Condylotomy to Reverse Temporomandibular Joint Osteoarthritis in Rabbits

ANESTHESIA/TMJ DISORDERS/FACIAL PAIN

Condylotomy to Reverse Temporomandibular Joint Osteoarthritis in Rabbits Edela Puricelli, DDS, PhD,* Felipe Ernesto Artuzi, DDS, MSc, PhD,y Deise Ponzoni, DDS, MSc, PhD,z and Alexandre Silva Quevedo, DDS, PhDx Purpose:

Osteoarthritis (OA) of the temporomandibular joint (TMJ) is characterized by local tissue degeneration and pain. Treatments have been aimed at reducing symptoms, and only some can interfere with the progression of pathophysiologic changes caused by OA. Condylotomy is an alternative treatment for patients with OA refractory to conventional treatment. The aim of the present animal study was to investigate the influence of condylotomy on TMJ OA in New Zealand rabbits.

Materials and Methods:

The 36 rabbits were divided into 3 groups: the osteoarthritis group (OG; induced OA), treatment group (TG; induced OA plus condylotomy), and control group (CG; neither OA nor surgery). OA was induced using intra-articular monosodium iodoacetate injection for 40 days. The rabbits in the TG underwent condylotomy and were killed 20, 40, and 60 days after treatment. The rabbits in the CG and OG were killed at the same points.

Results:

The articular joint condition was better in the TG 60 days after surgery (P = .032). A direct comparison revealed regression of TMJ OA over time among the treated rabbits (P = .008). Surgical treatment promoted mandibular condylar remodeling in the TG, reversing the tissue degeneration caused by OA. Conclusions: Our findings suggest that condylotomy could be an option for the treatment of OA and prevent damage to TMJ structures. This could be of particular importance for patients without satisfactory responses to more conservative treatment. Ó 2019 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg -:1-15, 2019

*Full Professor, Universidade Federal do Rio Grande do Sul; Oral and Maxillofacial Surgery Unit, Hospital de Clı´nicas de Porto

Clı´nicas de Porto Alegre (HCPA) under grant no. 100409. The funder

Alegre; Universidade Federal do Rio Grande do Sul School of

publish, or preparation of the manuscript.

had no role in study design, data collection and analysis, decision to

Dentistry, Porto Alegre, Rio Grande do Sul, Brazil.

Conflict of Interest Disclosures: None of the authors have any

ySurgeon, Oral and Maxillofacial Surgery Unit, Hospital de Clı´nicas de Porto Alegre; Graduate Program in Dentistry, Universidade Federal do Rio Grande do Sul, School of Dentistry,

relevant financial relationship(s) with a commercial interest.

Porto Alegre, Rio Grande do Sul, Brazil.

100409). The funder had no role in the study design, data collection

zAssociate Professor, Universidade Federal do Rio Grande do Sul; Head, Oral and Maxillofacial Surgery Unit, Hospital de Clı´nicas de

and analysis, decision to publish, or preparation of the manuscript.

Porto Alegre; Graduate Program in Dentistry, Universidade Federal

School of Dentistry, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2492, Santa Cecı´lia, Porto Alegre, RS 90035-

The present research was supported by the Fund for Research and Event Promotion of the Hospital de Clı´nicas de Porto Alegre (grant

Address correspondence and reprint requests to Dr Artuzi:

do Rio Grande do Sul, School of Dentistry, Porto Alegre, Rio Grande do Sul, Brazil.

004, Brazil; e-mail: [email protected]

xAssistant Professor, Universidade Federal do Rio Grande do Sul; Graduate Program in Neuroscience, Universidade Federal do Rio

Received November 19 2018 Accepted April 19 2019

Grande do Sul, School of Dentistry, Porto Alegre, Rio Grande do

Ó 2019 American Association of Oral and Maxillofacial Surgeons

Sul, Brazil.

