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An animal model of Kashin–Beck disease induced by a low-nutrition diet and exposure to T-2 toxin

Open ArchivePublished:June 28, 2013DOI:https://doi.org/10.1016/j.joca.2013.05.005

      Summary

      Objective

      We investigated the combined roles of a low-nutrition diet (low levels of protein, iodine, and selenium) and T-2 toxin in bone development and to establish an experimental animal model of Kashin–Beck disease (KBD) that reliably mimics the disease's pathological changes for further study of the pathogenesis and prevention of the disease.

      Methods

      Sprague–Dawley rats were randomly divided among four groups: group A, normal diet; group B, normal diet plus T-2 toxin; group C, low-nutrition diet; and group D, low-nutrition diet plus T-2 toxin exposure. The radiographic and histopathological changes in the tibial growth zone, plate cartilage and metaphysis were examined.

      Results

      In group D, all epiphyseal plates were blurred, thin, and irregular. Tibias were significantly shorter in group D than in groups A and B. After 4 weeks, epiphyseal plates showed chondrocyte necrosis, with the more obvious necrosis appearing in groups C and D. The positive rate of lamellar necrosis was significantly higher in group D than in groups B and A (P < 0.01). In group D, metaphyseal trabecular bone was sparse, disordered, and disrupted, and massive transverse trabecular bone appeared in the metaphysis at 12 weeks.

      Conclusions

      A rat model of KBD induced by a low-nutrition diet and T-2 toxin exposure demonstrated radiographic and histopathological abnormalities of the proximal epiphyseal plate and the tibial metaphysis that are very similar to the bone changes found in patients with KBD. This animal model will be helpful for further study of the pathogenesis and prevention of KBD.

      Keywords

      Introduction

      Kashin–Beck disease (KBD) is a chronic osteochondropathy that is endemic mainly to northeastern and southwestern China, involving 15 provinces and extending to southeast Siberia and North Korea, affecting approximately 2.5 million of the 30 million people living in the endemic area of China
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      . Although this disease has long been recognized and has been studied for more than 150 years, its etiology is still under debate. The current suspected causes mainly include cereal contamination by mycotoxin-producing fungi
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      The prevalence of mycotoxins in Kashin–Beck disease.
      , trace element deficiency in nutrition
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      Kashin–Beck osteoarthropathy in rural Tibet in relation to selenium and iodine status.
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      . Among many related trace elements, selenium and iodine have been considered the most likely factors in the occurrence of KBD.
      Populations from KBD-affected areas often show a deficiency of selenium and iodine. All selenium-deficient areas of China are also areas of iodine deficiency disease
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      Diagnostic, clinical and radiological characteristics of Kashin–Beck disease in Shaanxi Province, PR China.
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      . Low dietary levels of selenium and iodine are thought to be the most important environmental factors causing the disease
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      Sodium selenium for treatment of Kashin–Beck disease in children: a systematic review of randomized controlled trials.
      . Thus, selenium and iodine have been supplied for the past few decades to residents of affected areas in China for the prevention of KBD, and the results have been reported in many articles
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      Kashin–Beck disease and drinking water in Central Tibet.
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      • Huang Z.H.Y.
      Environmental Se-Mo-B deficiency and its possible effects on crops and Keshan–Beck disease (KBD) in the Chousang area, Yao County, Shaanxi Province, China.
      .
      However, it became apparent that trace elements alone cannot completely explain the occurrence of KBD
      • Yao Y.F.
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      • Kang P.D.
      Selenium, iodine and the relation with Kashin–Beck disease.
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      • Zuo H.
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      • et al.
      Effects of selenium and iodine deficiency on bone, cartilage growth plate and chondrocyte differentiation in two generations of rats.
      . A growing number of investigators believe that multiple heterogenous factors are involved
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      • et al.
      Articular cartilage metabolism in patients with Kashin–Beck disease: an endemic osteoarthropathy in China.
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      • et al.
      Epidemiological support for a multifactorial aetiology of Kashin–Beck disease in Tibet.
      . Epidemiological investigations of T-2 toxin, a trichothecene mycotoxin frequently found in a great number of field crops (such as maize, wheat, and oats), produced the hypothesis that KBD is mainly caused by severe contamination of food by fungal mycotoxins
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      • Shen Bin
      • Zhou Z.K.
      • et al.
      Study on the effect of T-2 toxin combined with low nutrition diet on rat epiphyseal plate growth and development.
      . Yang et al.
      • Yang J.B.
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      • Wen W.
      Necrosis in growth plate of cartilage of chickens fed with grain contaminated by Fusarium from Kashin–Beck disease area.
      reported a correlation between the presence of T-2 toxin in families' staple foods, dairy products and the presence of KBD in those families. The basic pathological feature of KBD is a focal necrosis (FN) of chondrocytes in the hypertrophic zone of growth plate cartilage and in the deep zone of articular cartilage, which can result in growth retardation, secondary osteoarthritis (OA), and disability in the advanced stages of the disease
      • Mo D.X.
      KBD cartilage necrosis histopathology and clinical significance.
      • Pritzker K.P.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      • Yang S.
      Chondronecrosis induced in rhesus monkeys fed with grains and water of Kashin–Beck's disease endemic area.
      . Because the growth plate cartilage is the growth center of bone, the developmental deformities are most likely a result of impaired chondrocyte differentiation and endochondral ossification.
      A detailed understanding of the roles of the relative risk factor(s) in the pathogenesis of KBD is necessary before any practical methods for disease prevention can be developed and implemented. However, the etiology of KBD is still under debate partly because of the lack of an experimental animal model that can reliably mimic the pathological changes of the human disease. Therefore, we conducted a study to determine whether giving rats selenium- and iodine-deficient food low in protein and made with barley from an area where KBD is endemic, in combination with exposing them to T-2 toxin, would produce the characteristic chondrocyte necrosis of KBD, to establish an experimental KBD animal model.

