Pyroptosis Related Molecules are Signicant for Cartilage Degeneration

Background: Pyroptosis is the highlight topic in inammation. However, there is rarely research on relationship between pyroptosis and temporomandibular joint osteoarthritis (TMJOA), The aim of this study is to explore whether pyroptosis related molecules are signicant for condylar cartilage degeneration and to verify the clinical effects of sodium hyaluronic acid (HA) treatment on TMJOA. Methods: Patients diagnosed as temporomandibular joint internal derangement (TMJID) without condylar defect and TMJOA with condylar defect were divided into two groups. Thirty patients in each group, and they were tested for pyroptosis related molecules via synovial uid included interleukin-1beta (IL-1β), IL-18, nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), cysteinyl aspartate specic proteinase 1 (CASP1) with Enzyme-linked immunosorbent assay (cid:0) Elisa (cid:0) . Eighteen cases in TMJOA group were tested again after twice HA treatment to evaluate HA’s therapeutic effects. Results: IL-1β, IL-18, NLRP3 and CASP1 were all positive in two groups, TMJOA patients with condylar defect had higher expressions of above molecules compared with TMJID patients (P (cid:0) 0.05). IL-1β, IL-18, NLRP3 were decreased with twice HA therapy (P (cid:0) 0.05), there was no signicant difference in CASP1 with twice HA injections (P=0.549). Conclusions: Cell pyroptosis may be involved in condylar degeneration. HA could reduce part of pyroptosis molecules to relieve inammation.


Background
Temporomandibular joint osteoarthritis (TMJOA), a dysfunction characterized by disc-condylar disorder, cartilage damage and extracellular matrix degeneration. Condylar degeneration is the direct result of TMJOA progression, threatens the quality of life in terms of highly occurrence and serious clinical symptoms [1]. Due to low blood and lymph supply of articular cartilage tissue, self-repair is unsuccessful once progressive destruction of condylar cartilage begins [2,3]. Despite of diversi ed medicine, surgery and tissue engineering techniques, these clinical therapeutic effects are not optimistic. Presently, the mechanism of cartilage damage remains unclear, its pivotal to nd the mechanism of condylar degeneration in order to target an optimal treatment. Previous studies have shown that malocclusion used to be an independent risk factor in temporomandibular joint disorder (TMD), which leads to an abnormal mechanical stimulation to condyle via proprioceptive mechanisms [4,5]. While in ammation as a common phenomenon accelerating condylar degeneration [6,7]. The imbalance of local in ammation and anti-in ammation leads to a disordered microenvironment of cartilage and ampli es local immune reactions.
Pyroptosis, a newly discovered programmed cell death, which depends on caspase family proteinase activity, con rmed as in ammation stimulated by danger signals included pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [8,9]. The morphological manifestations are cell swelling, membrane rupture, in ammatory factors releasing simultaneously. NLRP3 in ammasome is an crucial cytosolic signaling complex that perceives stimulus of DAMPs and activates potent in ammatory mediators [10]. which is comprising NLRP3, apoptosis-associated specklike protein containing a CARD(ASC), and the effector protease CASP1. Activated NLRP3 in ammasome can induce CASP1 mature, promoting IL-1β, IL-18 release into extracellular uid, these two in ammatory factors recruit leukocytes to sites of in ammation and participate ampli cation of in ammation. OA is an in ammatory disease, most of TMJOA patients reveal cartilage degeneration due to in ammatory response. However, there are rarely reports on the relationship between pyroptosis and TMJOA, we suppose pyroptosis may be involved in pathological progression of TMJOA, pyroptosis of chondrocytes or synoviocytes is likely to be a new mechanism of cartilage degeneration because its chronic in ammation essence.
We started with in ammation and compared the expression of IL-1β, IL-18, NLRP3 and CASP1 in synovial uid between TMJID patients and TMJOA patients to verify the signi cance of pyroptosis related molecules in condylar degeneration. What's more, We also tested for same molecules with twice HA therapeutic treatment in TMJOA group to evaluate its therapeutic effect.

Objects of study
The Ethical Committee of The First A liated Dental Hospital of Xinjiang Medical University approved this study with number K201910-04. All the patients were from The First A liated Hospital of Xinjiang Medical University, who were diagnosed as temporomandibular joint disorder (TMD) by the same expert of oral and maxillary surgery from October 2018 to April 2019. According to the clinical symptoms and cone-beam computed tomography (CBCT) or magnetic resonance imaging (MRI) examination, patients were screened out as the TMJID group without condylar bone defect and the TMJOA group with condylar bone defect, 30 cases in each group. Informed consent was obtained from all patients, Patients older than 18 years old, presented with temporomandibular joint (TMJ) symptoms and with malocclusion in oral examinations were included. Patients under 18 years old and those with history of jaw trauma, autoimmune diseases, and history of acute or chronic infections, serious cardiovascular, liver, kidney and other endocrine system diseases were excluded.

