| BACKGROUNDOsteoarthritis (OA), also referred to as degenerative joint disease, is one of the most prevalent chronic conditions in elderly people. OA mainly involves articular cartilage, bone and synovial membrane. The pathogenesis of OA consists of a general progressive loss of articular cartilage, remodeling and sclerosis of the subchondral bone, and the formation of subchondral bone cysts and marginal osteophytes. Arthrocele, pain, deformation and functional disturbance caused by OA severely disable patients' daily activities. With the merging of an aging society, the incidence of OA is increasing in an alarming pace, and OA is becoming a social and economic problem in our current society.The current management of OA is to relieve the symptom, to improve the joints function and movement, to postpone the articular degeneration. Recently Medical Ozone has been shown to have promising clinical results in treatment of various chronic diseases, especially in pain relief in conditions, such as pyriformis syndrome and OA. But the mechanism of medical Ozone in OA treatment is largely unknown. Above all, there are no consensus on the Ozone dosage and its safety standard among the medical professionals who practice Medical Ozone. This experimental study was designed for investigation of the above issues.PURPOSES1. To search for methods of producing OA animal models to match the need of large scale study of OA Ozone intra-cavity treatment.2. To study the possibility and safety of Ozone intra-cavity treatment of OA by pathologically observing the articular cartilage changes following Ozone intra-cavity injection of different dosages (those are accepted clinically in many countries of Asia and Europe).3. To study the Autohemotherapy of medical Ozone in treatment of OA by pathologically observing the articular cartilage changes following intravenous injection of model's blood blended with Ozone of different concentrations.4. To search the possible mechanisms of Ozone in treating OA with different administration route by detecting the level of oxidative stress index and the activities of serum cytokines.5. To observe and evaluate the effect of medical Ozone in treatment of OA by MRI.Part I. OA Animal Model EstablishmentIn this part, to find out the most effective method of OA animal model establishment for large scale studies, four methods were tested: 1.6% papain intra-articular injection and Collagenase intra-articular injection, immobilization of rabbit knee joints, femoral vein ligation.1. Model establishment by intra articular cavity injection of papain1.1 Materials and methods8 Healthy New Zealand white rabbits (6-8 month, weighting 2.0-2.5kg, 4 males and 4 females) were used. Right knee intra-articular injection of 0.3ml 1.6% papain solution was performed twice, at day 1 and day 4, to produce the OA model. The animals were sacrificed and dissected for pathological studies at 5 weeks after the initiation of the injection.1.2 ResultsVarious extents of joint swelling were observed after the injection of 1.6% papain solution. The rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 days and swelling faded away in 10 days. All rabbit's diets remain normal and alive without exceptional behavioral change.Gross and operative arthroscope inspections: Cartilage at the medial side of the tibia platform was grey yellow or grey white and dull. Fissures on cartilage surface were found. A few focuses of small and shallow erosion were noted. There was no obvious osteophyma.Light microscopy examination: Articular surface is rough and irregular. Relatively small and shallow fissures can be appreciated. The chondrocytes is decreased in cellularity and disarrayed in arrangement without cellular crowding. PAS and Toluidin blue stains show mild decrease in staining intensity in the chondroid matrix.1.3 ConclusionsThe pathological changes of the OA model produced by papain intra-articular injection are very similar to those of spontaneous OA. The experimental model is simple technically and easily reproducible with a high successful rate. These models are also suitable for quick and a large scale production for pathological and therapeutic studies.2. Model establishment by intra articular cavity injection of Collagenase2.1 Materials and Methods6 Healthy New Zealand white rabbits (6-8 month, weighting 2.0-2.5kg, both male and female animals) were used to produce the OA models. Right knee intra-articular injection of 0.5ml solution (I.e.2mg Collagenase II) was performed twice, at day 1 and day 4, to produce the OA model. The animals were sacrificed for pathological studies at 4 weeks after the initiation of the injection. 