The effect of MSM in the treatment of ankle arthrosis: Is MSM as effective as methylprednisolone or hyaluronic acid?

Posttraumatic ankle osteoarthritis (PTAO) causes severe ankle and adjacent joint morbidity. We aimed to compare the treatment efficacy of previously tried and still applied intra‐articular injections and oral methylsulfonylmethane (MSM) at functional and histopathological level in PTAO animal model. Thirty‐two adult female Sprague‐Dawley rats were divided into four groups (Group 1: Control, Group 2: 0.06 g/kg/day MSM, Group 3: 0.04 mg/µL methylprednisolone [MP], Group 4: 0.04 mg/µL hyaluronic acid [HA]). MSM was started orally between Day 0 to the end of 8 weeks. Intra‐articular injections were applied to the right ankles of the subjects after surgery. All subjects were killed after radiological evaluation at the 8th week. Subsequently, functional (range of motion) and histopathological evaluation was performed. Radiological evaluation showed better results of the MP (p < 0.001) and MSM (p < 0.001) groups than the control group. Severity of osteoarthritis (OA) in the MP group was significantly less than in the HA group (p = 0.032). When the total Osteoarthritis Research Society International score was compared, the severity of OA was higher in the KS and HA groups than in the control group (p < 0.001). No significant statistical difference was found in the histopathological comparison of MSM and control group (p = 0.466). There was no difference between the groups in range of motion measurement according to the contralateral ankle joint. The radiological progression of OA was slowed in the MSM and MP groups, but significant histopathological worsening was found in the MP and HA applied groups. We suggest that the treatment methods used in daily practice need to be reviewed.

therapies, orthoses, and physical therapy are primarily applied in the clinic in patients who develop ankle PTAO.Ankle arthrodesis and arthroplasty are available as surgical options. 1,24][5] On the other hand, despite developing technology and new prosthesis designs, ankle arthroplasty has a complication rate of up to 20% and is likely to cause disability after surgery. 4,6Therefore, recent studies focus on PTAO as a new clinical entity, emphasizing conservative treatment approaches.
In the conservative treatment of OA, hyaluronic acid (HA), and methylprednisolone (MP) injections have often been used for many years. 7,8In addition to these injections, glucosamine chondroitin complexes are frequently used to treat OA. 9 These supplements have been combined with anti-inflammatory and antioxidant agents to increase their effectiveness, of which methylsulfonylmethane (MSM) has been most commonly used recently. 10Although many studies have reported that all these methods reduce pain by reducing inflammation and have a protective effect on chondrocytes, the results are inconsistent, and no consensus has been reached.Moreover, almost all of these studies are conducted for knee and hip OA. [7][8][9] Considering their biomechanical structure, the cartilage metabolism and the thickness of the ankle joint are different from those of the knee and hip joints, and data on the effect of these treatments on ankle OA are limited. 2M is a popular dietary supplement that has been extensively studied in various areas of medical research in recent years. 102][13] Although MSM is frequently used daily, its precise contribution to the development process of OA has not been definitively demonstrated in the literature.This experimental study aimed to compare the treatment efficacy of intra-articular HA and MP injections and oral MSM at the functional and histopathological levels in the PTAO animal model.

| Study design and experimental groups
A power analysis was conducted before the formation of experimental groups.The analysis revealed that a minimum of 24 rats with an effect width of f = 1 for one-way analysis of variance would provide 90% test power at a 95% confidence level, with six rats in each group.Another calculation method, the "source equation" method, commonly used in animal studies, was also used.Based on this method, 32 female rats, with eight rats in each group, were deemed sufficient for the experiments.Therefore, this study was conducted using 32 Sprague-Dawley rats, aged 12−18 months, weighing 250−320 g, randomly assigned to one of four groups: control group (C), MSM group, MP group, and HA group (Table 1).
Separate color code was determined for each group, and subject numbers were written on their tails in the color appropriate to their group.The subjects were kept at a room temperature of 25°C in a 12 h light, 12 h dark cycle.No food or water restrictions were applied.