Groups of study
The subjects were divided into two groups, 30 cases in each group, TMJID group without condylar bone defect was con rmed according to the diagnostic criteria of RDC/TMD [11], and CBCT or MRI examination should reveal no condylar bone destruction, TMJOA group with condylar bone defect were revealed linear shadow, cyst or osteophyte formation in axial and coronal image with CBCT or MRI examination, not accompanied with symptoms of internal derangement in TMJ (Fig. 1). In order to evaluate HA's therapeutic effect, 18 patients out of 30 TMJOA patients were included in the self-control group, who were accepted with twice HA intra-articular injections.

Results
Clinical characteristics with two groups The clinical characteristics were summarized (Table 1). TMJID group included 18 females and 12 males, 3 patients with crossbite, 4 patients with sissors-bite, 6 patients with overbite in oral examination. TMJOA group was depicted with 20 females and 10 males, 5 patients with crossbite, 7 patients with sissors-bite, 7 patients with overbite in oral examination. Patients in two groups had different levels of temporomandibular joint dysfunction including joint pain, limited mouth opening and asymmetrical face.

Clinical data record
Clinical data was recorded by two professional doctors to make consistent judgement, comprising gender, age, symptoms of temporomandibular joint, types of malocclusion with oral examination, Visual analogy score, mouth opening and imaging manifestation. The follow-up information of the above datas were recorded in detail to evaluate clinical outcomes.
Collection of synovial uid with HA intra-articular injection Synovial uid was collected with HA intra-articular injection, Patients were instructed the importance of treatment and informed consent freely, head deviation on the seat, 3 milliliters (mL) lidocaine was punctured on the lesion at the anterior 1 centimeter (cm) of the tragus, the needle tip moved forward obliquely, up and inside, and retreated about 1 mL from the posterior oblique surface of the condylar to wash the immune substances in the articular cavity, and collected synovial uid 1.5 mL. HA was injected in same site to cure TMD. synovial uid was centrifuged timely by centrifuge (12000 r/min, 15 minutes, 4℃) and frozen in − 80℃ refrigerator lastly.
The test can assay the original content of unknown samples according to the variance of standard curve which is made based on the absorption value of standard sample. Statistical analysis SPSS21.0 software package was applied to statistical analysis and recording data. Values were present as mean and standard deviation (SD). Before the comparison, Shapiro-Wilks test was used to assess the normality and data distribution, the homogeneity of variance was tested with Leven's test. Statistical signi cance was assessed by Student's t test if the homogeneity of variance was equal or evaluated by Satterthwaite's if the homogeneity of variance was not consistent for two compared group. Data was determined with Paired t-test before and after twice treatment. there was a signi cance between two groups or self-control group if P 0.05. Scissors-bite (n) 4 7 Overbite (n) 6 7 Other types of malocclusion (n) 17 11 Joint pain (n) 7 6 Limited mouth opening (n) 9 11 Asymmetrical face (n) 1 4 Clinical effects on TMJOA group with twice HA injections Temporomandibular joint dysfunction was relieved after second treatment. There were increased mouth opening and decreased visual analogue scale (VAS) score in TMJOA patients with twice HA injections, P 0.001 ( Table 2).

