Effects of Balneotherapy in Jeju Magma-Seawater on Knee Osteoarthritis Model

Balneotherapy is a common non-pharmacological treatment for osteoarthritis (OA), however, the efficacy is controversial in knee OA. Jeju magma-seawater (JMS) has high contents of various minerals, which has anti-inflammatory and antioxidant properties via an oral route. Thus, we examined the effects of JMS bathing on knee OA and the combination effects with diclofenac sodium as an anti-inflammatory drug. Knee OA was induced by transection of the anterior cruciate ligament and the partial meniscectomy in rat. The rats were administered subcutaneously saline or diclofenac sodium in saline, followed by bathing in thermal distilled water or JMS for 8 weeks. The model represented the characteristic changes of the cartilage degradation, osteophyte formation and synovial inflammation, and the relevant symptoms of the joint swelling and stiffness. However, the JMS bathing reduced the joint thickness and improved the mobility. It also contributed to a well-preserved tissue supported by increases in bone mineral density of the joint and decreases in Mankin scores in the cartilages. The effects involved anti-inflammation, chondroprotection, anti-apoptosis, and chondrogenesis. Overall, the JMS bathing in combination with diclofenac sodium showed a similar trend associated with synergic effects. It suggests that JMS bathing can be promising for a clinical use in knee OA.


Results
Changes on thickness of knee joint and joint capsule. Our knee OA model exhibited joint swelling and palpable synovitis, however, the inflammation-related symptoms seemed to be alleviated by the JMS bathing with diclofenac sodium at 1 mg/kg and 2 mg/kg in saline (D1 + JMS and D2 + JMS groups, respectively) compared with the knee OA negative control (OA con) treated with saline injection (Sal) plus bathing in distilled water (DW). The kinetic change of knee thickness for 8 weeks was examined by two-way analysis of variance (ANOVA). There were significant main effects for the groups (F = 346.2; p < 0.01) and the week measured (F = 130.3; p < 0.01). There were also significant interactions between the groups and week (F = 255.9; p < 0.01). The post-hoc tests versus the sham group showed significant increases in the thickness of all knee OA model regardless of the treatments (p < 0.05, Fig. 1a). However, compared with the OA con, the thickness was significantly decreased in the D2 + DW, D1 + JMS and D2 + JMS groups on weeks 1 to 8 post-treatment and in the Sal + JMS on weeks 2 to 8 (p < 0.05). In particular, the thickness was decreased more in the D2 + JMS group than the D2 + DW on weeks 3 to 8 (p < 0.05). Although it increased in knee OA model compared with sham group after treatments for 8 weeks, it significantly decreased in all treatment groups, the D2 + DW, Sal + JMS, D1 + JMS and D2 + JMS (p < 0.01, Fig. 1b). The thickness was measured directly in the joint capsule exposed from surrounding tissues after all treatments. One-way ANOVA showed significant main effects for the groups (F = 20.5; p < 0.01). The post-hoc test versus the sham group revealed increases in the capsule thickness of the OA con; however, the test versus the OA con showed significant decreases in all treatment groups (p < 0.05, Fig. 1c). Consistently, the capsule thickness was decreased more in the D2 + JMS group than the D2 + DW (p < 0.05). There were no body weight changes among groups (see Supplementary Fig. S1).
Effects on the maximum extension angle of knee joint. The maximum extension angle was assessed for improvement of the knee joint stiffness. The angles appeared to be increased in the OA con group, indicating deteriorated knee mobility, but they seemed to be reduced in the treatment groups (Fig. 2a). One-way ANOVA showed significant main effects for the groups (F = 22.4; p < 0.01). The post-hoc test versus the sham group revealed significant increases in the maximum extension angles of the OA con; however, the test versus the OA con showed significant decreases in the treatment groups (p < 0.05, Fig. 2b). The angles were decreased more in the D2 + JMS group than the D2 + DW, suggesting the synergic effects of JMS bathing in combination with diclofenac sodium.
