Effects of Intra-articular Infiltration of Proteoglycan 4 / Lubricin in Temporomandibular Joint Osteoarthritis: An Experimental Study

Effects of Intra-articular Infiltration of Proteoglycan 4 / Lubricin in Temporomandibular Joint Osteoarthritis: An Experimental Study | 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 Effects of Intra-articular Infiltration of Proteoglycan 4 / Lubricin in Temporomandibular Joint Osteoarthritis: An Experimental Study Veronica Iturriaga, Bélgica Vásquez, Schilin Wen, Thomas Bornhardt, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6234556/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 : The proposed research aims to evaluate the histopathological effects of intra-articular administration of recombinant human proteoglycan 4 (rhPRG4) on articular cartilage and articular disc in an experimental model of induced temporomandibular joint osteoarthritis (TMJ-OA) in rabbits. Materials and Methods: An experimental study was conducted on twenty-four rabbits’ TMJs, distributed in: 1) TMJ-C, control group consisting of 4 healthy TMJs; 2) TMJ-OA, group consisting of 5 TMJs with OA; 3) TMJ-OA-WT, group consisting of 5 untreated TMJ-OAs; 4) rhPRG4-30, group consisting of 5 ATM with OA treated with lubricin 30 μg /ml; and 5) rhPRG4-100, group consisting of 5 ATM with OA treated with lubricin 100 μg/ml. A histopathological analysis was performed, considering the mandibular condyle, articular disc, and mandibular fossa, comparing the groups. In addition, a quantitative comparative analysis was performed using the Osteoarthritis Research Society International (OARSI) scale. The Kruskal-Wallis and Dunn's post hoc tests were used for statistical analysis, considering a statistical significance of p < 0.05. Results: Descriptive histological analysis of TMJ joint tissue reveals key differences between healthy, osteoarthritic, and rhPRG4-treated joints. The analysis also suggests beneficial treatment effects with intra-articular infiltration of rhPRG4, reducing the severity of osteoarthritis. Conclusions : Intra-articular infiltration of rhPRG4 in TMJ-OA has a cartilage and articular disc repair effect, reducing the severity of osteoarthritis and promoting a more organized cartilage structure, with slightly better results at the 30 μg/ml concentration. Clinical Relevance: Intra-articular infiltration of rhPRG4 is proposed as a new therapeutic alternative in TMJ-OA, enhancing joint tissue regeneration. temporomandibular joint temporomandibular joint disorders osteoarthritis lubricin proteoglycan 4 cartilage Figures Figure 1 Figure 2 Figure 3 INTRODUCTION The therapeutic use of regenerative medicine or tissue engineering has been increasing in pathologies such as osteoarthritis (OA) of the temporomandibular joint (TMJ). Tissue engineering is based on replacing, manufacturing, or regenerating human cells, tissues, or organs to restore or establish their normal function [ 1 , 2 ]. In its beginnings, regenerative medicine in TMJ-OA was based on implanting tissues into the joint to promote its repair. However, it was necessary to perform joint surgery to introduce the material. Over time, the technique evolved towards infiltrating liquid or semi-liquid substances of different viscosities, thus avoiding surgery [ 1 ]. Currently, the therapeutic use of regenerative medicine in TMJ-OA is mainly represented by the infiltration of hyaluronic acid (HA), platelet-rich plasma (PRP) or growth factors (GF), and stem cell-based therapies. Of these alternatives, HA and PRP present more significant evidence and are used in daily practice, while the evidence for stem cell therapy is still developing [ 3 – 7 ]. HA is a key lubricant component of synovial fluid in the TMJ. When used as a treatment, it presents essential evidence of its anti-arthritic effect on joint tissues [ 3 , 6 , 8 ]. Another lubricating molecule that may have reparative or regenerative effects within the components of synovial fluid in TMJ-OA is proteoglycan 4 (PRG4), also known as lubricin. PRG4 is a high molecular weight mucinous glycoprotein produced by B-type synoviocytes and chondrocytes of the superficial zone of articular cartilage. This protein is found in the synovial fluid (SF) of synovial joints [ 9 ]and plays a crucial role in joint lubrication, synovial homeostasis, immunomodulation, and suppression of inflammation [ 10 – 13 ]. PRG4 contains core 1 O glycosylations that provide its lubricating function, enabling it to perform multiple essential functions in joints [ 14 ]. In addition, it protects cartilage by preventing the deposition of abrasive proteins and facilitates SF energy absorption and dissipation, thus protecting cartilage from mechanical damage [ 15 ]. It has also demonstrated anti-proliferative effects, preventing synovial hyperplasia, although the specific mechanisms are not yet fully understood [ 16 ]. Recent studies have suggested that PRG4 deficiency is associated with cartilage destruction, and a decrease in PRG4 may be associated with the development of OA. This ability of PRG4 to reduce friction and protect cartilage indicates that it could be a promising therapeutic option for this pathology, providing adequate lubrication and long-term protection against joint degeneration [ 10 , 17 ]. Intra-articular administration of PRG4 has shown promising results in animal models of OA. Different ways of obtaining PRG4 have been described, some related to lubricin biosynthesis via synoviocytes, purified human lubricin, or mostly to full-length recombinant human PRG4 (rhPRG4). Early evidence was observed in rat knees, where intra-articular injection of PRG4 showed significant improvement in reducing joint friction and inhibiting cartilage degeneration [ 10 , 13 , 18 , 19 ]. Also, rhPRG4 has been shown to have anti-inflammatory effects in synovial joints. One of these pathways is associated with the inhibition of fibroblast-like synoviocyte proliferation [ 20 , 21 ] or the ability to bind and antagonize Toll-like receptors, decreasing the activation of nuclear factor kappa B (NF-κB) and inflammatory cytokines [ 22 ]. On the other hand, a correlation between increased plasma inflammatory cytokines, such as Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor alfa (TNF-α), and decreased plasma PRG4 has been demonstrated [ 23 ]. Elevated levels of inflammatory cytokines could trigger molecular processes that decrease PRG4 expression, thus favoring the development of OA. An IL-1 receptor antagonist somewhat restores PRG4 expression in articular cartilage, providing evidence for a correlation between proinflammatory cytokines and PRG4 expression [ 24 ]. In preclinical animal models of joint inflammation, a decrease in PRG4 concentration in the SF after joint injury is observed, which is associated with increased damage to the cartilage surface [ 25 ]. In patients with acute knee injuries, PRG4 levels in the SF decrease and return to homeostatic levels generally within one year after injury [ 17 ]. Concerning the TMJ, PRG4 expression has been characterized in healthy and OA articular cartilage. In healthy cartilage, PRG4 is mainly expressed in the superficial zone of the cartilage and, to a lesser extent, in the medial zone. In cartilage with TMJ-OA, no expression is observed in the medial zone, and expression in the superficial zone is decreased [ 26 – 29 ]. In addition, in PRG4-/- rats, changes in the TMJ tissue were seen from 2 months of development, whereas at 6 months, osteoarthritic degradation is evident in the joint tissue [ 30 ]. Despite advances in understanding OA and the therapeutic potential of PRG4, significant work remains to advance this potential in the clinic. Specifically, no studies evaluate the effects of PRG4 administration in TMJ-OA, a condition with unique pathophysiological characteristics due to the complexity of its structure and function. The proposed research aims to evaluate the histopathological effects of intra-articular administration of rhPRG4 on articular cartilage and articular disc in an experimental model of induced TMJ-OA in rabbits. MATERIALS AND METHODS Animals An experimental study was conducted on 12 healthy male Oryctolagus cuniculus rabbits weighing approximately 3 kg and eight months of age, according to the recommendations described by Poole et al. (2010) [ 38 ]. The animals were kept in a controlled environment regarding temperature, environmental noise, and a 12-hour light/12-hour dark cycle. The animals were housed in cages individually and randomly assigned to each group. Simple random sampling was used to allocate the animals to the experimental groups; each animal was assigned a number. The animals were under the care of a veterinarian, who was aware of the group assignment in the different phases of the experiment. Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and the National Research Council Guide for the Care and Use of Laboratory Animals [ 39 ] were followed. The study was performed at the experimental surgery unit of the Center of Excellence in Morphological and Surgical Studies of the Universidad de La Frontera, Chile, with the approval of the Scientific Ethics Committee of the Universidad de La Frontera (File Number 004/21, ACT NO. 027_21). The two TMJs of each animal were considered to reduce the sample size and meet the experimental criteria established by Russell and Burch [ 40 ]. A sample size calculation was performed considering up to 10% losses and a statistical significance of p < 0.05. According to the above, twenty-four TMJs were included, distributed in: 1) TMJ-C, control group consisting of 4 healthy TMJs; 2) TMJ-OA, group consisting of 5 TMJs with OA; 3) TMJ-OA-WT, group consisting of 5 untreated TMJ-OAs; 4) rhPRG4-30, group consisting of 5 ATM with OA treated with rhPRG4 at 30 µg /ml; and 5) rhPRG4-100, group consisting of 5 ATM with OA treated with rhPRG4 100 µg/ml. The distribution of the groups is shown in Fig. 1 . Protocols for TMJ-OA induction, intra-articular rhPRG4 administration, histological processing, and histological analysis were performed according to previously described protocols [ 3 , 3 , 4 , 35 , 41 , 42 ]. TMJ-OA induction Except for the TMJ-C group, TMJ-OA induction was performed in all groups. Animals were anesthetized intramuscularly with ketamine (40 mg/kg), xylazine (5 mg/kg), and acepromazine (1 mg/kg), and the TMJ area was shaved and disinfected with 70% ethyl alcohol. At that time, 50 µL of sodium mono-iodoacetate (MIA) at a concentration of 3 mg/mL was infiltrated into the joint space with a 22-gauge needle. A period of 50 days was allowed to elapse to develop TMJ-OA [ 41 , 43 ]. After a 50-day waiting period, the animals in the TMJ-OA group were euthanized and subsequently analyzed. In the TMJ-OA-WT, rhPRG4-30, and rhPRG4-100 groups, after the initial 50-day period, an additional 30 days were taken before euthanasia and analysis (Fig. 3 ). Intra-articular rhPRG4 administration Intra-articular rhPRG4 administration was conducted using the same protocol for the anesthesia and previously mentioned preoperative measures. The infiltration technique was standardized, considering the caudal margin of the orbital lamina as the anatomical reference point. It was directed 5 mm caudally and 1 mm ventrally, with the needle at a 45º angle towards the ventral concerning the skin [ 9 ]. In the rhPRG4-30 group, 0.1 mL of rhPRG4 at 30 µg/ml (Lµbris BioPharma®) was injected into the joint space with a 22 gauge needle. Similarly, in the rhPRG4-100 group, 0.1 mL of rhPRG4 at 100 µg/ml (Lµbris BioPharma®) was injected. Histological processing Once the experimental protocol was completed, the animals were sacrificed, and the joint tissue was dissected. No animal losses, adverse effects, or modifications were reported. Samples were coded to maintain process masking during evaluation. The tissue was fixed with 10% buffered formalin (1.27 mol/L formaldehyde in 0.1 M phosphate buffer, pH 7.2) for 48 hours. TMJs were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) (in 0.1 M phosphate buffer 7–8)35 in ultrasonic decalcification (Use 33, Medite, Burgdorf, Germany) for 30 days. Subsequently, samples were dehydrated in an ascending alcohol battery, rinsed in xylene, and embedded in Paraplast Plus (Sigma-Aldrich Co., St. Louis, MO, USA). Serial sections of the TMJs were cut in the parasagittal plane at a thickness of 5 µm using a Leica® RM 2255 microtome. To optimally grade the OA, successive sections of the deeper planes of the joint were stained and visualized under a light microscope. Then, one section per joint was selected for the more detailed analysis, considering the plane of the block that crosses the lesion to the greatest extent and shows the most pronounced alterations [ 34 ]. Histological sections were stained with Toluidine Blue. An optical microscope (Leica DM 2000 LED, Wetzlar, Germany) was used for visualization, and the slides were photographed with a digital camera (Leica high-definition MC 170, Wetzlar, Germany). To maintain blinding in the analysis, the slides were also coded. Histological analysis Histological slides were randomly analyzed for each TMJ to compare the different groups. A descriptive analysis of the mandibular condyle (MC), articular disc (AD), and mandibular fossa (MF) was performed. The cartilage was described from superficial to deep, beginning with the superficial zone (SZ), also known as the tangential zone, followed by the mid-zone (MZ), also called the transitional zone; the deep zone or radial zone (DZ); calcified cartilage; and subchondral trabecular bone. Regarding AD, the central zone (CZ) at its thinnest point and the anterior and posterior peripheral zone (PZ) areas were analyzed. Authors V.I. and B.V. independently and blindly performed the quantitative histological analysis of each sample. In the case of disagreements, these were discussed until reaching a consensus. The interobserver calibration was performed in three phases before analyzing the samples: theoretical training, laboratory training, and calibration (final inter-agreement kappa 0.865). The OARSI scale, was used to grade the articular cartilage grade and stage for both MC and MF. This scale classifies the different cartilage states, where grade 0 corresponds to normal tissue and grades 1 to 6 are relative to OA. Grades 1 to 4 of OA involve changes in cartilage only, while grades 5 and 6 also include subchondral bone. The OA stratification method, also defined by the OARSI scale, categorizes the disease into four stages based on the horizontal extent of the affected cartilage surface, regardless of the degree of underlying OA. Stage 1 represents less than 10% involvement; stage 2 represents 10 < 25% involvement; stage 3 represents 25–50% involvement; and stage 4 represents greater than 50% involvement. MC and MF cartilage were evaluated separately for each slide in each group [ 34 , 35 ]. Statistical analysis Quantitative analysis was performed by calculating the median and interquartile range of the scores obtained on the OARSI scale. Nonparametric inferential statistics were performed to compare the degree and stage of TMJ-OA between groups using Kruskal-Wallis and Dunn's post hoc tests. The analysis was performed with the STATA 18 program, considering a significance level of α = 0.05. RESULTS Descriptive histological analysis of articular cartilage in the rabbit temporomandibular joint reveals key differences between healthy and osteoarthritic joints. In the control group (TMJ-C), the cartilage was smooth, continuous, and had three well-demarcated zones: the superficial zone (SZ), the middle zone (MZ), and the deep zone (DZ), the latter being the thickest. In contrast, in the TMJ-OA group, a focal discontinuity on the surface and a generalized irregularity was observed, in addition to a thinning of the cartilage and a more fibrous matrix compared to the TMJ-C group. Deep fibrillations and proteoglycan depletion in the matrix were hallmarks of the TMJ-OA group, with hypertrophic chondrocytes and disorganization of collagen fibers. These structural differences suggest significant alterations associated with osteoarthritis in the TMJ-OA group (Table 1 ). Table 1 Histological characteristics of the temporomandibular joint tissue according to the groups under study. Structure TMJ-C TMJ-OA TMJ-OA-WT rhPRG4-30 rhPRG4-100 Mandibular condyle SZ Smooth and continuous surface. Flattened surface cells and a thin layer of fibrous connective tissue. Abrasion, matrix focal discontinuity. Fibrillations towards the deep zone. Abrasion, matrix focal discontinuity. Fibrillations towards the deep zone covering the entire articular cartilage. Slightly irregular surface. Abrasion of the surface layer in some areas. Small and flat or round chondrocytes, aligned parallel to the collagen fibers and the surface. Slightly irregular surface. In some sectors it is possible to observe abrasion. Small and flat or round cells. In some samples it is observed, focally, areas of collagen condensation whose fibers are directed to the middle and / or deep zone. MZ Undifferentiated cells and spherical chondrocytes in a proteoglycan matrix. Reduced cellularity with deep fibrillations. Reduced cellularity, more deep and higher density fibrillations. Proliferation of chondrocytes, arranged in isolation. Anisocytosis and proliferation of undifferentiated cells. Clusters of chondrocytes are seen in some samples. Matrix rarefaction, with areas of increased cationic staining around the chondrons. Condensation of collagen fibers. DZ It presents round and larger chondrocytes, organized in isogenic groups. Deep hypertrophic chondrocytes are observed. Less cellularity. Hypertrophic chondrocytes, forming clusters. Rarefaction and condensation of collagen fibers. Less cellularity and chondrocyte clusters. Rarefaction and condensation of collagen fibers. Vestiges of deep fibrillations. Focal rarefaction, increased collagen formation and cationic staining around the chondrons. Some hypertrophic chondrocytes. Loss of orientation of the chondrons in a disorganized matrix. Increased density of chondrocytes, with decreased cell size. Vestiges of deep fibrillations. Articular disc CZ Chondrocyte stacking and arrangement in parallel (rows) as to collagen fibers. Focal edema. Increased number and density of collagen fibers. Disorganized fibers. Less cellularity, with randomly arranged hypertrophic chondrocytes. Focal edema. Increased number and density of collagen fibers. Disorganized fibers. Less cellularity, with randomly arranged hypertrophic chondrocytes. Collagen fibers arranged in parallel with chondrocytes aligned to them. Chondrocytes are found within cartilage matrix. Disorganization of collagen fibers and edema. Less cellularity, with some chondrocytes arranged randomly and others parallel to the collagen fibers. PZ More abundant and dense chondrocyte rows. Randomly arranged hypertrophic chondrocytes within disorganized collagen fibers. Presence of connective tissue with abundant fibroblasts. Less peripheral connective tissue. Hypertrophy of the synovial membrane. Randomly arranged hypertrophic chondrocytes within disorganized collagen fibers. Some hypertrophic chondrocytes randomly arranged among disorganized collagen fibers. Mandibular fossa SZ Fibrous connective tissue, collagen fibers parallel to the surface with intermingled fibrocytes. Thickness, fiber disorganization, and edema. Rarefaction. Fibrillations that reach the deep zone of the cartilage. Thickness, fiber disorganization and rarefaction. Fibrillations that reach the deep zone of the cartilage. Slightly irregular surface. The limits between the SZ and MZ are not very evident. Hypocellularity. Slightly irregular surface. Unclear boundaries between SZ and MZ. Scarce cellularity with anisocytosis. MZ Undifferentiated