0278-2391/19/30455-0

Drs Puricelli and Artuzi contributed equally.

https://doi.org/10.1016/j.joms.2019.04.024

This research was supported by the Brazilian funding agency Fund for Research and Event Promotion (FIPE) of the Hospital de

1

2 Osteoarthritis (OA) is a degenerative joint disease presenting with a low-grade inflammatory reaction mainly characterized by joint cartilage erosion and abnormal turnover of subchondral bone tissue.1 In the temporomandibular joint (TMJ), OA will usually be clinically associated with disc displacement and a reduced intra-articular space.2 These changes result in functional overload and the initial loss of superficial anatomic structures of the TMJ, leading to signs and symptoms such as pain, clicking noises, and limited mouth opening.3,4 The articular surface of the TMJ plays a major role in distributing the strength resulting from joint function. However, an increased joint workload and structure imbalances can decrease tissue adaptive capacity and promote progression to pathologic conditions. Therefore, TMJ OA results primarily from trauma or acute or chronic joint overload,1 with excessive mechanical stress one of the most common causes of OA.5 These etiological factors elicit biochemical responses that produce pathologic changes in the tissues surrounding and forming the TMJ. Chondrocytes, in particular, will respond directly to continuous compressive stimuli by increasing inflammatory cytokine production, leading to degradation of extracellular matrix components, such as the collagen network and proteoglycans.6 To restrain these detrimental changes in the articular surfaces, different therapeutic techniques have been suggested.7 Intraoral devices to reposition the condyle anteriorly have been indicated, especially in cases of TMJ OA associated with chronic TMJ derangement.8 However, these devices have lacked efficacy in several patients.9 For patients for whom clinical approaches have proved ineffective, surgical procedures performed directly on the TMJ, such as arthroplasty, have been used as a therapeutic option.7 Nonetheless, concerns exist regarding the possible postoperative complications resulting from direct arthroplasty, including malocclusion, facial asymmetry, and TMJ ankylosis.1 In contrast, condylotomy enables an indirect surgical intervention in the TMJ, promoting anterior repositioning of the mandibular condyle. This mobilization of the condylar segment changes the forces acting on the TMJ, thus reducing the overload. Additionally, condylotomy has been related to disc recapture, increased articular space, and reduced painful symptoms.10 The therapeutic benefits of condylotomy have been attributed to the structural changes promoted by the procedure, making it an alternative treatment for temporomandibular dysfunction unresponsive to conventional treatment.10 Condylotomy will effectively reduce symptoms in patients with TMJ derangement, promoting progressive condylar remodeling, as observed on imaging tests.11,12 The TMJ has this

CONDYLOTOMY TO REVERSE TMJ OA IN RABBITS

adaptive remodeling capacity in both childhood and adulthood.13 The effects of condylotomy have also been investigated in experimental studies of intact joints.14,15 Experiments in young rats revealed that chondrocyte maturation will be accelerated and endochondral ossification strengthened when the condyle has been positioned anteriorly in the glenoid fossa.16 However, limited histologic data are available about the effect of condylotomy on the degenerative changes in the TMJ. Thus, systematic investigations using animal models are needed to better understand the mechanisms involved in surgical techniques for the treatment of TMJ OA. Understanding of the effects of condylar repositioning on the TMJ could be especially helpful in developing more effective treatments for TMJ OA. Therefore, the present study investigated, using systematic histologic analysis (at 20, 40, and 60 days), the effects of condylotomy on the prevention and reversal of changes in the articular surfaces of the TMJ in rabbits using a chemically induced OA model.

Materials and Methods ANIMALS

A total of 36 male New Zealand rabbits (Oryctolagus cuniculus L.) aged 6 months and weighing 3 to 4 kg were used in the present study. The rabbits were housed in individual cages in a temperature (21  1 C) and humidity controlled room and had ad libitum access to water and rabbit chow. All the rabbits were maintained in a standard 12:12 light/dark cycle (lights on at 7:00 AM and off at 7:00 PM). The present study was performed in accordance with Law no. 11794/2008, which establishes the procedures for use of animals in research, in compliance with current legislation in Brazil. Therefore, animal handling and all experiments were performed in accordance with international guidelines for animal welfare, and measures were taken to minimize animal pain and discomfort. The experiment used the number of animals necessary to produce reliable scientific data.3 Also, the animal care and use committee of Hospital de Clı´nicas de Porto Alegre approved the present study (protocol HCPA 100409). EXPERIMENTAL DESIGN