Effects on bone mineral density (BMD) and compressive strength. While dual-energy X-ray absorptiometry (DEXA) images of the knee joints depicted evident erosions and osteophyte formations in the OA con group, the lesions were mild in the treatment groups (Fig. 3a). For BMD of knee joint, one-way ANOVA showed significant main effects for group (F = 61.8; p < 0.01). The post-hoc test versus the sham group revealed decreases in the BMD of the OA con; however, the test versus the OA con showed significant increases in the treatment groups (p < 0.05, Fig. 3b). The data for BMD were further analyzed in both subchondral bones of the femur and tibia. One-way ANOVA showed significant main effects for the groups in both sides of the femur (F = 81.4; p < 0.01) and tibia (F = 147.0; p < 0.01). The post-hoc tests showed similar trends (Fig. 3c,d); lower in the OA con than the sham but higher in the treatment groups than the OA con (p < 0.05). Since BMD affects bone strength 35 , the compressive strength was assessed in both side cartilages (Fig. 3e,f). There were significant main effects for the groups in the femur (F = 63.4; p < 0.01, by ANOVA) and tibia (p < 0.01 by Kruskal-Wallis H). The post-hoc test versus the sham group revealed decreases in the compressive strength of both cartilages of the OA con, while the test versus the OA con showed significant increases in the treatment groups (p < 0.05). In Figure 1. Thickness of knee joint and joint capsule. Knee osteoarthritis (OA) rat model received an injection of saline (Sal) or diclofenac sodium in saline at 1 mg/kg (D1) or 2 mg/kg (D2), followed by bathing in thermal distilled water (DW) or Jeju magma-seawater (JMS). The group was designated according to the treatment of injection plus bathing. The OA control and the corresponding sham received Sal plus DW bathing. The kinetic changes on knee thickness and the relatives to Sham group at 8 week after treatment are indicated in (a,b), respectively. After all treatments, thickness of joint capsule is shown in (c). Values were expressed as means ± standard deviation (SD: eight samples/group). The kinetic changes were examined by two-way ANOVA as repeated measurements, and the others were by one-way ANOVA. **p < 0.01 versus sham group, ## p < 0.01 and # p < 0.05 versus OA con, and $$ p < 0.01 and $ p < 0.05 versus D2 + DW by LSD post-hoc tests.
www.nature.com/scientificreports www.nature.com/scientificreports/ particular, the data for BMD and compressive strength were higher in both cartilages of the D2 + JMS group than those of the D2 + DW (p < 0.05).
Effects on inflammatory and proteolytic activities. Levels of inflammatory mediators, prostaglandin E 2 (PGE 2 ) and 5-lipoxygenase (5-LO), and proteolytic enzymes, metalloproteinase (MMP)-2 and MMP-9, were assessed in both cartilages of the femur and tibia, and the synovial membrane (Fig. 4). The levels of PGE 2 in both cartilages and the levels of MMP-2 and MMP-9 in the tibial cartilages were examined by one-way ANOVA and the others were by non-parametric Kruskal-Wallis H. The multiple comparison tests showed significant Values were expressed as means ± SD (eight samples/group) (b). **p < 0.01 versus sham group, ## p < 0.01 versus OA con, and $$ p < 0.01 versus D2 + DW by LSD. main effects for the groups (p < 0.01). For the levels of PGE 2 and 5-LO, the post-hoc test versus the sham group revealed increases in both cartilages and the synovial membrane of the OA con (p < 0.05, Fig. 4a-f); however, the test versus the OA con showed significant decreases in the treatment groups (p < 0.05), excepting for PGE 2 in the synovial membrane of the Sal + JMS group. The levels were significantly decreased more in the D2 + JMS group than the D2 + DW (p < 0.05). Similarly, while the levels of MMP-2 and MMP-9 in the cartilages and the synovial membrane were higher in the OA con group than the sham group, they were lower in the treatment groups than the OA con (p < 0.05), excepting for MMP-2 in the synovial membrane of the Sal + JMS group (Fig. 4g-l). The levels were further lower in the D2 + JMS group than the D2 + DW (p < 0.05).