cells. Diffuse, with reduced thickness and scarce cellularity. Diffuse, reduced thickness and scarce cellularity. Less thickness and hypocellularity, with rarefaction and edema. Heterogeneous matrix, with focal edema, low cellularity and anisocytosis. DZ Chondrocytes immersed in matrix rich in collagen fibers. Clusters of hypertrophic chondrocytes. Rarefaction and condensation of collagen fibers. Presence of deep fibrillations. Deep fibrillations. rarefaction and collagen condensation. Clusters of hypertrophic chondrocytes. Osteoarthritic features are observed, similar to MZ. Heterogeneous matrix, traces of focal fibrillations. Small chondrocytes distributed mainly in isolation. TMJ-C: healthy temporomandibular joints as a control group; TMJ-OA: osteoarthritic temporomandibular joint; TMJ-OA-WT: osteoarthritic temporomandibular joint untreated and evaluated 30 days after treatment period; rhPRG4-30: osteoarthritic temporomandibular joint treated with lubricin 30 µg/mL and assessed 30 days after treatment; rhPRG4-100: osteoarthritic temporomandibular joint treated with 100 µg/mL and evaluated 30 days after treatment; SZ: superficial zone; MZ: mid zone; DZ: deep zone; CZ: central zone; PZ: peripheral zone. Descriptive histological analysis of the rabbit osteoarthritic temporomandibular joint suggests beneficial treatment effects by intra-articular administration of rhPRG4. Comparing the rhPRG4-30 and rhPRG4-100 groups with the TMJ-OA and TMJ-OA-WT groups, key differences in the condition of articular cartilage and synovial membrane in the TMJ are highlighted. In the TMJ-OA groups, discontinuity of the cartilage surface, thinning, a more fibrous matrix with deep fibrillations, and signs of proteoglycan depletion, chondrocyte loss, and disorientation were observed. The TMJ-OA-WT group showed greater disease progression with deeper and denser fibrillations. Both groups exhibited synovial membrane thickening due to the increased presence of inflammatory cells and hyperplasia of synovial cells with an increased size (Fig. 2 ). In contrast, in the rhPRG4-30 and rhPRG4-100 treated groups, repair processes were observed in the articular cartilage, manifesting in a more organized appearance with increased thickness, more uniform surface, and increased cationic staining of the matrix, along with less alteration in the synovial membrane compared to the TMJ-OA and TMJ-OA-WT groups. In the rhPRG4-30 group, some abrasion areas were observed in the SZ. In the MZ of the mandibular condyle, the proliferation of chondrocytes arranged in an isolated manner was observed, suggesting an ongoing repair process. In addition, traces of deep fibrillations were seen in the DZ, indicating an attempt at cartilage tissue regeneration. The articular cartilage of the rhPRG4-100 group also exhibited a repair process; however, increased collagen condensation and fibrillation were noted in deeper areas (Fig. 2 ). Both groups demonstrated a potential reparative effect with rhPRG4 treatment in the TMJ affected by OA. Notably, the rhPRG4-30 group showed a slight improvement in cartilage regeneration and organization compared to the rhPRG4-100 group (Table 1 ) (Fig. 2 ). Intra-articular administration of rhPRG4 as a treatment reduces the severity of osteoarthritis in the articular cartilage of the rabbit temporomandibular joint. The OARSI score of articular cartilage in TMJs revealed that both MC and MF exhibited the highest degree of OA in the TMJ-OA and TMJ-OA-WT groups (Fig. 3 ). Specifically, the rhPRG4-30 and PRG4 -100 groups showed a significant reduction in the severity of OA in the articular cartilage of the MC and MF compared to the TMJ-OA and TMJ-OA-WT groups (Fig. 3 A and B). The TMJ-OA group had no significant difference in OA severity compared to the TMJ-OA-WT group in all structures analyzed (Fig. 3 ). Likewise, the rhPRG4-30 and rhPRG4-100 groups also showed no significant differences in OA severity in both MC and MF (Fig. 3 A and B). Finally, significant differences in OA stage were only observed between the untreated and rhPRG4-100 groups (Fig. 3 D). These results suggest that intra-articular infiltration of rhPRG4 in osteoarthritic TMJs beneficially affects articular cartilage by reducing the severity of osteoarthritis and promoting a more organized cartilage structure. DISCUSIÓN The present study is the first to investigate the effects of intra-articular infiltration of rhPRG4 on induced TMJ-OA in rabbits. The results obtained are promising and suggest that rhPRG4 may significantly reduce the severity of OA. Histological analysis revealed significant improvements in the articular cartilage structure of rabbits with TMJ-OA treated with rhPRG4 compared to untreated groups. The rhPRG4-30 and rhPRG4-100 groups showed a more uniform cartilage surface, a more organized matrix, and increased cartilage thickness. These improvements are consistent with previous studies in the knee that have demonstrated beneficial effects of rhPRG4 in animal models of OA [ 10 , 18 ]. The articular disc also showed significant improvements in the rhPRG4-treated groups. In the untreated groups, the articular disc showed signs of degeneration, such as thinning, matrix disorganization, and proteoglycan loss, consistent with the TMJ-OA literature [ 31 ]. In contrast, the rhPRG4-treated groups showed a more uniform and thicker disc structure, suggesting a reparative effect of rhPRG4. The observed efficacy of rhPRG4 in improving TMJ articular cartilage structure and protecting the articular disc is attributed to its multiple biological functions. rhPRG4 plays a crucial role in lubricating articular surfaces, significantly reducing friction, which protects cartilage from mechanical wear and tear [ 32 ]. Furthermore, the ability of rhPRG4 to prevent chondrocyte apoptosis, as Waller et al. demonstrated, is essential for maintaining cell viability in OA-damaged cartilage [ 33 ]. rhPRG4 also possesses immunomodulatory properties that can reduce inflammation in the affected joint, a crucial factor in the progression of OA [ 13 ]. Chronic inflammation in OA contributes to cartilage degradation and joint dysfunction. By modulating the immune response and reducing inflammation, rhPRG4 protects existing cartilage and promotes a favorable environment for extracellular matrix repair and regeneration. An interesting observation is comparing rhPRG4 concentrations of 30 µg/ml and 100 µg/ml. Although both groups improved cartilage and articular disc structure, the rhPRG4-30 group showed slightly better regeneration and more coherent matrix organization than the rhPRG4-100 group. This finding suggests that a lower concentration of rhPRG4 may be more effective for cartilage repair, possibly due to a better balance between lubrication and stimulation of matrix synthesis [ 10 , 18 ]. However, further studies are required to find the optimal concentration. The results of this study show a reduction in the degree of OA according to the OARSI classification and a decrease in the extent of the lesion in a dose-dependent manner. The OARSI classification evaluates grade (cartilage quality) and stage (extent of damage), allowing us to differentiate between these aspects [ 34 , 35 ]. rhPRG4 appears to be effective in improving cartilage quality, reducing inflammation, and promoting extracellular matrix synthesis [ 20 , 21 , 36 ]. This targeted action is in line with previous studies [ 10 , 18 ] that have demonstrated the ability of rhPRG4 to improve the structural and functional integrity of existing cartilage. Currently, treatments for TMJ-OA focus primarily on symptom relief through non-steroidal anti-inflammatory drugs, corticosteroids, and physical therapies, but these approaches do not halt or reverse joint degeneration [ 37 ]. PRG4 administration represents a novel therapeutic strategy that could provide benefits beyond simple symptom relief. The results of this study suggest that rhPRG4 not only reduces joint friction and protects cartilage but also promotes repair and regeneration processes in damaged cartilage. This could have a significant impact on improving joint function and reducing long-term pain in patients with TMJ-OA. Although the results are promising, this study has some limitations that should be considered. Firstly, the experimental model was based on rabbits, and although these animals are suitable for preclinical studies, the results cannot be directly extrapolated to humans. As background, rhPRG4 has been used clinically to manage dry eye disease in humans. Therefore, the potential for rapid translational uptake is significant. On the other hand, a vehicle-only group was not included to restrict the size of the study and the number of animals used. In addition, the study focused on a relatively short period of treatment and follow-up, so long-term studies are required to assess the sustainability of the beneficial effects of PRG4. Further studies using advanced imaging techniques and molecular analysis are also needed to deepen the understanding of the mechanisms behind the efficacy of rhPRG4. These techniques may provide more detailed insight into how PRG4 affects cartilage architecture and cellular dynamics at the microstructural level. In addition, research exploring combinations of PRG4 with other therapeutic agents, such as hyaluronic acid or growth factors, could offer synergistic approaches to maximize joint repair and prevent OA progression. Declarations ROLE OF THE FUNDING SOURCE The study was financed by Project DI20-0018 and the Temporomandibular Disorder and Orofacial Pain Program, Universidad de La Frontera, Chile. The funding bodies did not participate in the conception, design, or execution of the project, which was carried out independently. CONFLICT OF INTEREST: The authors declare that Lµbris BioPharma® made recombinant human PRG4 available for research. However, it was not involved in the article's development, analysis, and preparation. DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS During the preparation of this work the authors used Grammarly Inc. in order to revise the drafting of the manuscript. After using this tool, the authors reviewed and edited the content as needed and takes full responsibility for the content of the publication. Competing Interests The authors declare that Lµbris BioPharma® made recombinant human PRG4 available for research. 