The rabbits were randomly divided into 3 groups: control group (CG), OA group (OG), and treatment group (TG). The CG rabbits did not undergo induction of OA and only received an intra-articular injection of 50 mL of saline into the right TMJ. The OG and TG rabbits received an intra-articular injection of a solution

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containing 0.5 mg of monosodium iodoacetate (MIA) (sodium iodoacetate, I2512-25G; Sigma-Aldrich, St. Louis, MO) dissolved in 50 mL of saline solution (10 mg/mL) into the right TMJ to induce OA. The injection procedure was as follows. The precise injection site was marked 5 mm posterior and superior to the zygomatic process before the procedure. The injection was performed using a disposable standard insulin syringe and needle. We had previously conducted a pilot study in which 50 mL of methylene blue was injected into the TMJ of a single rabbit as a dye marker of the anatomic space available for injection of the MIA solution. Penetration of the joint capsule occurred at a depth of 8 mm.17 After the development of OA, which occurred after 40 days, the rabbits in the TG underwent condylotomy on the right TMJ. The rabbits in the CG and OG did not undergo surgical intervention. For each group, the rabbits were assigned to 1 of 3 subgroups according to the time of sacrifice: 20, 40, and 60 days. Therefore, each experimental group was randomly subdivided as follows: OG20 (n = 5), OG40 (n = 5), and OG60 (n = 5) and TG20 (n = 5), TG40 (n = 5), and TG60 (n = 5). The control rabbits (n = 6) were equally divided and euthanized at the same points used for the experimental groups (20, 40, and 60 days; Fig 1).

incision was made over the inferior body of the mandible, extending from the angular process up to 4 cm anteriorly. Subsequently, the masseter muscle was raised to expose the mandibular ramus (Fig 2A). After exposure of the cortical bone, an osteotomy was performed in the ramus. Two vertical fracture lines were marked 2 mm apart from each other, extending from the anterior region of the condyle and coronoid process to the mandibular basal bone (Fig 2B). After fracture, the 2-mm bone fragment was removed, and the posterior segment was positioned close to the anterior segment in the upper region of the ramus with a clockwise motion, creating a 4-mm crack between these segments in the basal bone. The condyle was moved to a more anterior and inferior position. Next, the bone segments of the mandibular ramus were stabilized using rigid internal fixation with a 4-hole microplate measuring 1.5  20 mm and microscrews measuring 1.5  3.5 mm (Com
ercio de Implantes Cir
urgicos Ltda, Porto Alegre, RS, Brazil; (Figs 2C, 3). The tissues were closed with simple sutures using 4-0 polyglactin wire for the internal planes and 4-0 monofilament nylon wire for the skin. After surgery, the rabbits were maintained under normal feeding conditions and had their weight stabilized. The surgical treatment we used was a modified version of the technique proposed by Puricelli et al.18

SURGICAL TECHNIQUE

The rabbits were anesthetized with ketamine (15 mg/kg), midazolam (1 mg/kg), and meperidine (5 mg/kg) intramuscularly. General anesthesia was induced with 5% isoflurane. Enrofloxacin (5 mg/kg) was injected intramuscularly to prevent infection. After shaving the area, skin antisepsis was performed using a 0.5% chlorhexidine-alcohol solution. Next, an

HISTOLOGIC PREPARATION

At the predetermined points, the rabbits were euthanized by intravenous injection of propofol (7 mg/kg body weight), followed by 10% potassium chloride (1 mL/kg body weight). The skulls were bisected medially along the sagittal plane, stored in buffered formalin for 2 weeks, and decalcified in 10%

FIGURE 1. Flow chart showing study design. CG, control group; CG20, control group at 20 days; CG40, control group at 40 days; CG60, control group 60 days; OA, osteoarthritis; OG, osteoarthritis group; OG20, osteoarthritis group at 20 days; OG40, osteoarthritis group at 40 days; OG60, osteoarthritis group at 60 days; TG, treatment group; TG20, treatment group at 20 days; TG40, treatment group at 40 days; TG60, treatment group at 60 days. Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

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CONDYLOTOMY TO REVERSE TMJ OA IN RABBITS