2.2 ResultsLow spirit, visible inactivity was observed in rabbits after the injection of Collagenase II solution. Variable degree of joint swelling was present. The rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 weeks and swelling faded away in 10 days. All rabbits' diets remain normal and alive without exceptional behavioral change.Right knee DR study was performed 4 weeks after establishment. The radiograms showed both knee spaces were in concordance without obvious stenosis, and the articular surfaces were smooth, without osteophyma. The sub-patella fat pad was clear. Surrounding soft tissue was not swelling.MRI showed that condyleses of femurs and tibial plateaus were smooth, without thinning and defects; a small amount of hydrops in the suprapatellar bursa was found. The MR signals of the sub-cartilage bone were normal.Grossly, the surface of the femur condyles was still smooth and intact, but matted. The surface of the tibial plateau was rough and granulated. A few malacia foci but osteophyma were present.Histological study with HE staining revealed irregular fissures at the surface layer of the cartilage. The radial zone and calcify zone were architecturally normal. The chondrocytes was slightly decreased in cellularity and arrayed in normal arrangement. The tide mark was intact.2.3 conclusionsIn this study, it took the shortest time to achieve the production of the OA model which pathology was very much similar with that of humans. The method of Collagenase II intra articular injection was ideal for the production of early OA model. It was simple technically and took less time, and fit for quick producing OA models in great numbers.3. Other preliminary experiments for OA model productionOther two OA model production method had been tested in this study, immobilization of rabbit knee joints with plaster bandage and femoral vein ligation. But it's difficult to control the concordance of the OA-like pathological change and degree of the change in individual animal in the group due to the lack of control in immobilizing time. And During the traumatic procedures of model producing by femoral vein ligation, it's more easily for animals to die following in-operation bleeding and post-operation infections. And it's difficult to reproduce a typical OA model by simple ligation of the femoral vein. These establishment methods were not suitable for a quick and large scale model study.4. SummaryThe trail of production of OA in animal models pathologically similar to those happen spontaneously to humans had been based on thorough review of the literatures about OA model establishment. Our investigations proved that intra articular chemical substances injection was the most effective method among those tested. Comparatively, Collagenase II OA model was easier to reproduce in a rather short time and better for OA study than Papain model. Both immobilization of knee joints and femoral vein ligation were unsuitable for large scale establishment, as they were less tolerable to the animals and require more complicated and sophisticated animal handling and manipulation than our current situation allowed.Part II. Experimental Study of Medical Ozone Intra Articular Injection in Papain Induced OA Models1. PurposesIntra-articular injection of Ozone has recently been used to treat OA. However, the mechanism and safety of such treatment modality has not yet been fully investigated. This study was designed to evaluate the local effect of intra-articular Ozone injection to better understand the biologic mechanism and pathologic effect of Ozone therapy on articular cartilage.2. Materials and MethodsOA animal models were produced by intra-articular injection of Papain solution. Both Nitric oxide (NO) and Superoxide dismutase (SOD), the two most commonly used indexes for oxidative stress reaction, and the histopathology of the articular cartilage were analyzed.2.1 Animal grouping and model establishment32 New Zealand white rabbits were randomly divided in to normal group, OA model group without treatment, and OA model group with O3 Intra- articular cavity Injection (O3IACI). The O3IACI treated OA model group was further sub-divided in to two groups according to the Ozone dosage used in the experiment: O3IACI-20 group (Ozone dosage: 20μg/ml per injection), O3IACI-4O group (Ozone dosage: 40μg/ml per injection). 