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Arthritis Res Ther 23(1):241. 10.1186/s13075-021-02621-9 Elsaid KA, Fleming BC, Oksendahl HL, Machan JT, Fadale PD, Hulstyn MJ et al (2008) Decreased lubricin concentrations and markers of joint inflammation in the synovial fluid of patients with anterior cruciate ligament injury. Arthritis Rheum 58(6):1707–1715. 10.1002/art.23495 Jay GD, Fleming BC, Watkins BA, McHugh KA, Anderson SC, Zhang LX et al (2010) Prevention of cartilage degeneration and restoration of chondroprotection by lubricin tribosupplementation in the rat following anterior cruciate ligament transection. Arthritis Rheum 62(8):2382–2391. 10.1002/art.27550 Teeple E, Elsaid KA, Jay GD, Zhang L, Badger GJ, Akelman M et al (2011) Effects of supplemental intra-articular lubricin and hyaluronic acid on the progression of posttraumatic arthritis in the anterior cruciate ligament-deficient rat knee. Am J Sports Med 39(1):164–172. 10.1177/0363546510378088 Al-Sharif A, Jamal M, Zhang LX, Larson K, Schmidt TA, Jay GD et al (2015) Lubricin/Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of the Suppression of Proinflammatory Cytokine-Induced Synoviocyte Proliferation by Lubricin. Arthritis Rheumatol 67(6):1503–1513. 10.1002/art.39087 Alquraini A, Jamal M, Zhang L, Schmidt T, Jay GD, Elsaid KA (2017) The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes. Arthritis Res Ther 19(1):89. 10.1186/s13075-017-1301-5 Alquraini A, Garguilo S, Souza GD, Zhang LX, Schmidt TA, Jay GD et al (2015) The interaction of lubricin / proteoglycan 4 (PRG4) with toll-like receptors 2 and 4: an anti- inflammatory role of PRG4 in synovial fluid. Arthritis Res Ther1–12. 10.1186/s13075-015-0877-x Galicia K, Thorson C, Banos A, Rondina M, Hopkinson W, Hoppensteadt D et al (2018) Inflammatory Biomarker Profiling in Total Joint Arthroplasty and Its Relevance to Circulating Levels of Lubricin, a Novel Proteoglycan. Clin Appl Thromb Hemost 24(6):950–959. 10.1177/1076029618765090 Elsaid KA, Zhang L, Shaman Z, Patel C, Schmidt TA, Jay GD (2015) The impact of early intra-articular administration of interleukin-1 receptor antagonist on lubricin metabolism and cartilage degeneration in an anterior cruciate ligament transection model. Osteoarthritis Cartilage 23(1):114–121. 10.1016/j.joca.2014.09.006 Das N, Schmidt TA, Krawetz RJ, Dufour A (2019) Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation: Does proteoglycan 4 have the ability to buffer the inflammatory response? BioEssays 41(1):e1800166. 10.1002/bies.201800166 Leonardi R, Rusu MC, Loreto F, Loreto C, Musumeci G (2012) Immunolocalization and expression of lubricin in the bilaminar zone of the human temporomandibular joint disc. Acta Histochem 114(1):1–5. 10.1016/j.acthis.2010.11.011 Guo H, Fang W, Li Y, Ke J, Deng M, Meng Q et al (2015) Up-regulation of proteoglycan 4 in temporomandibular osteoarthritic synovial cells by hyaluronic acid. J Oral Pathol Med 44(8):622–627. 10.1111/jop.12273 Hill A, Duran J, Purcell P (2014) Lubricin protects the temporomandibular joint surfaces from degeneration. PLoS ONE 9(9):e106497. 10.1371/journal.pone.0106497 Leonardi R, Perrotta RE, Almeida LE, Loreto C, Musumeci G (2016) Lubricin in synovial fluid of mild and severe temporomandibular joint internal derangements. Med Oral Patol Oral Cir Bucal 21(6):e793–e799. 10.4317/medoral.21145 Koyama E, Saunders C, Salhab I, Decker RS, Chen I, Um H, Pacifici M, Nah HD (2014) Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ. J Dent Res 93(7):663–670. 10.1177/0022034514535807 Poole R, Blake S, Buschmann M, Goldring S, Laverty S et al (2010) Recommendations for the use of preclinical models in the study and treatment of osteoarthritis. Osteoarthritis Cartilage 18(3):S10–S16. 10.1016/j.joca.2010.05.027 Institute for Laboratory Animal Research (2011) Guide for the Care and Use of Laboratory Animals, Eighth Edition. National Academies, Washington, DC, USA Russell WMS, Burch RL (1992) The Principles of Humane Experimental Technique. Universities Federation for Animal Welfare. Wheathampstead UK Duygu G, Güler N, Cam B, Kürkcü M (2011) The effects of high molecular weight hyaluronic acid (Hylan G-F 20) on experi-mentally induced temporomandibular joint osteoartrosis: Part II. Int J Oral Maxillofac Surg 40:1406–1413. 10.1016/j.ijom. 2011.07.909 Laverty S, Girard CA, Williams JM, Hunziker EB, Pritzker KP (2010) The OARSI histopathology initiative recommendations for histological assessments of osteoarthritis in the rabbit. Osteoarthritis Cartilage 18:S53–65. 10.1016/j.joca.2010.05.029 Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA et al (2006) Osteoarthritis cartilage histopathology: Grading and staging. Osteoarthritis Cartilage 14:13–29. 10.1016/j.joca.2005.07.014 Pritzker KP, Aigner T (2010) Terminology of osteoarthritis cartilage and bone histopathology - a proposal for a consensus. Osteoarthritis Cartilage 18:S7–9. 10.1016/j.joca.2010.05.028 Takahashi T, Tominaga K, Takano H, Ariyoshi W, Habu M, Fukuda J et al (2004) A decrease in the molecular weight of hyaluronic acid in synovial fluid from patients with temporomandibular disorders. J Oral Pathol Med 33:224–229. 10.1111/j.0904-2512.2004.00024.x Cardoneanu A, Macovei LA, Burlui AM, Mihai IR, Bratoiu I, Rezus II et al (2022) Temporomandibular Joint Osteoarthritis: Pathogenic Mechanisms Involving the Cartilage and Subchondral Bone, and Potential Therapeutic Strategies for Joint Regeneration. Int J Mol Sci 24(1):171. 10.3390/ijms24010171 Li Y, Yuan Z, Yang H, Zhong H, Peng W, Xie R (2021) Recent Advances in Understanding the Role of Cartilage Lubrication in Osteoarthritis. Molecules 26(20):6122. 10.3390/molecules26206122 Waller KA, Zhang LX, Elsaid KA, Fleming BC, Warman ML, Jay GD (2013) Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis. Proc Natl Acad Sci U S A 110(15):5852–5857. 10.1073/pnas.1219289110 Estrella RP, Whitelock JM, Packer NH, Karlsson NG (2010) The glycosylation of human synovial lubricin: implications for its role in inflammation. Biochem J 429(2):359–367. 10.1042/BJ20100360 de Souza RF, Lovato da Silva CH, Nasser M, Fedorowicz Z, Al-Muharraqi MA (2012) Interventions for the management of temporomandibular joint osteoarthritis. Cochrane Database Syst Rev. ; 2012(4):CD007261. 10.1002/14651858.CD007261 Additional Declarations Competing interest reported. The authors declare that Lµbris BioPharma® made recombinant human PRG4 available for research. However, it was not involved in the article's development, analysis, and preparation. 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. 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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-6234556","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445320148,"identity":"d1f63586-d961-47b8-bde9-9bf5a6e950d1","order_by":0,"name":"Veronica Iturriaga","email":"data:image/png;base64,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","orcid":"","institution":"University of La Frontera","correspondingAuthor":true,"prefix":"","firstName":"Veronica","middleName":"","lastName":"Iturriaga","suffix":""},{"id":445320149,"identity":"2af69a00-e753-41e6-96db-895b23917680","order_by":1,"name":"Bélgica Vásquez","email":"","orcid":"","institution":"University of La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Bélgica","middleName":"","lastName":"Vásquez","suffix":""},{"id":445320150,"identity":"9c562ffd-7ab8-4f94-86ff-c7b322ee923c","order_by":2,"name":"Schilin Wen","email":"","orcid":"","institution":"Universidad Autónoma de Chile","correspondingAuthor":false,"prefix":"","firstName":"Schilin","middleName":"","lastName":"Wen","suffix":""},{"id":445320151,"identity":"554ce05a-38af-40f5-85b0-d39b0deffde8","order_by":3,"name":"Thomas Bornhardt","email":"","orcid":"","institution":"University of La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Bornhardt","suffix":""},{"id":445320152,"identity":"27508d6c-13a9-4ffb-83b0-ca0af4a4a11b","order_by":4,"name":"Mariano del Sol","email":"","orcid":"","institution":"University of La Frontera","correspondingAuthor":false,"prefix":"","firstName":"Mariano","middleName":"del","lastName":"Sol","suffix":""}],"badges":[],"createdAt":"2025-03-15 19:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6234556/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6234556/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81034762,"identity":"e9005894-20b2-4eb7-8dcb-113b65385f47","added_by":"auto","created_at":"2025-04-21 12:03:21","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":256935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagram of experimental groups\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6234556/v1/7d7507181f9b7695b938b36e.jpeg"},{"id":81036169,"identity":"2f050f95-b65d-4478-b341-45fda47f6e54","added_by":"auto","created_at":"2025-04-21 12:19:21","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3080543,"visible":true,"origin":"","legend":"\u003cp\u003eTemporomandibular joint of rabbit (Oryctolagus cuniculus). TMJ-C: healthy temporomandibular joints as a control group (A-D); TMJ-OA: temporomandibular joint with osteoarthritis (E-H); TMJ-OA-wt: osteoarthritic temporomandibular joint without treatment and evaluated at 30 days post-induction (I-L); rhPRG4-30: TMJ-OA joint treated with lubricin 30 μg /ml and assessed 30 days after the treatment (M-P); rhPRG4-100: TMJ-OA treated with lubricin 100 μg/ml and evaluated 30 days after treatment (Q-T); MC: mandibular condyle; AD: articular disc; M: mandibular fossa; SZ: superficial zone; MZ: middle zone; DZ: deep zone; CC: calcified cartilage; SB: subchondral bone; dotted line area: chondrocytes arranged in clusters parallel to the collagen fibers; grid area: chondron clustering near deep fibrillation; asterisk: heterogeneous matrix texture; arrowhead: disorganized collagen fibers; arrow: deep fibrillation. Toluidine Blue stain.