FIGURE 2. Condylotomy surgical procedure. A, Exposure of the mandibular ramus after elevation of the masseter muscle. B, Preparation osteotomy to release the bone segment. C, Placement of microplate and microscrews to fix the proximal segment anteriorly, in accordance with the study protocol. Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

nitric acid for 5 days. The decalcified TMJs were removed and sectioned in the sagittal plane. After preparation, the sections were stained with Safranin O/fast green. For histologic assessment of the degree of articular damage, a qualitative score was assigned in accordance with the Osteoarthritis Research Society International atlas, as described by Pritzker et al.19 This scoring system has been used to obtain an index of disease severity, with scores ranging from 0 (no OA) to 6 (maximum OA). The sections were examined by a single blinded observer, who had been trained and accredited by a pathologist specializing in pathologic anatomy and cytopathology. The intraobserver variability in the interpretation of histologic findings was assessed by comparing 2 histologic readings taken 10 days apart and computed using the kappa statistic.

STATISTICAL ANALYSIS

The Kolmogorov-Smirnov test was used to assess the normality of the data distribution. The disease severity in each group was analyzed using the Kruskal-Wallis nonparametric test, followed by Dunn’s post hoc test. The Mann-Whitney U test was used for overall comparisons of the median values between the CG and other groups (CG vs TG and CG vs OG), regardless of the time. Statistical analysis was performed using SPSS, version 19.0 (IBM Armonk, NY). The level of significance was set at 5% (P < .05).

Results ASSESSMENT OF OA SEVERITY

The intraobserver variability in the histopathologic grading of OA was low (kappa coefficient, 0.757).

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5

FIGURE 3. Schematic illustration of a rabbit skull representing the modified condylotomy technique. Note the projected osteotomy line in the mandibular ramus to move bone segments to a distance of 4 mm apart, with the proximal segment moved anteroinferiorly and a microplate fixed to stabilize the bone segments. Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

FIGURE 4. Box plot showing median values for assessment of osteoarthritis (OA) severity in different groups using the score proposed by Pritzker et al.19 The control group (CG) showed no signs of OA at any measurement period. The osteoarthritis group (OG) showed a high median value at all 3 points, showing that the disease severity remained stable throughout the study period. The median values for the treatment group (TG) were inversely proportional to the duration, showing that this group had responded to surgical treatment. The within-group effect was analyzed using the Kruskal-Wallis test, followed by Dunn’s post hoc test. Overall between-group analyses were performed using the MannWhitney U test. Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

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CONDYLOTOMY TO REVERSE TMJ OA IN RABBITS

FIGURE 5. Anatomic and histologic appearance of mandibular condylar surfaces. A, Control group. Temporomandibular joint (TMJ) with normal tissue structures, morphologically normal condyle, presence of a cartilage layer and intense Safranin O staining (black arrows), and intact articular disc (red arrow). B, Osteoarthritis (OA) group. TMJ with degenerative changes typical of OA, including flattening of joint surfaces, perforated articular disc (black arrows), areas of decreased Safranin O staining due to decreased proteoglycan content in cartilage matrix (red arrows), condylar surface erosion (blue arrow), and presence of articular intraosseous cyst (asterisk). C, Treatment group at 20 days showing significant regressive condylar remodeling with condylar flattening; the presence of a layer of fibrous connective tissue covering the subchondral bone tissue and the absence of a cartilage layer; no Safranin O staining (red arrows), markedly in the presence of proteoglycans; ruptured articular disc ends (black arrows); an important area of intraosseous degeneration, with an absence of trabeculae, suggesting the formation of an articular cyst (asterisk). (Fig 5 continued on next page.) Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

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FIGURE 5 (cont’d). D, Treatment group at 40 days showing decreased Safranin O staining in the cartilage surface (blue arrows), the presence of cartilage cracks (red arrows), ruptured articular disc ends (black arrows), and flattening of articular surfaces (asterisk). E, Treatment group at 60 days showing almost normal tissues, with an area of interruption of the cartilage layer in the anterior condyle (black arrow), normal cartilage layer covering almost the entire condylar surface (red arrows), an intact articular disc (asterisk), and Safranin O staining. Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