8 rabbits were in each group. OA model was produced by injecting 3ml of 1.6% Papain solution into the knee joint space twice three days apart.2.2 Animal processing7 days after the last injection of Papain solution, the rabbits of both O3IACI-20 group and O3IACI-40 group were treated with intra-articular injections of different Ozone dosage accordingly twice a week for 4 weeks. The normal and model groups were fed routinely. Synovial fluid and blood of all groups were collected, and all animals were sacrificed to collect articular cartilaginous tissue for further analysis 1 week after the last Ozone injection.2.3 Synovial fluid and serum analysisNO and SOD activity were detected in both serum and synovial fluid. NO was tested by Nitrate reductase chromatometry, SOD by Xanthine oxidase chromatometry.2.4 Histopathology evaluation of the cartilaginous tissueThe articular cartilage was resected from the knee joint in all animals and processed routinely for histologic sections. Paraffin sections of the cartilage were stained with HE, PAS and Toluidine Blue, respectively. The slides were analyzed for histologic changes under the light microscope. The cartilage changes were evaluated according to the Mankin Score.2.5 Statistical analysisSPSS 11.5 software package was used. Data for each group were presented as mean±SD. levene's test was applied to analyze the homoscedasticity of multi samples. One-way ANOVA as well as q test was applied to analyze every parameter of each region of interest as homogeneity of variances in multiple group samples means. LSD-t and Games-Howell tests were used for comparison among multiple samples means. A p <0.05 value was considered statistically significant.3. Results3.1 Animal behaviors observationAfter the first Papain solution injection, Variable extent of the right knee swelling was presented in all animals for producing OA model, the rabbits limped, struggled and kicked when their right knees were touched. Limping disappeared in 2 days and swelling faded away in 10 days. After Ozone injection, many rabbits in O3IACI-20 group and O3IACI-40 group behaved as usual and some appeared to be more active than before the Ozone treatment.3.2 Histomorphology study3.2.1 Gross inspectionPart examined: cartilage at the medial side of the tibia platformNormal group: the cartilage was shiny blue white and smooth. There were no fibrosis, fissure and ulcer.Model group: the cartilage was grey yellow or grey white and dullness. Fissures on cartilage surface were found. A few foci of small and shallow erosion were noted. There was no obvious osteophyma.O3IACI-20 group: the cartilage was grey yellow, rough and dullness. More fissures on the superficial cartilage were noted. Superficial erosions were obvious and multiple. There was no obvious osteophyma.O3IACI-40 group: the cartilage was rough. Much more fissures and erosions were seen in compared with O3IACI-2O group.3.2.2 Microscopic examinationNormal group: the surface of the cartilage was lubricated and intact. 4 zones of tangential layer, transitional zone, radial zone and calcified cartilage layer were in good arrangement. Tide mark was normal. Chondrocytes were arrayed in order and well-distributed but clustered. The chondroid matrix was uniformly red in PAS staining and dark blue in Toluidine blue staining.Model group: the surface of the cartilage was irregularly rough with tiny shallow cracks. The chondrocytes was decreased in cellularity and disarrayed in arrangement. PAS and Toluidine blue stains showed mild decrease in staining intensity in the chondroid matrix when compared to that of normal group.O3IACI-20 group: Articular surface was damaged with presence of empty lacunas due to disappearing of the chondrocytes. The layer of the cartilage decreased in thickness with apparent fissures. The tide mark was unclear or duplicated. The PAS and Toluidine blue stains were less intensive than that of the model group. Dosage-40 group: The articular cartilage damage was represented by the thinning of the cartilage layer, decreased cellularity of the chondrocytes, cellular disarray, clustering of hyperplastic chondrocyte and lack of normal zonal appearance. Compared to Dosage-20 group, fissures were increased and were more apparent. PAS and Toluidine blue staining intensity was further diminished in the chondroid matrix.3.2.3 Mankin Scores evaluationIn modified Mankin scoring system, the structure of the cartilage, cell