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6234556/v1/1af56e86b40bd5eda1268442.jpeg"},{"id":81034769,"identity":"a7c6fc83-7a81-41f2-bf58-b5e5e7460bd3","added_by":"auto","created_at":"2025-04-21 12:03:21","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":622692,"visible":true,"origin":"","legend":"\u003cp\u003eOARSI scale score to evaluate the histopathology of osteoarthritic cartilage in the condyle and mandibular fossa of the temporomandibular joint of the rabbit (Oryctolagus cuniculus). TMJ-OA: temporomandibular joint with osteoarthritis; TMJ-OA-WT: osteoarthritic temporomandibular joint without treatment and evaluated at 30 days post-induction; rhPRG4-30: TMJ-OA joint treated with lubricin 30 μg /ml and assessed 30 days after the treatment; rhPRG4-100: TMJ-OA treated with lubricin 100 μg/ml and evaluated 30 days after treatment. GraphPad Prism version 5.00. Significant statistical differences P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6234556/v1/65a90040813adaeeaa3c44d9.jpeg"},{"id":81392045,"identity":"71d1ffc1-90b4-4902-9edf-531229915dff","added_by":"auto","created_at":"2025-04-25 14:53:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4765854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6234556/v1/3b544204-9fd6-4e20-9999-b6a36b749311.pdf"}],"financialInterests":"Competing interest reported. The authors declare that Lµbris BioPharma® made recombinant human PRG4 available for research. However, it was not involved in the article's development, analysis, and preparation.","formattedTitle":"Effects of Intra-articular Infiltration of Proteoglycan 4 / Lubricin in Temporomandibular Joint Osteoarthritis: An Experimental Study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe therapeutic use of regenerative medicine or tissue engineering has been increasing in pathologies such as osteoarthritis (OA) of the temporomandibular joint (TMJ). Tissue engineering is based on replacing, manufacturing, or regenerating human cells, tissues, or organs to restore or establish their normal function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In its beginnings, regenerative medicine in TMJ-OA was based on implanting tissues into the joint to promote its repair. However, it was necessary to perform joint surgery to introduce the material. Over time, the technique evolved towards infiltrating liquid or semi-liquid substances of different viscosities, thus avoiding surgery [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the therapeutic use of regenerative medicine in TMJ-OA is mainly represented by the infiltration of hyaluronic acid (HA), platelet-rich plasma (PRP) or growth factors (GF), and stem cell-based therapies. Of these alternatives, HA and PRP present more significant evidence and are used in daily practice, while the evidence for stem cell therapy is still developing [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHA is a key lubricant component of synovial fluid in the TMJ. When used as a treatment, it presents essential evidence of its anti-arthritic effect on joint tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Another lubricating molecule that may have reparative or regenerative effects within the components of synovial fluid in TMJ-OA is proteoglycan 4 (PRG4), also known as lubricin.\u003c/p\u003e \u003cp\u003ePRG4 is a high molecular weight mucinous glycoprotein produced by B-type synoviocytes and chondrocytes of the superficial zone of articular cartilage. This protein is found in the synovial fluid (SF) of synovial joints [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]and plays a crucial role in joint lubrication, synovial homeostasis, immunomodulation, and suppression of inflammation [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PRG4 contains core 1 O glycosylations that provide its lubricating function, enabling it to perform multiple essential functions in joints [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In addition, it protects cartilage by preventing the deposition of abrasive proteins and facilitates SF energy absorption and dissipation, thus protecting cartilage from mechanical damage [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It has also demonstrated anti-proliferative effects, preventing synovial hyperplasia, although the specific mechanisms are not yet fully understood [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have suggested that PRG4 deficiency is associated with cartilage destruction, and a decrease in PRG4 may be associated with the development of OA. This ability of PRG4 to reduce friction and protect cartilage indicates that it could be a promising therapeutic option for this pathology, providing adequate lubrication and long-term protection against joint degeneration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntra-articular administration of PRG4 has shown promising results in animal models of OA. Different ways of obtaining PRG4 have been described, some related to lubricin biosynthesis via synoviocytes, purified human lubricin, or mostly to full-length recombinant human PRG4 (rhPRG4). Early evidence was observed in rat knees, where intra-articular injection of PRG4 showed significant improvement in reducing joint friction and inhibiting cartilage degeneration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Also, rhPRG4 has been shown to have anti-inflammatory effects in synovial joints. One of these pathways is associated with the inhibition of fibroblast-like synoviocyte proliferation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] or the ability to bind and antagonize Toll-like receptors, decreasing the activation of nuclear factor kappa B (NF-κB) and inflammatory cytokines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, a correlation between increased plasma inflammatory cytokines, such as Interleukin-1 beta (IL-1β) and Tumor Necrosis Factor alfa (TNF-α), and decreased plasma PRG4 has been demonstrated [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Elevated levels of inflammatory cytokines could trigger molecular processes that decrease PRG4 expression, thus favoring the development of OA. An IL-1 receptor antagonist somewhat restores PRG4 expression in articular cartilage, providing evidence for a correlation between proinflammatory cytokines and PRG4 expression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn preclinical animal models of joint inflammation, a decrease in PRG4 concentration in the SF after joint injury is observed, which is associated with increased damage to the cartilage surface [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In patients with acute knee injuries, PRG4 levels in the SF decrease and return to homeostatic levels generally within one year after injury [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConcerning the TMJ, PRG4 expression has been characterized in healthy and OA articular cartilage. In healthy cartilage, PRG4 is mainly expressed in the superficial zone of the cartilage and, to a lesser extent, in the medial zone. In cartilage with TMJ-OA, no expression is observed in the medial zone, and expression in the superficial zone is decreased [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, in PRG4-/- rats, changes in the TMJ tissue were seen from 2 months of development, whereas at 6 months, osteoarthritic degradation is evident in the joint tissue [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite advances in understanding OA and the therapeutic potential of PRG4, significant work remains to advance this potential in the clinic. Specifically, no studies evaluate the effects of PRG4 administration in TMJ-OA, a condition with unique pathophysiological characteristics due to the complexity of its structure and function. The proposed research aims to evaluate the histopathological effects of intra-articular administration of rhPRG4 on articular cartilage and articular disc in an experimental model of induced TMJ-OA in rabbits.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAn experimental study was conducted on 12 healthy male \u003cem\u003eOryctolagus cuniculus\u003c/em\u003e rabbits weighing approximately 3 kg and eight months of age, according to the recommendations described by Poole et al. (2010) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The animals were kept in a controlled environment regarding temperature, environmental noise, and a 12-hour light/12-hour dark cycle. The animals were housed in cages individually and randomly assigned to each group. Simple random sampling was used to allocate the animals to the experimental groups; each animal was assigned a number.\u003c/p\u003e \u003cp\u003e The animals were under the care of a veterinarian, who was aware of the group assignment in the different phases of the experiment. Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and the National Research Council Guide for the Care and Use of Laboratory Animals [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] were followed. The study was performed at the experimental surgery unit of the Center of Excellence in Morphological and Surgical Studies of the Universidad de La Frontera, Chile, with the approval of the Scientific Ethics Committee of the Universidad de La Frontera (File Number 004/21, ACT NO. 027_21).