The distribution of the data was found to be not normal (P < .05), and the data were analyzed using nonparametric tests. The results of the assessment of OA severity are shown in Figure 4. General differences were found among CG, OG, and TG groups (c2 = 13.273; P = .001). Regardless of the point of evaluation (20, 40, or 60 days), the OA scores were lower in the CG compared with the OG (U = 0.000; P < .0001) and TG (U = 6.000; P < .001). None of the rabbits in the CG showed signs of TMJ disc degeneration, and the OA scores equaled 0 at all measurement points (c2 = 0.000; P = 1.00). In the OG, no difference was found in OA severity among the measurement points (c2 = 3.545; P = .170), showing that the effects of OA induction were stable over time, with no significant tissue changes during the study period. However, significant differences were found in OA severity in the subgroup of rabbits treated with condylotomy (TG; c2 = 7.682; P = .021). At the 20-day period, the TG rabbits had a greater OA index compared with the OG rabbits (OG20 < TG20; c2 = 2.500; P = .032). This might indicate acceleration in the initial remodeling process

owing to the new condylar position. However, no significant difference was found between the TG and OG at 40 days after condylotomy (OG40 = TG40; c2 = 11.500; P = .841). Similarly, no differences were observed between the TG20 and TG40 (c2 = 5.000; P = .151), indicating stabilization of the initial tissue changes promoted by the new condylar position. The articular joint condition was better in the TG rabbits at 60 days after surgery (OG60 > TG60; c2 = 2.500; P = .032). A direct comparison revealed a regression of TMJ OA over time among the treated rabbits (TG20 vs TG60; P = .008). Although an increase occurred in OA index during the 20-day period after surgery (OG20 vs TG20), improvements in the articular conditions over time (TG20 vs TG60) showed that condylotomy initially promotes joint remodeling and then reverses OA progression. HISTOLOGIC ANALYSIS

In rabbits, the normal condylar surface of the TMJ is entirely covered with fibrocartilage and has a convex morphology in its anterior region (Fig 5A). In the

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CONDYLOTOMY TO REVERSE TMJ OA IN RABBITS

FIGURE 6. Histologic appearance of mandibular condylar surfaces. A, Control group. Cartilage with intense Safranin O staining, indicating proteoglycan content (black arrows). Asterisk indicates the articular disc. B, Control group. Thin layer of fibrous connective tissue and a regular surface contour (red arrows); a normal arrangement of hypertrophic layer cells (black arrows). (Fig 6 continued on next page.) Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

CG, no degenerative changes were observed at any of the study measurement points. The articular layer showed intense Safranin O staining, indicating that the proteoglycan content of the extracellular matrix was normal (Fig 6A). The cellular components of cartilage subdivisions were at different stages of ossification and were morphologically normal, indicating tissue preservation (Fig 6B). Histologic analysis of the rabbits in the OG revealed major degenerative changes in the TMJ (Fig 5B). Morphologically, the artic-

ular surfaces were severely flattened. In addition, erosion was observed in the central portion of the condyle, in particular, with cartilage matrix loss (Fig 6C). A decrease in matrix thickness and Safranin O staining was observed at the condylar end, indicating decreased proteoglycan content. In addition, fibrillations, matrix cracks, and cell clustering were present (Fig 6D). Intraosseous articular cysts had formed in the subchondral region. Two joints showed perforation of the articular disc.

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FIGURE 6 (cont’d). C, Osteoarthritis group. Erosion with loss of demarcation of the cartilage outline and the absence of fibrous connective tissue (arrows). D, Osteoarthritis group. Areas of fibrillation reaching the deep zone of the cartilage (black arrows); a crack and absence of fibrous connective tissue (red arrow); and decreased Safranin O staining (asterisk).(Fig 6 continued on next page.) Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

The TG20 exhibited the most pronounced changes compared with any of the OG subgroups and was considered the group with the greatest degree of OA. Important regressive condylar remodeling and complete loss of hyaline cartilage were evidenced by the absence of Safranin O staining (Fig 5C). Intense bone remodeling was suggested by the presence of numerous osteoblasts and Howship’s lacunae containing active osteoclasts (Fig 6E). The bone surface was covered only with a layer of dense fibrous connective tissue, often surrounded by

blood vessels. Areas of heterotopic ossification were observed amidst the fibrous tissue (Fig 6F). The thickness of the tissue varied depending on the condylar region. Perforation of the articular disc was also observed. In the TG40, different degrees of OA severity were observed, ranging from cartilage matrix loss, flattening and condylar remodeling to the presence of cartilage (Fig 5D) and reduced Safranin O staining (Fig 6G). Superficial cracks and areas of fibrous connective tissue surrounded by blood vessels were also observed (Fig