appearance, staining of the cartilage matrix, tidemark was evaluated. Total score arranged from 0 to above. Normal cartilage was scored as 0. A low score indicated mild changes in articular cartilage while a high one indicated severe degeneration.The normal group was scored 0.0±0.0 while the model group was 4.5±0.5 which was significantly higher than that of the normal group (P<0.01). The scores of the two O3IACI groups together were 6.0±0.8-8.1±1.2, they were significantly higher than that of the model group (P<0.01). When compared the two O3IACI groups, the score of the O3IACI-40 group (8.1±1.2) was significantly higher than that of the O3IACI-2O group (6.0±0.8). (P<0.01). The results of the Mankin score evaluation indicated that multiple injections of Ozone into the articular space could further induce damage in the cartilage tissues of osteoarthritis, and the degree of the Ozone induced cartilage damage appeared to be dosage dependent.3.3 laboratories AnalysisSerum NO level: In comparison with the level of the normal group (91.88±26.94μmol/L), the NO level of the model group (146.75±16.43μmol/L) or each of the two O3IACI groups (O3IACI20, 156.01±24.53μmol/L; O3IACI40, 166.23±22.01μmol/L) was significantly higher, respectively, (P<0.01). However, there was no significant difference among the two of O3IACI groups and the model group (p>0.05)Serum SOD level: In comparison with the SOD level of normal group (246.18±34.65 U/ml), the level of the model group (330.78±46.68 U/ml) or each of the two O3IACI groups (374.96±44.02 U/ml, 411.59±30.00 U/ml) was significantly higher, respectively(P<0.01); the SOD level of the two O3IACI groups were higher than that of the model group, the differences were statistically significant, P<0.05. However, the difference between the O3IACI-20 group (374.96±44.02 U/ml) and the O3IACI-40 group (411.59±30.00 U/ml) was not statistically significant(P> 0.05).Synovia NO level: In comparison with the NO level of the normal group (31.43±5.06μmol/L), the NO level of the model group (111.37±35.92umol/L) or each of the two O3IACI groups (108.28±20.45umol/L, 105.16±38.18umol/L) was significantly higher, respectively (P<0.01); Differences between the two O3IACI groups and between the O3IACI groups and the model group were not significant statistically; (P>0.05).Synovia SOD level: The difference between SOD level of the model group (86.02±48.47 U/ml) and that of the normal group (63.51±39.81 U/ml ) was not significant, P>0.05; the level of either of the two of O3IACI groups (119.05±34.96 U/ml, 140.14±42.76 U/ml) were higher than that of the normal group, respectively; (P <0.05). The SOD levels of the O3IACI-40 group (140.14±42.76 U/ml) was significantly higher than that of the model group (P<0.05); but there was no statistically significant difference between the O3IACI-20 group (119.05±34.96 U/ml) and the model group (P> 0.05), and between the two 03IACI groups, (P> 0.05), respectively.4. ConclusionsOzone, when introduced into the articular cavity, could cause increased secretion of SOD. Up-regulation in synthesis of antioxidant enzymes, such as SOD, is known to be able to lead to block chronic oxidative stress reaction which may be one of the mechanisms of Ozone therapeutic effect in OA. However, intra-articular injection of Ozone did not reduce NO levels of both serum and synovial fluid. Regardless of these, we have documented that repeated injections of Ozone to joint cavity have an adverse deleterious effect on OA-like articular cartilage. Our findings raise a serous concern regarding the safety of intra-articular injection of Ozone in treating OA. The mechanism of its deleterious effect on OA-like cartilage is not clear and further investigation is needed. Physicians should be aware of this potential adverse effect on OA patients with a conservative approach to withhold the use medical Ozone as one of the treatment choices for OA until its therapeutic benefit can be approved against its adverse effect in further animal study and clinical trial.Part III. Experimental Study of Collagenase II produced OA models treated with Medical Ozone by intra articular injection and Autohemotherapy1. PurposesThis study was designed to study the variation of serum cytokines before and after introduction of Ozone of different dosages in two ways, intra-articular injection and autohemotherapy, to better understand the biologic mechanism of Ozone therapy on OA, and to further confirm the pathologic effect of Ozone on OA articular