      Materials and methods

      Experimental animals and groups

      One hundred twenty weaned Wistar rats were purchased from Huaxi Animal Experimental Centre of Sichuan University. Half of them were male and half were female, and each of them weighed between 60 and 70 g. The rats were housed in cages in our institution's animal room, where the constant temperature was 22–23°C, the relative humidity was 40–70%, and there was a fixed 12-h light–dark cycle. All of the rats were handled and housed according to the guidelines and manuals established by Animal Ethics Committee of the West China School of Medicine, Sichuan University.
      The rats were randomly divided into four groups, with 30 rats in each group. Group A was fed a normal diet as a control, group B was fed a normal diet and was given T-2 toxin, group C was fed a low-nutrient diet (low levels of protein, iodine, and selenium), and group D was fed a low-nutrient diet (low levels of protein, iodine, and selenium) and given T-2 toxin. All rats were given free access to distilled water at all times.

      Diets and T-2 toxin administration

      Two diets, one normal and the other low-nutrition (low levels of protein, iodine, and selenium), were prepared and tested respectively by the Huaxi Animal Experimental Centre of Sichuan University and the Sichuan Center for Disease Control and Prevention (Table I). Nutrient components of the normal diet were determined according to Chinese national standards (GB14924.2-2001: protein, 18.64%; selenium, 0.14 mg/kg; iodine, 0.5–0.6 mg/kg). The normal diet came from a non-KBD-endemic area; the low-nutrition diet came from Rangtang County, a KBD-endemic area, and consisted of mainly of hull-less barley. The component test for the low-nutrition diet showed that protein accounted for 10.2%; selenium, 0.031 mg/kg; and iodine, 0.18 mg/kg, values that were much lower than those for the normal diet.
      Table IResults of analysis of rat diet
      ItemNormal dietLow-nutrition diet
      Protein (g/100 g)18.6415.23
      Calcium (g/100 g)0.680.27
      Selenium (mg/kg)0.14<0.10
      Iodine (mg/kg)208100.10
      T-2 toxin was purchased from Trilogy Analytical Laboratory (Washington, MO, USA) at a purity of 99%. Crystalline T-2 toxin was dissolved in absolute ethanol and diluted with 0.9% normal saline. We administered T-2 toxin intragastrically 5 days a week at dose levels of 0.1 mg/kg per day.

      Sample preparation

      We collected data on hair luster, activity, size, and weight of the rats. At 4, 8, and 12 weeks, eight rats were randomly taken from each group, and 3–4 ml of blood was sampled from each rat by cutting the cervical vessel. The blood samples were left undisturbed for 20 min, and then they were centrifuged at 1500 rpm for 10 min to separate out the serum.
      In those eight rats, the left knees, including the distal femur and the proximal tibia, were harvested and were fixed in 4% wt/vol. polyformaldehyde for 2 days and decalcified in 10% wt/vol. ethylene diamine tetraacetic acid for 4 weeks. The knees were then transferred to phosphate buffer (pH 7.4), embedded in paraffin, and cut into sections 6–7 μm thick for staining with hematoxylin and eosin and for Masson trichrome staining.