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CONDYLOTOMY TO REVERSE TMJ OA IN RABBITS

FIGURE 6 (cont’d). E, Treatment group at 20 days. Anterior mandibular condyle with Howship’s lacunae containing active osteoclasts (black arrows) and osteoblasts (red arrows), evidencing intense bone remodeling; a blood vessel amidst the fibrous connective tissue coving the remodeled condyle (blue arrow). F, Treatment group at 20 days. Areas of ossification (black arrows); blood vessels (red arrows) amidst fibrous connective tissue (asterisk).(Fig 6 continued on next page.) Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

6H). In the TG60, most cases of joint degeneration were compatible with low-grade OA (Fig 5E), evidencing tissue recovery after condylotomy, in contrast to the OG60, in which the rabbits still presented with severe long-term joint degeneration. In the TG60, certain portions of the condyle showed a decrease in Safranin O staining in the joint cartilage, suggesting decreased proteoglycan content (Fig 6I). Some chondrocyte clusters were also found. Surface irregularities in advanced stages

of repair were observed, associated with reduced areas of cartilage matrix loss (Fig 6J).

Discussion The present data have shown that changing the condylar position through TMJ condylotomy can modify OA progression, promoting greater benefits at longer follow-up periods (60 days). Thus, this

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FIGURE 6 (cont’d). G, Treatment group at 40 days. Regions with no chondrocytes and loss of Safranin O staining (arrows). H, Treatment group at 40 days. Presence of fibrous connective tissue deeply surrounding the areas of cartilage cracks (red arrows); cracks (blue arrows); blood vessels amidst the connective tissue, suggesting the existence of a repair area (black arrows); cartilage with cell degeneration and decreased proteoglycan content (asterisk).(Fig 6 continued on next page.) Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

surgical procedure can be considered a therapeutic option for patients with OA unresponsive to conventional treatment. Another important aspect of the present study was the choice of animal model, because rabbits were found to be suitable for testing the surgical technique we investigated. The age of 6 months for

rabbits is approximately equivalent to 16 years in humans. Male rabbits reach puberty and sexual maturity at 5 to 6 months of age, although they are still not considered fully adult at this stage.20 The rabbit is one of several species in which sexual maturity coincides with age at cessation of bone growth. Thus,

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FIGURE 6 (cont’d). I, Treatment group at 60 days. Regularly arranged fibrous connective tissue (asterisk) and reduced cell density and decreased proteoglycan content (arrows). J, Treatment group at 60 days. Normal cartilage layer (red arrows); area of discontinuity of cartilage, suggesting the existence of a repair process (black arrow), Safranin O staining. Puricelli et al. Condylotomy to Reverse TMJ OA in Rabbits. J Oral Maxillofac Surg 2019.

the decision to use rabbits aged 6 months practically ensured the absence of additional longitudinal bone growth during the study period.21 However, maxillomandibular immobilization could not be maintained as recommended for condylotomy.22 Therefore, as an alternative to immobilization, internal rigid fixation was used to maintain the predetermined condylar position.23 This avoided any surgery-related bias, making it possible to assess articular function and provide the rabbits with adequate

nutrition. In the present study, wide undermining of the tissues for submandibular access made the condylar segment unstable. This technical issue, combined with the unfeasibility of maxillomandibular immobilization, made rigid fixation of the condyle necessary to ensure a standard position in all animals. In the traditional or conventional technique in humans, the condylar segment will be slightly lateralized, which was not performed in our experiment. Another difference between the modified condylotomy used in