cartilage.2. Materials and MethodsOA animal models were produced by intra-articular injection of Collagenase II solution. Nitric oxide (NO), Superoxide dismutase (SOD), and two most important cytokines in OA development, TNF-αand IL-1β, and the histopathology of the articular cartilage were analyzed.2.1 animal grouping and model establishment36 New Zealand white rabbits were randomly divided in to normal group, OA model group without treatment, OA model group with O3 Intra- articular cavity Injection (O3IACI) and OA model group with ozonized blood Autohemotherapy (O3AHT). The O31 ACI treated OA model group and the O3AHT treated OA model group were further sub-divided in to two groups separately according to the Ozone dosage used in the experiment: O3IACI-10 group (Ozone dosage: 10μg/ml per injection), O3IACI-30 group (Ozone dosage:30μg/ml per injection), O3AHT-10 group (Ozone dosage: 10μg/ml per injection), O3AHT-30 group (Ozone dosage:30μg/ml per injection).6 rabbits were in each group. OA model was produced by injecting 0.5ml of collagenase II solution into the knee joint space twice three days apart.2.2 animal processing4 weeks after the last injection of Collagenase II solution, the rabbits of both O3IACI-10 group and O3IACI-30 group were treated with intra-articular injections of different Ozone dosage accordingly twice a week for 4 weeks.Otherwise, the rabbits of both O3AHT-10 group and O3AHT-30 group were treated with autohemotherapy. During the autohemotherapy procedure, 4ml venous blood was taken form the vein in the ear of the rabbit and thoroughly mixed with 4ml Ozone of different dosage accordingly by a Y tube unit, and re-infused into the vein, twice a week for 4 weeks.The normal and model groups were fed routinely. Blood of all groups were collected and all animals were sacrificed to collect articular cartilaginous tissue for further analysis 1 week after the last Ozone treatment.2.3 Animal behaviors observation2.4 Imaging studies:Knee images of rabbits of each group were studied by DR as well as MRI2.5 Serum analysisNO,T-SOD ,TNF -αand IL-1βwere detected. NO was tested by Nitrate reductase chromatometry, SOD by Xanthine oxidase chromatometry, and both TNF-αand 1L-1βby ELISA.2.6 Histopathology study of the cartilaginous tissueThe articular cartilage was resected from the knee joint in all animals and processed routinely for histologic sections. Paraffin sections of the cartilage were stained with HE, PAS and Toluidine Blue, respectively. The slides were analyzed for histologic changes under the light microscope. The cartilage changes were evaluated according to the Mankin Score.2.7 Statistical treatmentSPSS 11.5 software package was used. Data for each group were presented as mean±SD. levene's test was applied to analyze the homoscedasticity of multi samples. Completely random designed One-way ANOVA was applied to analysis every parameter of interest as homogeneity of variances in multiple group samples means. Welch test was applied to analyze every parameter of interest as Heterogeneity of variance in multiple group samples means. LSD-t and Games-Howe 11 were used for comparison among multiple sample means. A p <0.05 value was considered statistically significant.3. Results3.1 Animal behaviors observationAfter the first Collagenase II solution injection, inactivity, gait difficulty was observed in all rabbits for producing OA model; variable extent of the right knee was presented. Rabbits did limp apparently, struggle and kick when their right knees were touched. About 6 weeks after the first Collagenase II injection, Osteophyma was palpated at the medial part of the right knee in 50% of the OA models. The rabbits of the Ozone treated groups became active and recovered quicker than those of the model group.3.2Imaging studies3.2.1 DR imagingNormal group: both knee spaces were in concordance without obvious loss, and the articular surfaces were smooth, without osteophyma. The sub-patella fat pad was clear. Surrounding soft tissue was not swelling.Model group: obvious hyperostosis of the right tibial plateau was found, the edge of tibial plateau was sharp. The medial articular capsule was swelling. The sub-patella fat pad was not so clear as normal. The knee space was lost.O3IACI-10 group: hyperostosis of the right tibial plateau was noted. Its edge was sharp and the sclerotic articular surface was lightly hyperostosis and eburnated. The right knee space was not narrowed. The sub-patella fat pad was clear.O3IACI-30 