      Serum selenium levels and concentrations of triiodothyronine and thyroxine

      The serum concentrations of the trace elements were measured by fluorescent atomic absorption spectrometry. Total protein and albumin in the serum were measured with an automatic biochemical analyzer (DDP, Roche Diagnostics, Penzberg, Germany). Serum triiodothyronine and thyroxine levels were determined by radioimmunoassay.

      Radiographic examination

      At 4, 8, and 12 weeks, six rats from each group were underwent radiographic examination. The rats were positioned with their hips in 90° of abduction and with both knees flexed at 130°, and a molybdenum X-ray machine (Senographe 2000D, GE, Fairfield, CT, USA) was used to evaluate the margins of the rats' bones for the presence of blurring, thinning, irregularity with and without sclerosis, interruption, and destruction. Bilateral tibial bones were assessed for length and diameter at proximal epiphyseal plates using Canvas 9.01 software (Scientific Imaging Edition; Deneba Systems, Miami, FL, USA).

      Histological evaluation

      Necrotic tissue showed vacuolation, pyknosis and lysis of nuclei, as well as loss of cell morphology. According to standards for necrosis categories
      • Mo D.X.
      KBD cartilage necrosis histopathology and clinical significance.
      , necrosis was grouped as follows: FN, lamellar necrosis (LN), penetration necrosis (PN), and zonal necrosis (ZN). FN involved two to three cell columns with two to three necroses in each column in one cell zone, LN involved more than two FNs in one cell zone, PN involved FN penetrating more than two cell zones, and ZN involved necrosis across the whole cell zone.
      All histological measurements were performed within the central two-thirds of the tibial growth plate and the metaphyseal region. All measurements were obtained by a single observer who was unaware of which experimental subgroup each specimen belonged to.

      Assessment of OA cartilage histopathology

      From the collection of approximately 1200 scored slides, a set of 240 slides was selected (60 slides for each group). All sections were scanned with a digital slide scanner (Aperio ScanScope System, Aperio Technologies, Vista, CA, USA) at a magnification of 40× (pixel size = 0.25 μm2), and the scans were evaluated online with WebScope (Spectrum Digital Information Management System, Aperio Technologies). We recruited three observers who were familiar with cartilage histopathology and had different levels of experience with both grading systems. Histological scoring was performed with at least two scorers being unaware of the identity of the each specimen's treatment group. Three observers scored the 240 slides twice, at least 3 weeks apart, with both grading systems. We used the Osteoarthritis Research Society International (OARSI) system, described by Pritzker et al.
      • Pritzker K.P.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      , as our grading template. For all of the grades and stages, it is assumed that the tissue reaction observed has microscopic features characteristics of OA activity
      • Pritzker K.P.
      • Gay S.
      • Jimenez S.A.
      • Ostergaard K.
      • Pelletier J.P.
      • Revell P.A.
      • et al.
      Osteoarthritis cartilage histopathology: grading and staging.
      . The score is an index of combined grade and stage:
      Score=grade×stage


      This produces an OA score between 0 and 24 that is based on the most advanced grade and most extensive stage present. The staging parameters of the OARSI system were applied to the entire tissue section on each slide. A score of 0 points represents normal cartilage, whereas a score of 24 points represents a severely degenerated joint surface. The scores of the two observers were averaged, and outliers with a difference of >1 point were scored again, until consensus was reached. The higher the score, the more the damaged the cartilage.

      Statistical analysis

      Data are expressed here as means and standard deviation of the mean. For nonparametric data, we used the Friedman test. For parametric data, we used the paired t-test or one-way ANOVA to analyze the differences between groups. Statistical analysis was performed using SPSS for Windows software (version 14; IBM, Armonk, NY, USA).
      All results were adjusted for multiple testing using the Bonferroni correction. For all analyses, P ≤ 0.05 was defined as a significant difference.