Effects on expressions of mRNA related to cartilage composition. Expression levels of mRNA for collagen type II (collagen2) and aggrecan as key components for the cartilage ECM, and sex-determining region Y-box (SOX)9 as a transcription factor for chondrogenic differentiation, were assessed in both side cartilages . Bone mineral density (BMD) and compressive strength. BMD was assessed by dual energy X-ray absorptiometry digital radiography. The representative image and its reverse image are shown in (a) (upper and lower, respectively). Arrows indicate osteophytes. A region of interest is represented as solid and dotted boxes for the knee joint and both subchondral bones of the femur and tibia, respectively, and the BMDs were measured (b-d). Compressive strength was assessed in the cartilages of the femur (e) and tibia (f). Values were expressed as means ± SD (eight samples/group). **p < 0.01 and *p < 0.05 versus sham group, ## p < 0.01 and # p < 0.05 versus OA con, and $$ p < 0.01 and $ p < 0.05 versus D2 + DW by LSD (b-e) or MW (f). and the synovial membrane (Fig. 5). One-way ANOVA examined data for the levels of collagen2 in both cartilages, aggrecan in the femoral cartilage and synovial membrane, and SOX9 in the synovial membrane, while non-parametric analyses examined for the others. There were significant main effects for the groups (p < 0.01), followed by the post-hoc tests. In both cartilages, the expressions of collagen2, aggrecan and SOX9 were down-regulated in the OA con compared with the sham group; however, they were significantly up-regulated in the treatment groups compared with the OA con group (p < 0.05). In the synovial membrane, while the expression of collagen2 was significantly higher in the OA con than the sham group, the other expressions of aggrecan and SOX9 were lower (p < 0.05). However, the expressions were reversed in the treatment groups compared with the OA con group (p < 0.05). Furthermore, the levels of collagen2, aggrecan and SOX9 were significantly higher in the cartilages of the D2 + JMS group than the D2 + DW, excepting for SOX9 in the femoral cartilage. The levels of aggrecan and SOX9 were also higher in the synovial membrane of the D2 + JMS than that of the D2 + DW (p < 0.05).
Histopathological changes on cartilage degradation. In Safranin O stain, the OA con group exhibited evident damages in a surface of the cartilages with the decreased chondrocyte, clone formation and stain intensity, however, the changes were mild in all treatment groups (Fig. 6a). The thickness of the tibial cartilage and synovial membrane and the Mankin scores in both side cartilages were examined by one-way ANOVA and the others were by Kruskal-Wallis H. The multiple comparison tests showed main effects for the groups (p < 0.01). The post-hoc versus the sham group revealed significant decreases in the thickness of both cartilages of the OA Figure 4. Expressions of inflammatory mediators and metalloproteinases (MMPs). Tissue levels of inflammatory cytokines, prostaglandin E 2 (PGE 2 , a-c) and 5-lipoxygenase (5-LO, d-f), and MMPs, MMP-2 (gi) and MMP-9 (j-l), were assessed in cartilages of the femur and tibia, and the synovial membrane (m.). Values were expressed as means ± SD (eight samples/group). **p < 0.01 and *p < 0.05 versus sham group, ## p < 0.01 and # p < 0.05 versus OA con, and $$ p < 0.01 and $ p < 0.05 versus D2 + DW by LSD (a,b,h,k) or MW (c-g,i,j,l).
www.nature.com/scientificreports www.nature.com/scientificreports/ con; however, the test versus the OA con showed significant increases in the treatment groups, excepting for the femoral cartilages of the Sal + JMS (Fig. 6b,c). While total Mankin scores of both cartilages were higher in the OA con than the sham group, they were lower in the treatment groups than the OA con (p < 0.05, Fig. 6e,f). Conversely, the synovial membrane-lining epithelial thickness and inflammatory cells were increased in the OA con compared with the sham group; however, they were decreased in the treatment groups compared with the OA con (p < 0.05, Fig. 6d,g). In particular, the D2 + JMS group versus D2 + DW showed significant increases in the thickness of both cartilages and decreases in the Mankin scores and the thickness of the synovial membrane (p < 0.05).

Immunohistochemical analyses on inflammation, apoptosis and cellular proliferation.