group: hyperostosis of the right tibial plateau was found, the edge of tibial plateau was sharp. The medial articular capsule was swelling. The sub-patella fat pad was not so clear as normal. The knee space was still normal.03AHT-10 group: osteophyma was found at the medial tibial plate of the right knee. The medial articular capsule was swelling. There was a slight reduction in lucency of the sub-patella fat pad. The knee space was lost.O3AHT-30 group: osteophyma was found at the medial tibial plate of the right knee. The medial articular capsule was swelling. There was a slight reduction in lucency of the sub-patella fat pad. The knee space was a little lost.3.2.2 MRI studiesNormal group: in 3DWATSc sequence, the articular cartilage surface of the internal condyle of femur and the medial tibial plateau was smooth and intact without thinning and defect. Signals were normal. In STIRLongTE sequence, the signals of the anterior and posterior angles of the medial meniscus and that of subchondral bone were normal. No articular hydrops was presented.Model group: in 3DWATSc sequence, the articular cartilage of the medial tibial plateau was remarkable thinning with a low signal. The thickness of the cartilage in condyles of femur was normal. Its signal was normal. In STIRLongTE sequence, small amount of articular hydrops in the suprapatellar bursa was presentedO3IACI-10 group: in 3DWATSc sequence, the articular cartilage of the medial condyle of femur was thinning with a low signal. The thickness of the cartilage of the tibial plateau was different and the surface was rough. The posterior angle of the medial meniscus was unclear. In STIRLongTE sequence, Small amount of articular hydrops in the supra & infra patellar bursas were presented. O3IACI-30 group: in 3DWATSc sequence, the cartilage of the right medial condyle of femur and medial tibial plateau was thinning with signal reduction. In STIRLongTE sequence, small amount of articular hydrops in the supra & infra patellar bursas were presented.O3AHT-10 group: in 3DWATSc sequence, the cartilage of the right medial tibial plateau was thinning with signal reduction. The thickness of the cartilage of the right medial condyle of femur was nearly normal with a normal signal. In STIRLongTE sequence, small amount of articular hydrops in the supra & infra patellar bursas were presented. O3AHT-30 group: 3DWATSc showed the cartilage of the right medial condyle of femur and medial tibial plateau was thinning with reduction in signal. Small amount of articular hydrops in the supra & infra patellar bursas were presented in STIRLongTE.3.3 laboratories Analysis3.3.1 Serum NO and T-SOD levelsSerum NO level: In comparison with the level of the normal group (79.44±15.31μmol/L), the NO level of the model group (152.23±17.88μmol/L) or each of the two O3IACI groups (O3IACI-10, 163.20±22.31μmol/L; O3IACI-30, 158.05±36.47μmol/L),and each of the two O3AHT groups (O3AHT-10,183.21±23.12; O3AHT-30,183.95±50.72) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).Serum SOD level: In comparison with the SOD level of normal group (233.84±30.66U/ml), the level of the model group (359.71±66.73 U/ml) or each of the two O3IACI groups (O3IACI-10, 351.23±46.46 U/ml, O3IACI-30, 375.85±75.96 U/ml) and each of the two O3AHT (O3AHT-10, 397.01±65.81; O3AHT-30, 379.05±37.67) was significantly higher, respectively (P<0.05); However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).3.3.2 Serum TNF-αand IL-1βlevelsSerum TNF-αlevel: In comparison with the level of the normal group (8.50±3.46 pg/Ml), the NO level of the model group (28.37±17.40 pg/Ml) or each of the two O3IACI groups (O3IACI-10, 88.72±72.88 pg/Ml; O3IACI-30, 198.98±310.97 pg/Ml),and each of the two O3AHT groups (O3AHT-10, 56.07±73.83 pg/Ml; O3AHT-30, 39.12±40.74 pg/Ml) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).Serum IL-1βlevel: In comparison with the level of the normal group (43.37±18.27 pg/Ml), the NO level of the model group (146.89±98.93 pg/Ml) or each of the two O3IACI groups (O3IACI-10, 416.30±319.45 pg/Ml; O3IACI-30, 691.71±798.24 pg/Ml),and each of the two O3AHT groups (O3AHT-10, 287.24±363.95 pg/Ml; O3AHT-30, 219.72±229.94 pg/Ml) was significantly higher, respectively, (P<0.05). However, there was no significant difference among those of Ozone treated groups and the model group (p>0.05).3.4 Histomorphology study3.4.1 Gross inspectionPart examined: cartilage at the medial side of the tibia platformNormal group: the cartilage was