\u003c/p\u003e \u003cp\u003eThe two TMJs of each animal were considered to reduce the sample size and meet the experimental criteria established by Russell and Burch [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A sample size calculation was performed considering up to 10% losses and a statistical significance of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. According to the above, twenty-four TMJs were included, distributed in: 1) TMJ-C, control group consisting of 4 healthy TMJs; 2) TMJ-OA, group consisting of 5 TMJs with OA; 3) TMJ-OA-WT, group consisting of 5 untreated TMJ-OAs; 4) rhPRG4-30, group consisting of 5 ATM with OA treated with rhPRG4 at 30 \u0026micro;g /ml; and 5) rhPRG4-100, group consisting of 5 ATM with OA treated with rhPRG4 100 \u0026micro;g/ml. The distribution of the groups is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Protocols for TMJ-OA induction, intra-articular rhPRG4 administration, histological processing, and histological analysis were performed according to previously described protocols [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTMJ-OA induction\u003c/h3\u003e\n\u003cp\u003eExcept for the TMJ-C group, TMJ-OA induction was performed in all groups. Animals were anesthetized intramuscularly with ketamine (40 mg/kg), xylazine (5 mg/kg), and acepromazine (1 mg/kg), and the TMJ area was shaved and disinfected with 70% ethyl alcohol. At that time, 50 \u0026micro;L of sodium mono-iodoacetate (MIA) at a concentration of 3 mg/mL was infiltrated into the joint space with a 22-gauge needle. A period of 50 days was allowed to elapse to develop TMJ-OA [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. After a 50-day waiting period, the animals in the TMJ-OA group were euthanized and subsequently analyzed. In the TMJ-OA-WT, rhPRG4-30, and rhPRG4-100 groups, after the initial 50-day period, an additional 30 days were taken before euthanasia and analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eIntra-articular rhPRG4 administration\u003c/h3\u003e\n\u003cp\u003eIntra-articular rhPRG4 administration was conducted using the same protocol for the anesthesia and previously mentioned preoperative measures. The infiltration technique was standardized, considering the caudal margin of the orbital lamina as the anatomical reference point. It was directed 5 mm caudally and 1 mm ventrally, with the needle at a 45\u0026ordm; angle towards the ventral concerning the skin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the rhPRG4-30 group, 0.1 mL of rhPRG4 at 30 \u0026micro;g/ml (L\u0026micro;bris BioPharma\u0026reg;) was injected into the joint space with a 22 gauge needle. Similarly, in the rhPRG4-100 group, 0.1 mL of rhPRG4 at 100 \u0026micro;g/ml (L\u0026micro;bris BioPharma\u0026reg;) was injected.\u003c/p\u003e\n\u003ch3\u003eHistological processing\u003c/h3\u003e\n\u003cp\u003eOnce the experimental protocol was completed, the animals were sacrificed, and the joint tissue was dissected. No animal losses, adverse effects, or modifications were reported. Samples were coded to maintain process masking during evaluation. The tissue was fixed with 10% buffered formalin (1.27 mol/L formaldehyde in 0.1 M phosphate buffer, pH 7.2) for 48 hours. TMJs were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) (in 0.1 M phosphate buffer 7\u0026ndash;8)35 in ultrasonic decalcification (Use 33, Medite, Burgdorf, Germany) for 30 days. Subsequently, samples were dehydrated in an ascending alcohol battery, rinsed in xylene, and embedded in Paraplast Plus (Sigma-Aldrich Co., St. Louis, MO, USA). Serial sections of the TMJs were cut in the parasagittal plane at a thickness of 5 \u0026micro;m using a Leica\u0026reg; RM 2255 microtome. To optimally grade the OA, successive sections of the deeper planes of the joint were stained and visualized under a light microscope. Then, one section per joint was selected for the more detailed analysis, considering the plane of the block that crosses the lesion to the greatest extent and shows the most pronounced alterations [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Histological sections were stained with Toluidine Blue. An optical microscope (Leica DM 2000 LED, Wetzlar, Germany) was used for visualization, and the slides were photographed with a digital camera (Leica high-definition MC 170, Wetzlar, Germany). To maintain blinding in the analysis, the slides were also coded.\u003c/p\u003e\n\u003ch3\u003eHistological analysis\u003c/h3\u003e\n\u003cp\u003eHistological slides were randomly analyzed for each TMJ to compare the different groups. A descriptive analysis of the mandibular condyle (MC), articular disc (AD), and mandibular fossa (MF) was performed. The cartilage was described from superficial to deep, beginning with the superficial zone (SZ), also known as the tangential zone, followed by the mid-zone (MZ), also called the transitional zone; the deep zone or radial zone (DZ); calcified cartilage; and subchondral trabecular bone. Regarding AD, the central zone (CZ) at its thinnest point and the anterior and posterior peripheral zone (PZ) areas were analyzed.\u003c/p\u003e \u003cp\u003eAuthors V.I. and B.V. independently and blindly performed the quantitative histological analysis of each sample. In the case of disagreements, these were discussed until reaching a consensus. The interobserver calibration was performed in three phases before analyzing the samples: theoretical training, laboratory training, and calibration (final inter-agreement kappa 0.865).\u003c/p\u003e \u003cp\u003eThe OARSI scale, was used to grade the articular cartilage grade and stage for both MC and MF. This scale classifies the different cartilage states, where grade 0 corresponds to normal tissue and grades 1 to 6 are relative to OA. Grades 1 to 4 of OA involve changes in cartilage only, while grades 5 and 6 also include subchondral bone. The OA stratification method, also defined by the OARSI scale, categorizes the disease into four stages based on the horizontal extent of the affected cartilage surface, regardless of the degree of underlying OA. Stage 1 represents less than 10% involvement; stage 2 represents 10\u0026thinsp;\u0026lt;\u0026thinsp;25% involvement; stage 3 represents 25\u0026ndash;50% involvement; and stage 4 represents greater than 50% involvement. MC and MF cartilage were evaluated separately for each slide in each group [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eQuantitative analysis was performed by calculating the median and interquartile range of the scores obtained on the OARSI scale. Nonparametric inferential statistics were performed to compare the degree and stage of TMJ-OA between groups using Kruskal-Wallis and Dunn's post hoc tests. The analysis was performed with the STATA 18 program, considering a significance level of α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eDescriptive histological analysis of articular cartilage in the rabbit temporomandibular joint reveals key differences between healthy and osteoarthritic joints.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn the control group (TMJ-C), the cartilage was smooth, continuous, and had three well-demarcated zones: the superficial zone (SZ), the middle zone (MZ), and the deep zone (DZ), the latter being the thickest. In contrast, in the TMJ-OA group, a focal discontinuity on the surface and a generalized irregularity was observed, in addition to a thinning of the cartilage and a more fibrous matrix compared to the TMJ-C group. Deep fibrillations and proteoglycan depletion in the matrix were hallmarks of the TMJ-OA group, with hypertrophic chondrocytes and disorganization of collagen fibers. These structural differences suggest significant alterations associated with osteoarthritis in the TMJ-OA group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistological characteristics of the temporomandibular joint tissue according to the groups under study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStructure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTMJ-C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTMJ-OA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTMJ-OA-WT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003erhPRG4-30\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003erhPRG4-100\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMandibular condyle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSmooth and continuous surface. Flattened surface cells and a thin layer of fibrous connective tissue.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbrasion, matrix focal discontinuity. Fibrillations towards the deep zone.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbrasion, matrix focal discontinuity. Fibrillations towards the deep zone covering the entire articular cartilage.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSlightly irregular surface. Abrasion of the surface layer in some areas. Small and flat or round chondrocytes, aligned parallel to the collagen fibers and the surface.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSlightly irregular surface. In some sectors it is possible to observe abrasion. Small and flat or round cells. In some samples it is observed, focally, areas of collagen condensation whose fibers are directed to the middle and / or deep zone.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUndifferentiated cells and spherical chondrocytes in a proteoglycan matrix.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReduced cellularity with deep fibrillations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReduced cellularity, more deep and higher density fibrillations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProliferation of chondrocytes, arranged in isolation.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAnisocytosis and proliferation of undifferentiated cells. Clusters of chondrocytes are seen in some samples. Matrix rarefaction, with areas of increased cationic staining around the chondrons. Condensation of collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIt presents round and larger chondrocytes, organized in isogenic groups. Deep hypertrophic chondrocytes are observed.