In immunostains for tumor necrosis factor (TNF)-α and COX-2 as pro-inflammatory markers and cleaved poly (ADP-ribose) polymerase (PARP) as an apoptosis marker, the expressions were less in all treatment groups than the OA con (Fig. 7). The immunoreactivities for bromodeoxyuridine (BrdU) as a cellular proliferation marker, were different in the cartilages and synovial membrane of the treatment groups compared with the OA con; increased in the cartilages but reduced in the synovial membrane. The COX2-and BrdU-positive cells in the femoral cartilage and tibial cartilage, respectively, were examined by one-way ANOVA, and the others were by Kruskal-Wallis H (Table 1). There were significant main effects for the groups (p < 0.01). The post-hoc versus the sham group revealed increases in the immunoreactive cells for TNF-α, COX-2 and PARP in both cartilages and the synovial membranes of the OA con; however, the test versus the OA con showed significant decreases in those of the treatment groups (p < 0.05), excepting for the tibial cartilage of the Sal + JMS group. Comparing to the D2 + DW group, the D2 + JMS group showed significant decreases in the immunoreactive cells in both cartilages and the TNF-α-and PARP-positive cells in the synovial membrane (p < 0.05). For the BrdU-positive cells, the post-hoc versus the sham group showed decreases in both cartilages of the OA con but increases in the synovial membrane (p < 0.05). However, the immunoreactivities were reversed in the Sal + JMS, D1 + JMS and D2 + JMS groups compared with the OA con (p < 0.05). The BrdU-positive cells were not different in both cartilages between the D2 + DW and the OA con groups; however, they were reduced in the synovial membrane of the D2 + DW compared with that of the OA con (p < 0.05). www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ of proinflammatory cytokines (PGE 2 , 5-LO, TNF-α and COX-2) and the cartilage ECM-degrading enzymes (MMP-2 and MMP-9). In addition, while the apoptotic (PARP-positive) cells were reduced in the articular tissues, the proliferative (BrdU-positive) cells were increased with up-regulation of chondrogenic genes (SOX9, collagen2 and aggrecan). The overall effects of JMS bathing were significantly more so when combined with diclofenac sodium. It suggests the therapeutic potential of JMS bathing for knee OA.
The characteristic changes of knee OA lesions were observed in the DEXA and histopathological analyses, which were mild in treatments including the JMS bathing. An inflammatory mechanism has been noted as an important factor for promoting the structural damages in OA joints, as well as pain and immobility 36 . The initial change is characterized by infiltration of leukocytes in the osteoarthritic synovium 5,36 . The progress increases COX-2 and 5-LO in coordination with released cytokines including interleukin-1 and TNF-α, followed by production of PGE 2 and leukotriene. It can cause pains and subchondral sclerosis progressively over time. Here, the JMS bathing inhibited the release of inflammatory mediators in the knee joint, which might contribute to alleviating the joint swelling and increasing the range of motion. The anatomical and physiological changes could be triggered by metabolic and molecular alterations in the joint. Along with cytokines, MMPs are also responsible for degeneration of joint cartilage through the ECM degradation 4 . On the other hands, SOX9 is a master regulator of chondrogenesis, and it regulates other chondrogenic genes, collagen2 and aggrecan 37 . Particularly, MMP-2 and -9 degrade collagen denatured by collagenases and cleave aggrecan molecules 38 . In this context, the facts that JMS bathing reduced the MMPs and up-regulated the chondrogenic genes, might inhibit the physiological remodeling and pathological degeneration of the cartilage tissue. These provide useful information for protective effects of the JMS bathing on the pathological progress of knee OA.
The protective effects of the JMS bathing may contribute to a well-preserved cartilage tissue serving as a scaffold for mineral deposition and enhancing the proliferating and antiapoptotic activities. The increased subchondral BMD and chondrogenesis have positive correlations with the mechanical loading 35 . Further, the chondrogenic genes, collagen2 and aggrecan, can provide bony tensile and the compressive strength, respectively, as well as chondrocyte differentiation 39 . NSAIDs including diclofenac sodium produce pain relief and improvement of joint motion in knee OA, but some can be detrimental to bone regeneration by suppressing synthesis of proteoglycans 40 . Here, the proliferating cells in the cartilage were not changed by the treatment of diclofenac sodium followed by DW bathing. However, the proliferation was further increased by a combination with the JMS bathing. In other results as well, the combination treatments showed a similar trend associated with synergic effects. This suggests that JMS bathing in combination with a proper dose of diclofenac sodium can be promising as an ideal regimen for knee OA.
Some clinical trials provide compelling evidences for significant mineral effects on knee OA in balneotherapy in comparison with the pretreatment or tap water 41,42 . In addition, balneotherapy containing various minerals including bicarbonate, calcium and sulfur, involves reducing serum inflammatory cytokines (i.e., IL-1β, TNF-α, IL-6, IL-8, TGF-β), exerting inhibition of the cartilage degradation and pain relief [24][25][26][27][28]43 . The direct mineral effect on OA progression has been examined via the dietary intakes. Supplements including Mg, Mn, V and Se, alleviate the OA lesions and the relevant symptoms in the human patients and animal models [44][45][46] . In particular, Mg has received attention because it promotes chondrocyte viabilities and its deficiency involves outbreaks of knee OA  Table 1. Immunohistochemical analysis for inflammation, apoptosis and cellular proliferation. The immunoreactive cells in Fig. 7 were counted and expressed as means ± standard deviation (eight samples/ group) in the femoral and tibial cartilages (cart.) and the synovial membrane (m.). **p < 0.01 and *p < 0.05 versus sham group, ## p < 0.01 and # p < 0.05 versus OA con, and $$ p < 0.01 and $ p < 0.05 versus D2 + DW by LSD (for positive cells for COX2 of the femoral cartilages and BrdU of the tibial cart.) or MW (for others). TNF-α = tumor necrosis factor, COX2 = cyclooxygenase 2, PARP = poly (ADP-ribose) polymerase, and BrdU = bromodeoxyuridine.