| Surgical procedure
All animals underwent the same standard surgical operation performed by the same surgeon.Before surgery, general anesthesia was induced using 60 mg/kg Ketamine Hydrochloride (Keta-Control ® ; Mefar Pharmaceuticals) and 7 mg/kg Xylazine Hydrochloride (VetaXyl ® ; VET-AGRO Multi-Trade Company Sp.) administered intraperitoneally.The surgical site on the right ankle was shaved using a surgical clipper.The rats were placed in the supine position, and their right front and hind legs were secured with tape to a foam board.The surgical site was cleaned with a 10% povidoneiodine antiseptic solution (Batticon ® ).Medial malleolus osteotomy was performed with a surgical chisel on the right ankle of all subjects in each group, following the principles of Liang et al. for creating the PTAO model in rats. 2 During surgery, a surgical loupe was used, and a 20 mm incision was made over the medial malleolus of the right ankle.After subcutaneous dissection, the medial malleolus was exposed.A region 15 mm proximal to the distal end of the medial malleolus was marked.An  oblique osteotomy was performed in this region with an angle of 30-40°and involving at least one-third of the tibia using an osteotomy tool.The osteotomy was confirmed during the surgery from the anterior surface of the tibia.No fixation was applied to the osteotomy line after the procedure (Figure 1).Intraperitoneal enrofloxacin 10 mg/kg (Baytril; Bayer HealthCare AG) was administered for surgical infection prophylaxis.The rats were given intraperitoneal fentanyl at 0.02 mg/kg (fentanyl; Janssen-Cilag Pty. Ltd.) immediately after surgery as an analgesic.There was no specific restriction on their movements or activities after surgery.
After 8 weeks, the rats in all four groups were killed using ketamine 100 mg/kg (Keta-Control ® ; Mefar Pharmaceuticals).Both the osteotomized legs and the contralateral legs were amputated at the level of the tibia.After the comparative functional analysis, the extremity that underwent osteotomy was taken into the preparation process for histopathological analysis.

| MSM treatments
After ankle osteotomy, depending on the development of OA at both molecular and radiological levels within 8 weeks, MSM was administered orally to the subjects in the MSM group using the gavage method at a dose of 0.06 g/kg/day from Day 0 to Week 8.The same dose of MSM was given to the animals in this group at the same time every day for 8 weeks.No side effects were observed after MSM administration.

| Injection treatments
Due to the 2-4 week period being the most effective for the impact of intra-articular injections, the MP and HA groups were administered intraarticular injections under analgesia to the ankles, where osteotomy was performed on the first day of the 2nd, 3rd, and 4th weeks. 14The injections were administered to the anteromedial region of the ankle joint using a 30-G fine needle under fluoroscopy control (Figure 2).In the MP group, 50 µL of Prilocaine hydrochloride (Priloc ® ; Vem Pharmaceuticals) plus 0.04 mg/µL MP acetate (Depo-Medrol ® ; Pfize) was injected into each ankle joint.In the HA group, 50 µL of isolated HA (Ostenil ® ; Trb Chemedica SA) was injected into each ankle joint without dilution.After each dose, ankle joint movements and foot circulation were checked.

| Radiological analysis
Lateral and AP radiographs were taken for the osteotomized ankle on the sixth day of the 8th week.The radiographs were obtained from the hospital's Picture Archiving and Communication Systems and scored using the Ankle and Hindfoot Radiographic Osteoarthritis (AHRO) scoring system (Figure 3).

| Histopathological analysis
The tissues, fixed in 10% formalin solution, were decalcified and then embedded in paraffin blocks after the follow-up process.
Section (5-µm-thick) from all samples were stained with hematoxylineosin (H&E) and prepared for microscopic examination.All samples were evaluated by a pathologist who was blind to the nature of the groups.The modified Mankin grading system and the Osteoarthritis Research Society International (OARSI) scoring system were used in the microscopic evaluation of joint cartilage in OA models (Figure 4).

| Functional analysis
Both osteotomised and non-osteomised ankle joints were amputated from the middle of the tibia to compare the range of motion between the pathological and non-pathological ankle joints.Range of motions were measured and recorded using a standard measurement template drawn with an angle-measuring device in maximum dorsiflexion and plantarflexion movements (Figure 5).

| Statistical analysis
The Table 2 shows the AHRO scores obtained after radiological evaluation of experimental animals at 8 weeks postsurgery and their comparison between groups.It was determined that there was a statistically significant difference between the groups (p < 0.001).The difference between the control group and the MP and MSM groups was statistically significant (p < 0.001; p = 0.001).In addition, severity of OA in the MP group was significantly less than in the HA group (p = 0.032).
H&E staining was used for the existing parameters in the OARSI scoring.The total scores of the groups were compared, and their descriptive statistics were defined in based on histological evaluation.The histological evaluation scores show a significant difference between the groups (p < 0.001).The difference between the control group and the HA (p = 0.001) and MP (p < 0.001) groups was found to be significant while there was no statistically significant difference between control group and MSM group (p = 0.466) (Table 3).
The results of the measurements of the difference in joint range of motion obtained for functional assessment are defined in Table 4.
In the comparison of joint range of motion in functional evaluation, no significant difference was found between the groups regarding dorsiflexion (p = 0.103) and plantarflexion (p = 0.771) movements.

