomandibular joint disc","lastPublishedDoi":"10.21203/rs.3.rs-5392138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5392138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eCurrent animal models of anterior disc displacement (ADD) of the temporomandibular joint (TMJ) have primarily been developed in small animals, using sutures or elastic bands to pull the disc anteriorly. These models often exhibit severe osteoarthritis (OA) and rapid progression to late-stage ADD, which do not accurately reflect the natural course of ADD in humans. This study aimed to establish a unilateral ADD-induced surgical protocol in a minipig model and evaluate the early changes of ADD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSix Bama minipigs (12 joints) were divided into 3 groups: control group (CG), ADD experimental group (ADD-EG), and contralateral experimental group (CL-EG). Nickel-titanium coil springs were used to induce mild, sustained traction to anteriorly displace the TMJ discs. After a 5-week observation period, in-vivo magnetic resonance imaging (MRI) scans confirmed the anterior position of the TMJ discs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMicro-computed tomography showed minimal condylar bone changes, while histological analysis revealed mild OA-like condylar cartilage changes. MRI identified hypointense lesions posterior to the TMJ discs, indicative of pseudo-disc changes in the posterior attachments (PAs). Histological staining further confirmed chondroid metaplasia in the PAs surrounded by transitional zones in both ADD-EG and CL-EG.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study successfully established a unilateral ADD-induced minipig model and identified pseudo-disc changes in the PAs under abnormal mechanical stress, which may offer new perspectives for ADD therapies. To our knowledge, this is the first study to observe bilateral pseudo-disc changes in a unilateral ADD-induced model.\u003c/p\u003e","manuscriptTitle":"Bilateral pseudo-disc changes in a minipig model for unilateral anterior disc displacement of the temporomandibular joint","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-03 14:30:51","doi":"10.21203/rs.3.rs-5392138/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":"6aab1422-10b2-4ffd-8544-1335e25ecde2","owner":[],"postedDate":"December 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-19T22:38:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-03 14:30:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5392138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5392138","identity":"rs-5392138","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}