Scientific RepoRtS | (2020) 10:6620 | https://doi.org/10.1038/s41598-020-62867-2 www.nature.com/scientificreports www.nature.com/scientificreports/ or inflammatory responses 47,48 . Other animal mechanism studies have shown that dietary deep seawater containing NaCl, Mg, and Ca increases BMD and stimulates bone formation and resorption for a bone turnover 49,50 . Although the JMS contains high contents of minerals such as Na, Mg, Ca, K, Zn and strontium, the mechanism regarding how minerals absorbed during the bathing affects knee OA, is still uncertain, and further studies are needed.
To better understand bathing effects in the JMS on the pathophysiological process of knee OA, surgically induced rat model that reflects a post-traumatic early OA was employed. While the model has an advantage presenting reproducible disease progression, it can't be entirely representative of human knee OA including the naturally degenerative changes. In addition, there has been inconsistent efficacy between animal and human studies, probably because of the differences in the anatomy, histology and physiology 51 . However, limitations in the clinical studies showing variations in the disease onset and the progression, necessitate a preclinical study on the early OA development for the fundamental cure. Even though just one animal model is not sufficient to study the effects of JMS bathing for all of knee OA, this may subsequently contribute to the development of treatment regimens. The current finding suggests beneficial effects of the JMS bathing mainly by inhibiting inflammation as a possible target. Given the global burden of OA disease requiring long-term management with various treatment options, this provides useful information supporting that JMS bathing can be a potent alternative therapy for improving quality of life in patients with knee OA.  (20-25 °C) and humidity (45-55%) controlled room with a light/dark cycle of 12/12 h. Food and water was available ad libitum. After acclimatization for 7 days, OA was surgically induced in the left knee as described previously 52 . Briefly, after an exposure of the medial joint capsule, the anterior cruciate ligament (ACL) was transected, and the medial meniscus was partially removed. The sham group received the same surgery except for the ACL transection and the partial meniscectomy.

Methods
Experimental design. Four weeks after the surgical induction, the sham (n = 8) and OA model (n = 40) were selected based on body weights. Knee thickness was then measured by an electronic digital caliper (CD-15CPS, Mytutoyo, Tokyo, Japan), and the OA model was divided into five groups (n = 8 per group) with no significant differences in knee thickness (13.87 ± 0.18 mm in OA model, ranged in 13.61~14.22 mm; 10.96 ± 0.15 mm in sham, ranged in 10.76~11.17 mm). The sham and one group of OA model received an injection with saline as a vehicle of diclofenac sodium plus bathing in DW referring hydrotherpy (sham and OA control, respectively). The other four groups of OA model were allocated as treatment groups; an injection with diclofenac sodium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 2 mg/kg in saline plus DW bathing (D2 + DW), saline injection plus JMS bathing (Sal + JMS) or injection of diclofenac sodium at 2 mg/kg and 1 mg/kg plus JMS bathing (D2 + JMS and D1 + JMS, respectively). The saline and diclofenac sodium were injected subcutaneously in a volume of 5 ml/ kg, and the dosage was based on a previous study 52 . Thirty min after the administration, the rats underwent the bathing for 20 min in polycarbonate cages (280 × 420 × 180 mm) containing pre-warmed DW or JMS (4 L) at a depth of 5 cm under monitor to prevent accidental drinking of JMS. Rats received the treatment once a day for 8 weeks, and then euthanized using CO 2 gas. The body weighs and knee thickness were measured every week by a technician blinded to the groups. Three days before euthanasia, the rats were intraperitoneally administered with BrdU (Sigma-Aldrich, St. Louise, MO, USA) at 50 mg/kg in a volume of 2 ml/kg 52 .

Measurement of functional knee extension.