| DISCUSSION
[17] Although, there are many studies on knee and hip OA, successful results have not been obtained in either conservative or surgical treatment of ankle OA.As the surgical methods are limited to arthrodesis, distraction arthroplasty, and joint arthroplasty, and the results obtained from these surgeries are less satisfactory than the knee and hip joints, this has led to a prevailing preference for conservative approaches to slow down or prevent the progression of the disease in contemporary practice. 4,6,10,18,19 this study, we examined the effect of MSM on ankle OA.MSM is added to supplements containing chondroitin sulfate, HA, and glucosamine and is frequently used in conservative treatment.1][22] We are observed. 2Although the number of animal studies evaluating the radiological effects of intra-articular injection therapies is limited in the literature, there is a study evaluating the effect of HA and MP application on the radiological findings of OA in the knee joint.
However, no study has evaluated the radiological findings in the ankle joint.Tanideh et al. used 70 Sprague-Dawley male rats and induced knee OA by cutting the anterior cruciate ligament.Their study showed that 1 mg HA and 1 mg MP, started immediately after surgery, significantly increased radiological recovery compared to the control group. 23In our study, despite administering 2 mg MP and 2 mg HA to the ankle joints, we did not observe a positive radiological effect of HA.However, we did find potential positive effects of MP and MSM.This may be because we started the injections in the 2nd week.On the other hand, it is possible that the biomechanical properties, cartilage thickness, and metabolism of the ankle joint are different from those of the knee joint.Moreover, we could not evaluate whether their combination has an additive effect on the treatment.In future studies, it would also be appropriate to examine the effects of combined applications.
It is known that PTAO affects ankle function and leads to functional loss as the severity of OA worsens.Thus, the most significant finding of OA after joint pain is the reduction in joint range of motion, which reflects functional impairment.In the case of the ankle joint, the range of motion is typically evaluated through dorsiflexion and plantarflexion of the talocrural joint.A study by Eerdekens et al. indicated that the loss of joint range of motion in the ankle negatively affects walking kinematics and cannot be compensated. 24Therefore, the joint's range of motion is an important parameter in assessing OA progression.In this study, to overcome potential differences among subjects in joint range of motion, we compared the intervened ankles with the contralateral ankles of the same subject in the evaluation of joint range of motion.The evaluation results did not reveal any statistically significant differences between the groups, suggesting that functional outcomes may not necessarily parallel the histopathological and radiological findings.The utilization of the ankle joint's range of motion alone in functional assessment has imposed a limitation on our evaluation.Therefore, it is crucial to consider incorporating parameters related to pain assessment or walking kinematics to enhance the meaningfulness of functional assessment.
These additional parameters should be considered for a more comprehensive and informative evaluation of functional outcomes.
There are limited studies evaluating potential treatment options for cartilage damage resulting from traumatic ankle injuries and subsequent OA models like our study.However, Jimbo et al. demonstrated-in a rat model of ankle OA induced by monosodium iodoacetate-that intraarticular administration of HA reduced pain sensation and histopathologically inhibited PTAO development to a mild extent. 25Additionally, a review study conducted by McCrum et al. on MP injections showed that anti-inflammatory agents could adversely affect fracture healing in the resulting inflammatory environment. 26In the same review, it was also emphasized that repeated injections can lead to secondary cartilage loss.
In our study, based on the histopathological data obtained, MSM did not yield different results compared to the control group, while MP and HA injection treatments were shown to induce more severe OA than the control group.Due to the ankle being a small joint compared to the knee, the injection application can be anticipated to cause worsening histopathological changes in the MP and HA groups, leading to secondary cartilage loss.We hypothesized that other reasons for the different results in our study could include the detrimental effect of the anti-inflammatory environment created after the fracture on cartilage healing and the early triggering of the degenerative process in the ankle OA model induced by monosodium iodoacetate.Therefore, in future studies, the potential negative effects of early administration of antiinflammatory agents in the postfracture setting should not be overlooked to gain a clearer understanding of the etiology of the process.
The major limitation of our study is the inability (due to financial constraints) to examine the effects of MP, HA, and MSM at the molecular level and to evaluate the relationship between these effects and the molecular steps suggested in the literature.On the other hand, the fact that the ankle joint is smaller than the knee and hip joint in rats may have increased the risk of iatrogenic injury during injection treatments and thus may have affected the results.Moreover, it was not possible to evaluate the cartilage damage with advanced imaging methods, such as MRI or microCT.In addition, gait and pain analysis could be included in the study in addition to measurements of the joint's range of motion, making it more comprehensive for functional assessment.Finally, in this study, the effects of MP, HA, and MSM treatments were evaluated alone, but the effects of their combinations could not be evaluated.
In conclusion, PTAO is a complication that can arise-despite proper management by a surgeon-due to the unique physiology and dynamics of the ankle.Currently, many treatments are used to prevent or slow down this process, but studies in the literature on these preparations are limited.Therefore, it is challenging to come to a conclusion regarding their definitive benefits based on the available data to date.Based on the data obtained from our study, it was determined that MSM, which has a lower side effect profile, may be equally effective as HA and corticosteroids.However, further studies incorporating more combinations are needed to obtain more definitive results.