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLess cellularity. Hypertrophic chondrocytes, forming clusters. Rarefaction and condensation of collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLess cellularity and chondrocyte clusters. Rarefaction and condensation of collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVestiges of deep fibrillations. Focal rarefaction, increased collagen formation and cationic staining around the chondrons. Some hypertrophic chondrocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLoss of orientation of the chondrons in a disorganized matrix. Increased density of chondrocytes, with decreased cell size. Vestiges of deep fibrillations.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArticular disc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChondrocyte stacking and arrangement in parallel (rows) as to collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFocal edema. Increased number and density of collagen fibers. Disorganized fibers. Less cellularity, with randomly arranged hypertrophic chondrocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFocal edema. Increased number and density of collagen fibers. Disorganized fibers. Less cellularity, with randomly arranged hypertrophic chondrocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCollagen fibers arranged in parallel with chondrocytes aligned to them. Chondrocytes are found within cartilage matrix.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDisorganization of collagen fibers and edema. Less cellularity, with some chondrocytes arranged randomly and others parallel to the collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMore abundant and dense chondrocyte rows.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRandomly arranged hypertrophic chondrocytes within disorganized collagen fibers. Presence of connective tissue with abundant fibroblasts.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLess peripheral connective tissue. Hypertrophy of the synovial membrane.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRandomly arranged hypertrophic chondrocytes within disorganized collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSome hypertrophic chondrocytes randomly arranged among disorganized collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMandibular fossa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFibrous connective tissue, collagen fibers parallel to the surface with intermingled fibrocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThickness, fiber disorganization, and edema. Rarefaction. Fibrillations that reach the deep zone of the cartilage.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThickness, fiber disorganization and rarefaction. Fibrillations that reach the deep zone of the cartilage.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSlightly irregular surface. The limits between the SZ and MZ are not very evident. Hypocellularity.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSlightly irregular surface. Unclear boundaries between SZ and MZ. Scarce cellularity with anisocytosis.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUndifferentiated cells.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiffuse, with reduced thickness and scarce cellularity.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDiffuse, reduced thickness and scarce cellularity.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLess thickness and hypocellularity, with rarefaction and edema.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHeterogeneous matrix, with focal edema, low cellularity and anisocytosis.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChondrocytes immersed in matrix rich in collagen fibers.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClusters of hypertrophic chondrocytes. Rarefaction and condensation of collagen fibers. Presence of deep fibrillations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeep fibrillations. rarefaction and collagen condensation. Clusters of hypertrophic chondrocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOsteoarthritic features are observed, similar to MZ.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHeterogeneous matrix, traces of focal fibrillations. Small chondrocytes distributed mainly in isolation.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTMJ-C: healthy temporomandibular joints as a control group; TMJ-OA: osteoarthritic temporomandibular joint; TMJ-OA-WT: osteoarthritic temporomandibular joint untreated and evaluated 30 days after treatment period; rhPRG4-30: osteoarthritic temporomandibular joint treated with lubricin 30 \u0026micro;g/mL and assessed 30 days after treatment; rhPRG4-100: osteoarthritic temporomandibular joint treated with 100 \u0026micro;g/mL and evaluated 30 days after treatment; SZ: superficial zone; MZ: mid zone; DZ: deep zone; CZ: central zone; PZ: peripheral zone.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDescriptive histological analysis of the rabbit osteoarthritic temporomandibular joint suggests beneficial treatment effects by intra-articular administration of rhPRG4.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eComparing the rhPRG4-30 and rhPRG4-100 groups with the TMJ-OA and TMJ-OA-WT groups, key differences in the condition of articular cartilage and synovial membrane in the TMJ are highlighted. In the TMJ-OA groups, discontinuity of the cartilage surface, thinning, a more fibrous matrix with deep fibrillations, and signs of proteoglycan depletion, chondrocyte loss, and disorientation were observed. The TMJ-OA-WT group showed greater disease progression with deeper and denser fibrillations. Both groups exhibited synovial membrane thickening due to the increased presence of inflammatory cells and hyperplasia of synovial cells with an increased size (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, in the rhPRG4-30 and rhPRG4-100 treated groups, repair processes were observed in the articular cartilage, manifesting in a more organized appearance with increased thickness, more uniform surface, and increased cationic staining of the matrix, along with less alteration in the synovial membrane compared to the TMJ-OA and TMJ-OA-WT groups. In the rhPRG4-30 group, some abrasion areas were observed in the SZ. In the MZ of the mandibular condyle, the proliferation of chondrocytes arranged in an isolated manner was observed, suggesting an ongoing repair process. In addition, traces of deep fibrillations were seen in the DZ, indicating an attempt at cartilage tissue regeneration. The articular cartilage of the rhPRG4-100 group also exhibited a repair process; however, increased collagen condensation and fibrillation were noted in deeper areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBoth groups demonstrated a potential reparative effect with rhPRG4 treatment in the TMJ affected by OA. Notably, the rhPRG4-30 group showed a slight improvement in cartilage regeneration and organization compared to the rhPRG4-100 group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIntra-articular administration of rhPRG4 as a treatment reduces the severity of osteoarthritis in the articular cartilage of the rabbit temporomandibular joint.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe OARSI score of articular cartilage in TMJs revealed that both MC and MF exhibited the highest degree of OA in the TMJ-OA and TMJ-OA-WT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Specifically, the rhPRG4-30 and PRG4 -100 groups showed a significant reduction in the severity of OA in the articular cartilage of the MC and MF compared to the TMJ-OA and TMJ-OA-WT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The TMJ-OA group had no significant difference in OA severity compared to the TMJ-OA-WT group in all structures analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Likewise, the rhPRG4-30 and rhPRG4-100 groups also showed no significant differences in OA severity in both MC and MF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Finally, significant differences in OA stage were only observed between the untreated and rhPRG4-100 groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThese results suggest that intra-articular infiltration of rhPRG4 in osteoarthritic TMJs beneficially affects articular cartilage by reducing the severity of osteoarthritis and promoting a more organized cartilage structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSIÓN","content":"\u003cp\u003eThe present study is the first to investigate the effects of intra-articular infiltration of rhPRG4 on induced TMJ-OA in rabbits. The results obtained are promising and suggest that rhPRG4 may significantly reduce the severity of OA.\u003c/p\u003e \u003cp\u003eHistological analysis revealed significant improvements in the articular cartilage structure of rabbits with TMJ-OA treated with rhPRG4 compared to untreated groups. The rhPRG4-30 and rhPRG4-100 groups showed a more uniform cartilage surface, a more organized matrix, and increased cartilage thickness. These improvements are consistent with previous studies in the knee that have demonstrated beneficial effects of rhPRG4 in animal models of OA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The articular disc also showed significant improvements in the rhPRG4-treated groups. In the untreated groups, the articular disc showed signs of degeneration, such as thinning, matrix disorganization, and proteoglycan loss, consistent with the TMJ-OA literature [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In contrast, the rhPRG4-treated groups showed a more uniform and thicker disc structure, suggesting a reparative effect of rhPRG4.