The induced left hindlimb from the coxofemoral to the ankle region was sampled after all treatments. Thickness of the joint capsule and the maximum articular extension angle were measured as described previously 53 . The lower angle meant more functional improvement of the joint extension. This was performed by a veterinarian blinded to the groups.
Measurement of BMD and compressive strength. BMD was assessed using DEXA (InAlyzer, MEDIKORS Inc., Seungnam, Korea) as described previously 54 , and expressed as g/cm 2 . It was measured in the knee joint and both subchondral bones of the femur and tibia by drawing boxes surrounding the approximate regions using the image analyzing soft-ware. Focal compressive strength was assessed in a central region of the medial femoral and tibial condyle at a depth of 0.2 mm using a computerized testing machine with a digital force gauge (JSV-H1000 and HF-10, Japan Instrumentation System Co., Tokyo, Japan) as Newton (N).

Measurement of PGE2, 5-LO and MMPs.
A part of the joint samples was homogenized in RIPA solution using a bead beater (Taco TM Prep, GeneReach Biotechnology Corp., Taichung, Taiwan) and an ultrasonic cell disruptor (KS-750, Madell Technology Corp., Ontario, CA, USA). The homogenates were centrifuged at 1,200 × g at 4 °C, and the supernatants were used for levels of PGE 2 , 5-LO, and MMPs. The PGE 2 was measured at 450 nm using a PGE 2 assay kit (PKGE004B, Parameter TM , R&D Systems, Minneapolis, MN, USA; detection of 39 to 2,500 pg/ml and sensitivity of 41.4 pg/ml), 5-LO activity was measured at 490 nm using a lipoxygenase Histopathological analysis. A joint sample was fixed in 10% formalin and decalcified in a mixture of 24.4% formic acid and 0.5 N sodium hydroxide for 5 days. The samples were paraffin-embedded and serial-sectioned at a thickness of 3 μm. The sections were stained with Safranin O and evaluated according to the modified Mankin scores for four subgroups (0-3 points, each); surface of the cartilage, hypocellularity, cloning of cells and stain intensity 52 . The higher the score, the more severe the level of OA (semi-quantitative scores; max = 12). In addition, thicknesses of the articular cartilages (μm/cartilage), the synovial membrane-lining epithelial thickness (μm/knee joint) and inflammatory cells (cells/mm2) were assessed using a computerized image analyzer (iSolution FL v. 9.1, IMT i-solution Inc., Vancouver, BC, Canada). The histopathologist was blinded to the groups.
Immunohistochemistry. Another serial section was pretreated with trypsin (Sigma-Aldrich) and 2 N hydrochloric acid for an antigen retrieval. The endogenous peroxidase was removed by 0.3% hydrogen peroxide, and the non-specific binding protein was treated with normal horse serum for 1 h. Then, the sections were incubated with primary antibodies at 4 °C overnight. The antibodies for COX-2 (160126, Cayman; 1:200), PARP (9545, Cell Signaling Technology Inc., Danvers, MA, USA; 1:100), TNF-α (sc-52746, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200) and BrdU (ab1893, Abcam, Cambridge, UK; 1:100) were used. Next day, the sections were incubated with a biotinylated secondary horse anti-mouse/rabbit IgG antibody and Vectastain Elite ABC reagents (Vector Laboratories Inc., Burlingame, CA, USA) for 1 h each. The immunoreactivity was visualized by a peroxidase substrate kit (Vector Laboratories Inc.). All sections were incubated in a humidity chamber, and rinsed with 0.01 M phosphate-buffered saline three times between each step. Cells occupying immunoreactive regions over 20% were regarded as positive, and the number was assessed in 10 regions of interest in each section by a histopathologist blinded to the groups.
Statistical analyses. Data are expressed as means ± standard deviation (SD). First, data were analyzed by the Levene test for homogeneity of variance. If it is not significant, the values were examined by one way-ANOVA, followed by the least significant differences (LSD) post-hoc test. Otherwise, Kruskal-Wallis H test was conducted for non-parametric comparisons, followed by the Mann-Whitney U (MW) post-hoc with Bonferroni correction method. The kinetic data for the body weight and knee thickness were analyzed by two way-ANOVA with main factors of the groups and the time-point measured, and the time-point was treated as a repeated measurement. The multiple comparisons were focused on the effects of treatment groups including JMS bathing. A p-value <0.05 was considered statistically significant.