T A B L E 1
Study groups.
statistical analysis was performed using IBM SPSS Statistics for Windows Version 25.0.Normality tests were performed to check the F I G U R E 1 Creating an osteoarthritis model by medial malleolus osteotomy in an experimental animal.(A) Medial malleolus exposure with surgical dissection.(B) Determining the medial malleolus osteotomy point.(C) Medial malleolus osteomy.(D) Intraoperative view of the osteotomy line.F I G U R E 2 Application of injection therapy to the ankle of rats.F I G U R E 3 AHRO scoring system.AHRO, ankle and hindfoot radiographic osteoarthritis; HA, hyaluronic acid; MP, methylprednisolone; MSM, methylsulfonylmethane.normal distribution of variables using the Kolmogorov−Smirnov and Shapiro−Wilk tests.Descriptive statistics were presented for quantitative variables, including the mean, standard deviation, median, minimum, and maximum values.The Kruskal−Wallis test was used for nonparametric variables to compare groups, and the Bonferroni multiple-comparison test was used to determine significant differences between groups.Categorical variables were summarized using count and percentage values and analyzed using the Pearson's χ 2 or Fisher exact tests.A p-value less than 0.05 was considered statistically significant.F I G U R E 4 (A) Control group (H&E, 5xpng.magnification).(B) MSM group showing grade 0 chondrocyte necrosis.There is minimal inflammatoryinfiltration, vascular proliferation, and edema in the synovial membrane.Thereare minimal changes in the subchondral bone (H&E, 5xpng.magnification).(C) MP group showing grade 4 chondrocyte necrosis.Increased number of necroticcells (empty lacunes) in all layers of the cartilage structure in the jointspace.Severe mixed type inflammatory infiltration and vascular proliferationin the synovial membrane.Note the destruction, osteochondral tissue loss andulceration in the subchondral bone (H&E, 5xpng.magnification).(D) HA group showing grade 4 chondrocyte necrosis.Increased number of necroticcells (empty lacunes) in all layers of the cartilage structure in the jointspace.There is no inflammatory infiltration in the synovial membrane.Mildvascular proliferation and intimal hyperplasia and edema were observed.Thereare minimal changes in the subchondral bone (H&E, 5xpng.magnification).F I G U R E 5 Assessment of ankle range of motion.(A) Creating the measurement ground using a goniometer.(B) Measurement of joint range of motion after sacrification.The study was carried out between June and August 2022.In this study, no animals from any group died or were excluded from the study in the postoperative period.No complications were observed, such as wound infection, hematoma, circulatory disorders, or nerve damage associated with the related osteotomy.No treatment-related complications were observed among the groups.
All experiments concerning animal protection for experimental use were carried out in accordance with the European Communities Council Directive of 24 November 1986, 60869-0 EEC.The experiments received approval from our institution's Ethics Committee for Animal Research (DA22/16).All the experiments were carried out at our institution's Animal Experiments Laboratory and were supervised by veterinarians and researchers licensed to use experimental animals.
the efficacy of MSM by comparing it with the results obtained from HA and MP injections, which are frequently used in daily clinical practice in the conservative treatment of OA.Our study showed that MSM and MP application reduced the radiological findings of OA.Bonferroni-corrected pairwise comparisons, there is no difference between methods with the same letter; p < 0.05 is statistically significant.Comparison of total OARSI scores.
However, neither MSM nor MP and HA affected the joint's range of motion.Interestingly, it was determined that MP and HA injections exacerbated synovial inflammation and worsened histological findings, but MSM application had no effect at the histopathological level.Radiology plays a vital role in the clinical evaluation of OA.Liang et al., who developed a PTAO model in rats with the method of medial malleolus osteotomy for the ankle, showed in their study that OA develops after osteotomy, both radiologically and histologically, like human ankle OA.In the 8th week after osteotomy, the ankle joint space narrows, and subchondral sclerosis and osteophyte formations T A B L E 2 Comparison of AHRO scores.*Kruskal−Wallis test.a,b,c,d Abbreviations: HA, hyaluronic acid; MP, methylprednisolone; MSM, methylsulfonylmethane; OARSI, Osteoarthritis Research Society International.*Kruskal−Wallis test.a,b Bonferroni-corrected pairwise comparisons, there is no difference between methods with the same letter; p < 0.05 is statistically significant.