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humans and the technique used in our study was detachment of the stylomandibular ligament and medial pterygoid muscle from the condylar segment, through the submandibular route, which allowed for wide exposure and mobilization of the condyle. In humans, because surgical access is obtained via the intraoral route, this detachment will be subtotal to prevent subluxation or medial displacement of the condyle. Furthermore, osteotomy was performed anterior to the coronoid process, owing to the small size of this structure in rabbits and its proximity to the mandibular condyle to prevent atypical fractures. With modified condylotomy, the fracture line will be in the region of the mandibular notch.22,24 Tissue remodeling is a biologic mechanism to maintain the balance between articular morphology and function. When functional efforts exceed the remodeling capacity, the TMJ conditions will progress to degenerative disease.25 In this process, chondrocytes are responsible for increasing metabolic activity and activating pathologic processes, leading to cartilage degeneration.26 Based on this principle, models of mechanical OA have been proposed.27 However, in the present study, we decided to use an MIA-induced OA model owing to the efficacy of this model in inducing OA without surgical intervention and promoting the long-term maintenance of the degenerative process.17 One of the advantages of the MIA model is that the stage and severity of OA can be controlled by modulating the concentration or dose injected.28 Initially, a substantial inflammatory response will occur in the synovial membrane. This response becomes less pronounced within 3 days and will have essentially resolved by day 7. Hence, this model elicits an acute inflammatory response that rapidly regresses and, in the medium and long-term, induces major degenerative changes in the joint.29 However, it causes only low-grade inflammatory changes. The MIA model differs markedly from other joint diseases such as rheumatoid arthritis, which is a systemic condition caused by an autoimmune process and features chronic high-grade inflammation.30 MIA induces OA in a dose- and time-dependent manner. The use of MIA for this purpose was first described by Kalbhen31 in 1987. Considering the design of the present study, with the late analysis of the surgical treatment group (#100 days), it was imperative that the disease remain both severe and highly stable for a prolonged period. Thus, owing to the demonstrated efficacy of the MIA model in previous studies,32,33 including research by our group,17 we selected this substance for induction of OA. Direct injection prevented the need for a surgical procedure to induce OA, which could have led to complications such as disc displacement34 or perforation.35 Furthermore, in the rabbit TMJ, the glenoid fossa is incom-

13 plete in the region posterior to the condyle, which provides an important and convenient access route to the articular cavity for precise experimental manipulation.36,37 In other studies,17,32 an adequate level of disease severity was observed 40 days after induction. Thus, condylotomy was performed at that point, when degenerative changes were already appreciable in the rabbit TMJ. Saline solution injected into the TMJ did not induce histologic changes in the joint tissues of the CG. At the first evaluation point, the treated rabbits (TG20) exhibited complete loss of cartilage matrix throughout the condyle, and bone trabeculae were covered with fibrous connective tissue (Figs 5C, 6E,F). At the same point, this process was less intense in the OG20 rabbits, in which areas of remaining cartilage were observed (Figs 5B, 6C,D). Similarly, previous studies have demonstrated an association between the loss or decrease of cartilage thickness and changes in the condylar position.23,38 This cartilage degeneration is caused by reduced synthesis of extracellular matrix components and increased chondrocyte degeneration.4 Sato et al39 found that areas of the articular eminences that experienced bone remodeling were covered with fibrous connective tissue after articular disc perforation in the TMJ of rabbits. The fibrous tissue covering the remodeling subchondral bone has the ability to absorb articular forces and, thus, can be used as a replacement for perforated articular discs,39 which could explain the present findings in the rabbits in TG20. Similar to tendons and ligaments, the layer of fibrous connective tissue consists mainly of fibroblasts, type I collagen fibers, and proteoglycans.15,40 Owing to the fibrous nature of its tissue composition, the elastic modulus of the mandibular condylar surface will be greater than that of the underlying cartilage, which is similar to that of hyaline, thus increasing the capacity of this surface to transmit and withstand tensile forces.40 However, when fibrosis and connective tissue deposition occur, such as in the present study, a change occurs in tissue function, making tissues more rigid and less extensible. This process can reveal the occurrence of a previous chronic inflammatory stage, characterized by the synthesis of type III collagen, which is typical of joints with OA. Nevertheless, this stage of predominant type III collagen is transient, and type III collagen will be replaced with large amounts of type I collagen, promoting changes in the size and arrangement of collagen fibers.41 In addition to proliferative, hypertrophic, and calcification zones, and unlike the hyaline cartilage, the TMJ cartilage is covered with fibrous connective tissue.40 According to a study by Hochman and Laskin,42 this tissue component might provide undifferentiated cells that could generate new chondrocytes in the case of