      Results

      Animal characteristics and weight gain

      Details regarding changes in general condition for all groups are presented in Table II. There was no statistically significant difference in the initial body weight between the four groups (P > 0.05). By week 4, no rats died in group A, two rats had died in group B, two in group C, and three in group D. Weight gain was the greatest in group A, followed by group B (P < 0.05); it was lowest in group D (P < 0.05).
      Table IIChanges in hair, activity level, faeces, and body weight in each group
      IndexControl

      (n = 30)
      Normal diet and T-2 toxin

      (n = 30)
      Low-nutrition diet

      (n = 30)
      Low-nutrition diet and T-2 toxin

      (n = 30)
      Hair lackluster (n)
      Week 40000
      Week 80000
      Week 120568
      Decreased activity (n)
      Week 40001
      Week 80013
      Week 120146
      Loose stools (n)
      Week 40001
      Week 80013
      Week 120114
      Weight (g)
      Week 065 ± 4.367 ± 5.466 ± 3.967 ± 4.8
      95% CI(52.5, 65.9)(55.2, 65.4)(55.9, 65.5)(58.5, 66.5)
      Week 482 ± 5.481 ± 4.279 ± 4.178 ± 5.3
      95% CI(79.3, 85.5)(79.3, 83.9)(74.6, 81.2)(75.3, 81.4)
      Week 899.8 ± 4.197 ± 5.294 ± 3.6
      P < 0.05 versus group A.
      85 ± 4.7
      P < 0.05 versus groups A and B.
      95% CI(97.3, 102.4)(94.5, 1.2.5)(90.7, 95.2)(81.4, 89.8)
      Week 12148.1 ± 19.2124.6 ± 12.2
      P < 0.05 versus group A.
      108.6 ± 10.2
      P < 0.05 versus groups A and B.
      101.8 ± 11.4
      P < 0.05 versus groups A and B.
      95% CI(134.5, 156.8)(119.6, 132.7)(1.1.8, 115.5)(95.7, 110.5)
      Mortality (n)
      Week 40223
      Weight values given as mean ± SD and 95% CI.
      P < 0.05 versus group A.
      P < 0.05 versus groups A and B.

      Serum selenium levels and concentrations of triiodothyronine and thyroxine

      Serum selenium analysis was performed to make sure that restricted availability of selenium in the low-nutrition diet was reflected in the body (Table III). We confirmed that dietary selenium deficiency effectively limited the mineral's intake, because serum selenium levels were significantly lower during the entire experimental period in groups C and D than in groups A and B. In groups C and D, triiodothyronine and thyroxine concentrations were also significantly lower than in groups A and B during the entire experimental period (Table III).
      Table IIIBlood selenium, serum protein, and hormone profile by group
      WeekGroup A