shiny blue white and smooth. There were no fibrosis, fissure and ulcer. There was hydrops in the space, neither hyperplasia nor thickening of the synovium.Model group: the surface of the right condyles was still smooth and intact. The right tibial plateau was dull grey and granulo-changed. Thinning and erosion of the medial articular cartilage were seen. Osteophyma was formed at the medial edge of the right tibial plateau.O3IACI-10 group: the surface of the right condyles was rough and dull. Its cartilage was thinning; the right lateral and medial tibial plateaus were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.O3IACI-30 group: the synovial membrane of the right knee articular cavity was hyperaemic and thickening. The cartilage of the condyles of femur was dissipated and dull. The lateral and medial plateaus of the right tibial were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.O3AHT-10 group: the cartilage surface of the condyles of the right femur was rough with focal erosions; The lateral and medial plateaus of the right tibial were dark grey yellow and rough with focal erosions and malacia, Osteophyma was found at the internal angle of the right tibia.O3AHT-30: the synovial membrane of the right knee articular cavity was hyperaemic. The cartilage of the condyles of femur was dissipated with focal erosions. The lateral and medial plateaus of the right tibial were dark grey yellow and rough with rugosity. Osteophyma was found at the internal angle of the right tibia.3.4.2 Microscopic examination3.4.2.1 Articular cartilageNormal group: the surface of the cartilage was lubricated and intact. 4 zones of tangential layer, transitional zone, radial zone and calcified cartilage layer were in good arrangement. Tide mark was normal. Chondrocytes were arrayed in order and well-distributed but clustered. The cartilage matrix was uniformly red in PAS staining and dark blue in Toluidine blue staining.Model group: the surface layer of the cartilage was thinning and rough, and villiform in some part. There were a lot of deep fissures extending deeply to the calcified layer from the surface. The tidemark was destroyed. The cartilage layer was peeled away from the subchondral bone. Chondrocyte degenerated and decreased in cellularity; Toluidine blue stains showed obvious decrease in staining intensity in the chondroid matrix. The cartilage was of fibrosis.O3IACI-10 group: the surface of the cartilage was rough with many fissures extending deeply to the radial zone from the superficial layer. The chondrocytes in the transitional zone and radial zone were disarrayed in arrangement and decreased in cellularity. Cellular clustering hyperplasia was presented. The tidemark was still intact. Toluidine blue stains showed obvious decrease in staining intensity in the chondroid matrix and cracked cartilage.O3IACI-30 group: villiform like changes were found at the superficial cartilage with great amounts of fissures extending to the radial zone from the superficial layer. Chondrocytes were clustering and disarrayed in arrangement. The cartilage layer was exfoliated. Toluidine blue stains showed medium decrease in staining intensity in the chondroid matrix and profuse deep cracks.O3AHT-10 group: there were many fissures extending deeply to the calcified layer from the superficial layer. Chondrocytes Chondrocyte degenerated and decreased in cellularity; the tidemark was un-complete. The cartilage layer was separated from the subchondral bone. Toluidine blue stains showed decrease in staining intensity in the chondroid matrix, and also cracks and fibrosis.O3AHI-30 groups: a lot of fissures extending deeply to the radial zone from the superficial layer of the cartilage. Matrix was dissolved in the radial zone as well as calcified layer. The cartilage structure was destroyed and out of order. Chondrocytes were necrotic. The tidemark was un-complete. The cartilage layer was exfoliated. Toluidine blue stains showed severe decrease in staining intensity in the chondroid matrix, and also many cracks and fibrosis3.4.2.2 SynoviumNormal group: Neither thickening nor hyperplasy was found in the synovium tissue. There was no vascularization and inflammatory cell infiltrationModel group: capillary engorgement in the synovial tissue was found. Inflammatory cells were seen surrounding the vesselsO3IACI-10 group: slight capillary engorgenment in the synovial tissue was found. Small amounts of inf...
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