\u003c/p\u003e \u003cp\u003eThe observed efficacy of rhPRG4 in improving TMJ articular cartilage structure and protecting the articular disc is attributed to its multiple biological functions. rhPRG4 plays a crucial role in lubricating articular surfaces, significantly reducing friction, which protects cartilage from mechanical wear and tear [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, the ability of rhPRG4 to prevent chondrocyte apoptosis, as Waller et al. demonstrated, is essential for maintaining cell viability in OA-damaged cartilage [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. rhPRG4 also possesses immunomodulatory properties that can reduce inflammation in the affected joint, a crucial factor in the progression of OA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Chronic inflammation in OA contributes to cartilage degradation and joint dysfunction. By modulating the immune response and reducing inflammation, rhPRG4 protects existing cartilage and promotes a favorable environment for extracellular matrix repair and regeneration.\u003c/p\u003e \u003cp\u003eAn interesting observation is comparing rhPRG4 concentrations of 30 \u0026micro;g/ml and 100 \u0026micro;g/ml. Although both groups improved cartilage and articular disc structure, the rhPRG4-30 group showed slightly better regeneration and more coherent matrix organization than the rhPRG4-100 group. This finding suggests that a lower concentration of rhPRG4 may be more effective for cartilage repair, possibly due to a better balance between lubrication and stimulation of matrix synthesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, further studies are required to find the optimal concentration.\u003c/p\u003e \u003cp\u003eThe results of this study show a reduction in the degree of OA according to the OARSI classification and a decrease in the extent of the lesion in a dose-dependent manner. The OARSI classification evaluates grade (cartilage quality) and stage (extent of damage), allowing us to differentiate between these aspects [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. rhPRG4 appears to be effective in improving cartilage quality, reducing inflammation, and promoting extracellular matrix synthesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This targeted action is in line with previous studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] that have demonstrated the ability of rhPRG4 to improve the structural and functional integrity of existing cartilage.\u003c/p\u003e \u003cp\u003eCurrently, treatments for TMJ-OA focus primarily on symptom relief through non-steroidal anti-inflammatory drugs, corticosteroids, and physical therapies, but these approaches do not halt or reverse joint degeneration [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. PRG4 administration represents a novel therapeutic strategy that could provide benefits beyond simple symptom relief. The results of this study suggest that rhPRG4 not only reduces joint friction and protects cartilage but also promotes repair and regeneration processes in damaged cartilage. This could have a significant impact on improving joint function and reducing long-term pain in patients with TMJ-OA.\u003c/p\u003e \u003cp\u003eAlthough the results are promising, this study has some limitations that should be considered. Firstly, the experimental model was based on rabbits, and although these animals are suitable for preclinical studies, the results cannot be directly extrapolated to humans. As background, rhPRG4 has been used clinically to manage dry eye disease in humans. Therefore, the potential for rapid translational uptake is significant. On the other hand, a vehicle-only group was not included to restrict the size of the study and the number of animals used. In addition, the study focused on a relatively short period of treatment and follow-up, so long-term studies are required to assess the sustainability of the beneficial effects of PRG4. Further studies using advanced imaging techniques and molecular analysis are also needed to deepen the understanding of the mechanisms behind the efficacy of rhPRG4. These techniques may provide more detailed insight into how PRG4 affects cartilage architecture and cellular dynamics at the microstructural level. In addition, research exploring combinations of PRG4 with other therapeutic agents, such as hyaluronic acid or growth factors, could offer synergistic approaches to maximize joint repair and prevent OA progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eROLE OF THE FUNDING SOURCE\u003c/h2\u003e\n\u003cp\u003eThe study was financed by Project DI20-0018 and the Temporomandibular Disorder and Orofacial Pain Program, Universidad de La Frontera, Chile. The funding bodies did not participate in the conception, design, or execution of the project, which was carried out independently.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that L\u0026micro;bris BioPharma\u0026reg; made recombinant human PRG4 available for research. However, it was not involved in the article\u0026apos;s development, analysis, and preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors used Grammarly Inc. in order to revise the drafting of the manuscript. After using this tool, the authors reviewed and edited the content as needed and takes full responsibility for the content of the publication.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare that L\u0026micro;bris BioPharma\u0026reg; made recombinant human PRG4 available for research. However, it was not involved in the article\u0026apos;s development, analysis, and preparation.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the conception design of the study, interpretation of data for the work, drafting of the article, critically revising the manuscript for important intellectual content, and, giving final approval, agreeing to be accountable for all aspects of this work. V.I. and S.W. contributed to the experimental procedures, data analysis, and interpretation. B. V. contributed with data analysis and interpretation. M. d. S. and T. B. contributed by providing study materials and data interpretation.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors thank Dr. Francisca Villagr\u0026aacute;n for her collaboration in the statistical analysis of the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMason C, Dunnill P (2008) A brief definition of regenerative medicine. Regen Med 3(1):1\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2217/17460751.3.1.1\u003c/span\u003e\u003cspan address=\"10.2217/17460751.3.1.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerthiaume F, Maguire TJ, Yarmush ML (2011) Tissue engineering and regenerative medicine: history, progress, and challenges. 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Cochrane Database Syst Rev. ; 2012(4):CD007261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/14651858.CD007261\u003c/span\u003e\u003cspan address=\"10.1002/14651858.CD007261\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"temporomandibular joint, temporomandibular joint disorders, osteoarthritis, lubricin, proteoglycan 4, cartilage","lastPublishedDoi":"10.21203/rs.3.rs-6234556/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6234556/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: The proposed research aims to evaluate the histopathological effects of intra-articular administration of recombinant human proteoglycan 4 (rhPRG4) on articular cartilage and articular disc in an experimental model of induced temporomandibular joint osteoarthritis (TMJ-OA) in rabbits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods:\u003c/strong\u003e An experimental study was conducted on twenty-four rabbits’ TMJs, distributed in: 1) TMJ-C, control group consisting of 4 healthy TMJs; 2) TMJ-OA, group consisting of 5 TMJs with OA; 3) TMJ-OA-WT, group consisting of 5 untreated TMJ-OAs; 4) rhPRG4-30, group consisting of 5 ATM with OA treated with lubricin 30 μg /ml; and 5) rhPRG4-100, group consisting of 5 ATM with OA treated with lubricin 100 μg/ml. A histopathological analysis was performed, considering the mandibular condyle, articular disc, and mandibular fossa, comparing the groups. In addition, a quantitative comparative analysis was performed using the Osteoarthritis Research Society International (OARSI) scale. The Kruskal-Wallis and Dunn's post hoc tests were used for statistical analysis, considering a statistical significance of p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eDescriptive histological analysis of TMJ joint tissue reveals key differences between healthy, osteoarthritic, and rhPRG4-treated joints. The analysis also suggests beneficial treatment effects with intra-articular infiltration of rhPRG4, reducing the severity of osteoarthritis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Intra-articular infiltration of rhPRG4 in TMJ-OA has a cartilage and articular disc repair effect, reducing the severity of osteoarthritis and promoting a more organized cartilage structure, with slightly better results at the 30 μg/ml concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance: \u003c/strong\u003eIntra-articular infiltration of rhPRG4 is proposed as a new therapeutic alternative in TMJ-OA, enhancing joint tissue regeneration.\u003c/p\u003e","manuscriptTitle":"Effects of Intra-articular Infiltration of Proteoglycan 4 / Lubricin in Temporomandibular Joint Osteoarthritis: An Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 12:03:16","doi":"10.21203/rs.3.rs-6234556/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":"86b10b2a-216c-44cf-b802-ffbd5fc50edb","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-25T14:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 12:03:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6234556","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6234556","identity":"rs-6234556","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}