Concentrations of pyroptosis related molecules
The positive expressions of IL-1β, IL-18, NLRP3, CASP1 were all detected in two groups, However, there were higher concentrations of above molecules in TMJOA group compared with TMJID group, P 0.05 (Table 3; Fig. 2).  Discussion Condyle cartilage defect can hardly self-heal, which is related to in ammation. Pyroptosis participates chronic in ammation, so we hypothesized that pyroptosis might exist in TMJOA. Our research proved that IL-1β, IL-18, NLRP3 and CASP1 were higher expressed in degenerative TMJOA synovial uid, which were consistent with pyroptosis molecules. Thus, pyropyosis of chondrocytes or synovial cells may be exist in condylar degeneration. Current data also indicated that HA protected condylar from progressive degradation by means of relieving clinical symptoms and decreasing some pyroptosis molecules. These results provide a basis for further study on the mechanism of TMJOA.
In ammation is a signal in progressive articular tissue damage. Previous studies have shown that the expressions of in ammatory mediators included interleukin (IL), matrix metalloproteinase (MMP) and prostaglandin E2 (PGE2) in the synovial uid of OA patients are higher than that of the normal [12,13]. Besides, in ammation also breaks down the catabolism of matrix and leads deterioration of internal environment. Speci c anti-in ammation therapy may provide a new clue to prevent cartilage from damaging. Meanwhile, pyroptosis is the highlighted topic in in ammation disease recently, which can connect with cartilage degradation scienti cally because its in ammation essence. Osseous changes generally occur in response to TMJ in ammation while CBCT image shows hard-tissues clearly [14,15]. Our research is scienti c in grouping according to CBCT or MRI to distinguish condylar destruction accurately.
IL-1β and IL-18 are cell lysis products in cell pyroptosis, participating a series of in ammatory and autoimmune diseases, these two cytokines can amplify in ammation through multiple pathways and contribute to the occurrence of pyroptosis [16][17][18]. Some studies have proved a higher expression on IL-1β in TMJOA, However, IL-18 was reported rarely. Our study are consistent with above studies at higher concentration of IL-1β and justi ed a new result of higher IL-18 concentration. NLRP3, one of the most speci c upstream signal protein in cell pyroptosis, participating canonical in ammasome of pyroptosis mediated by CASP1, then eliciting plasma membrane pore formation, mitochondrial dysfunction and lysosomal rupture. A study revealed collagen-induced arthritis had a high expression of NLRP3, antiin ammatory therapy targeting NLRP3 might become a new method for the treatment of OA [19].
Moreover, Synovial in ammation is closely related to NLRP3 and CASP1 [20], Pyroptosis of synovial broblasts in knee osteoarthritis mediates synovial in ammation. But the cytological mechanism of temporomandibular joint may be different from primary knee joint because its special structure charts included brous layer. Thus, we explore pyroptosis in temporomandibular joint with its unique brocartilage structure in this research. Despite of a low positive concentration of NLRP3 in two groups, but higher concentrations on IL-1β, IL-18, NLRP3 and CASP1 in TMJOA patients compared with TMJID patients provided possibility to pyroptosis.
The occurrence of pyroptosis depends on a precise signal regulation, it does not occur unless the DAMPs or PAMPs were stimulated, intracellular signal is activated subsequently, DAMPs as endogenous molecular structures released to extracellular environment through tissue injury or hypoxia stimulation, participate canonical pathway of pyroptosis controlled by CASP1 [21]. PAMPs comprise molecular structures that are found in microbes included lipopolysaccharide (LPS), which can take part in canonical and non-canonical pathway of pyroptosis controlled by CASP1 or CASP-4/5/11. A recent study proved that the expression of CASP1 increased signi cantly in knee osteoarthritis cartilage compared with normal cartilage tissue. TMJ is a rotating-hinge joint, which is suitable for occlusal mechanical environment, so we believed that DAMPs were main stimulation because temporomandibular joint lived in a sterile environment and accepted with biomechanical stimulation such as stress, uid shear stress transmitted by abnormal occlusal contact. Previous reports indicated unilateral anterior crossbite model built in sprague-dawley (SD) rats could induce OA lesions via apoptosis or necrosis of chondrocytes and matrix loss [22]. In this study, the malocclusion is most probably source of DAMPs because all patients in two groups were accompanied with oral malocclusion.
Additionally, We tried to evaluate therapeutic effect of twice HA injection therapy on pyroptosis molecules. Application of HA as empirical intra-articular injection plays an important role in the protection, nutrition and function of joints [23]. HA also regulates electrolyte and water in extracellular uid, lubricates joints, resists infection. Our current research also veri ed its anti-in ammatory effect due to a relieved symptoms and decreased concentrations of IL-1β, IL-18, NLRP3. Although there is no statistical signi cance on concentration of CASP1 between twice HA injections, which may be because a short time of follow-up can not establish a complete immune mechanism.
Currently, Targeted drug therapy tends to be mature. Inhibitor therapy targeting above pyroptosis molecules may become a new resolution for progressive osteoarthritis once mechanical stress can not be relieved immediately, which also makes individualized articular joint destruction treatment posssible.
In conclusion, pyroptosis related molecules were higher expressed in OA patients. pyroptosis of chondrocytes or synovial cells might be a new reason for cartilage degradation, and empirical HA' intraarticular injection was effective in reliving clinical symptoms and decreasing some in ammatory factors.