14 damage to the cartilage.43 Therefore, arthritic changes, especially traumatic ones, can be reversed as long as etiological factors are corrected.42 When the condylar surface is covered only with fibrous connective tissue surrounded by blood vessels, changes in the pattern of articular forces will promote tissue reorganization and a reduction in OA severity, as observed in the TG60 (Figs 5E, 6I,J). Resorption and regressive condylar remodeling (RCR) were observed at the first time point (20 days), with severe flattening of the articular surfaces. RCR covers the typical descriptions of degenerative diseases, such as OA and osteoarthrosis.44 At the same time point, similar to what occurred in the cartilage tissue, the treated rabbits showed greater loss of bone tissue than did the rabbits in the OA group (OG20 vs TG20). This process results from changes in the location and pattern of forces acting on the condylar surface after surgical treatment, leading the condyles to adapt to the new physiology. Yang and Hwang23 observed that increased joint overload after vertical osteotomy of the mandibular body exacerbated OA in rabbit condyles 4 weeks after the procedure. However, they found an increase in the forces acting on these structures, rather than the decrease observed in the present study.23 OA progression can stop at any stage, depending on the physiologic changes occurring in the joint.25 Condylotomy might enable this change by moving the condyle to a more anterior position.45 Thus, although an initial increase in tissue destruction occurred (OG20 vs TG20), a reduction in the changes caused by OA was found in the long-term. For example, OA severity was significantly reduced in the treated rabbits when comparing the OG60 and TG60. More importantly, disease reversal was observed, with the treated rabbits showing improvement over time (TG20 vs TG60). Therefore, it seems reasonable to suggest that joint tissue regeneration occurs, leading to reversal of the degenerative process. The effects of recovery from the articular changes caused by OA might not be attributable to spontaneous tissue improvement during the disease course because the chemically induced OA model remained stable. Also, in a previous study, this model remained stable for up to 100 days after induction.17 The mechanism of rehabilitation to compressive workloads involves activation of surface osteoblasts, increasing the formation of new appositional bone, and the osteoclasts remodel the bone tissue to better adapt to changes in the pattern of articular forces.46 The TMJ has an especially high capacity for growth and remodeling owing to chondrogenesis and endochondral ossification and will adapt to mechanical and positional changes.47 The processes of chondrogenesis and consequent endochondral bone formation observed in the mandibular condyle are regulated by

CONDYLOTOMY TO REVERSE TMJ OA IN RABBITS

several extracellular growth factors,48 such as insulin-like growth factor, transforming growth factor, fibroblast growth factor, and bone morphogenetic protein. When activated, these factors will promote important signaling for regulation of cell proliferation, differentiation, and maturation during chondrogenesis, which involves developing mesenchymal cells and chondrocytes.49 However, under normal conditions, TMJ fibrocartilage will be physiologically avascular. Therefore, the cartilage tissue has limited reparative potential, which might require the combined use of supplemental therapies, such as condylotomy.26,50 The more anterior position and condylar surface decompression might trigger a cascade of molecular reactions signaled by growth factors, allowing changes in the proliferation and growth of articular cartilage.13 The present findings suggest that this process might have been involved in the recovery of the rabbits in the TG. In conclusion, the present study has demonstrated an increase in the remodeling capacity of the mandibular condyle with OA after condylotomy. Although the procedure increased the initial levels of bone degeneration, it was able to induce long-term improvement. Regarding clinical activity, some therapeutic protocols, in which conservative treatment has proved ineffective, have shown that a delayed surgical indication can cause greater joint tissue loss. Therefore, when the response to treatment has not been adequate, it is important to reassess the options to decide when to abandon conventional techniques and recommend surgical intervention. The use of surgery could prevent greater functional changes, which would increase the complexity of subsequent treatment. Thus, further studies that include other variables, such as age, gender, and time of disease progression, are needed to increase our knowledge regarding the benefits and limitations of condylotomy as a surgical treatment of TMJ OA.

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