      Control (n = 8)
      Group B

      Normal diet and T-2 toxin (n = 8)
      Group C

      Low-nutrition diet (n = 8)
      Group D

      Low-nutrition diet and T-2 toxin (n = 8)
      4
      Selenium (μg/L)862.6 ± 78.2a (833.7, 891.5)397 ± 104.5b (387.3, 401.8)365 ± 91.5b (354.5, 376.1)347 ± 88.3b (343.1, 353.8)
      TP (g/L)68.2 ± 7.3a (65.3, 70.7)63.6 ± 10.7a (60.5, 65.4)59.1 ± 8.7a (57.2, 60.2)57.8 ± 7.8a (56.4, 59.6)
      ALB (g/L)42.5 ± 5.6a (40.4, 46.7)38.7 ± 7.2a (37.4, 39.9)28.9 ± 9.6b (26.3, 29.7)26.6 ± 9.2a (25.3, 27.8)
      T3 (nmol/L)2.0 ± 0.17a (1.8, 2.1)1.86 ± 0.16a (1.74, 1.88)1.69 ± 0.1a (1.55, 1.74)1.39 ± 0.17a (1.27, 1.42)
      T4 (nmol/L)52.5 ± 5.22a (50.3, 55.2)51 ± 4.15a (50.3, 53.2)47.95 ± 5.78a (43.2, 49.7)44.9 ± 5.55a (43.1, 47.7)
      8
      Selenium (μg/L)843.1 ± 56.4a (823.2, 855.3)323.4 ± 82.5b (321.7, 325.6)314 ± 23.6b (306.8, 321.8)302.5 ± 26.2b (298.5, 310.7)
      TP (g/L)66.4 ± 7.1a (61.4, 68.7)55.1 ± 5.4b (51.4, 57.6)51.1 ± 8.2b (46.8, 58.8)49.8 ± 7.9b (46.3, 49.2)
      ALB (g/L)43.6 ± 4.5a (42.1, 45.7)31.5 ± 6.1b (29.6, 33.9)25.6 ± 7.7c (21.4, 28.9)21.7 ± 7.9b (19.6, 24.6)
      T3 (nmol/L)1.98 ± 0.08a (1.87, 2.03)1.55 ± 0.09b (1.34, 1.68)1.29 ± 0.08b (1.11, 1.45)1.09 ± 0.0.6b (0.89, 1.23)
      T4 (nmol/L)46.5 ± 3.18a (42.3, 48.3)43 ± 4.64a (42.1, 47.2)36 ± 3.88b (31.5, 39.6)29.6 ± 4.1b (25.4, 27.8)
      12
      Selenium (μg/L)859.1 ± 105.2a (844.6, 863.2)217.5 ± 98.5b (211.7, 223.3)201 ± 114.6b (197.7, 213.7)197 ± 128.9b (175.3, 213.7)
      TP (g/L)65.5 ± 5.8a (62.1, 68.3)49.5 ± 5.1b (45.7, 52.6)44.6 ± 7.8b (41.2, 47.8)40.8 ± 5.9b (37.4, 45.3)
      ALB (g/L)41.4 ± 3.9a (39.4, 45.7)26.7 ± 6.5b (23.2, 28.9)20.3 ± 4.5c (18.3, 23.4)18.4 ± 4.3b (15.7, 21.5)
      T3 (nmol/L)1.98 ± 0.08a (1.78, 2.21)1.55 ± 0.09b (1.44, 1.67)1.29 ± 0.08b (1.17, 2.41)1.09 ± 0.0.6b (0.94, 1.13)
      T4 (nmol/L)49 ± 3.8a (43.7, 52.8)46.43 ± 5.21a (42.3, 48.7)38.32 ± 3.17b (31.43, 43.72)31.47 ± 4.12b (28.4, 36.8)
      Values are shown as means ± SD and 95% CI. Values in the same horizontal row with different letters were significantly different (P < 0.05; analysis of variance, followed by Student–Newman–Keuls test). ALB = albumin; TP = total protein; T3 = triiodothyronine; T4 = thyroxine.

      Growth of bone and cartilage

      There was no sharp distinction in tibial radiographs among the four groups at 4 weeks. However, there were abnormal proximal epiphyseal plate findings with obvious changes to the proximal metaphyseal area at 8 and 12 weeks. Radiological changes to the proximal epiphyseal plates and the metaphyseal area were most commonly observed in group D. Abnormal radiological signs included blurring, thinning, irregularity with and without sclerosis, interruption, and a depression in or destruction of the epiphyseal plate and the proximal metaphyseal area of the tibia. Compared with group A, group D had radiological changes in both bone density and epiphyseal plates (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Radiological changes to the proximal epiphyseal plates and the metaphyseal area were most commonly observed in group D (d, red arrows) compared with group A (a, red arrows), group B (b, red arrows) and group C (c, red arrows) had radiological changes in both bone density and epiphyseal plates at 12 weeks. Abnormal radiological signs included blurring, thinning, irregularity with and without sclerosis, interruption, and a depression in or destruction of the epiphyseal plate and the proximal metaphyseal area of the tibia.
      At 12 weeks, the proximal diameter of the epiphyseal plate and tibia length were measured. The average proximal diameter at the epiphyseal plate did not significantly differ among the groups at three time points in the study (P > 0.05). At 12 weeks, the average proximal diameter was 6.57 ± 0.37 mm in group A, 6.52 ± 0.29 mm in group B, 6.49 ± 0.31 mm in group C, and 6.55 ± 0.34 mm in group D (Fig. 2). Although there were no significant differences in proximal diameter among the four groups, there was a significant difference between groups A and B, compared with groups C and D, in tibia length (P = 0.013 and 0.037, respectively). However, regarding proximal diameter, there was no significant difference between groups A and B (P = 0.121) or between groups C and D (P = 0.067). At 12 weeks, tibia lengths were 36.69 ± 3.12 mm in group A, 35.42 ± 3.44 mm in group B, 32.15 ± 2.86 mm in group C, and 30.87 ± 3.15 mm in group D.
      Figure thumbnail gr2
      Fig. 2Radiological measurement of proximal diameter of the epiphyseal plate and tibia length. The average proximal diameter at the epiphyseal plate did not significantly differ among the groups at 12 weeks in the study (P > 0.05), compared with group A, the 95% CI of groups B, C, and D were (0.0378, 0.0692), (0.0536, 0.1044) and (−0.0002, 0.0302) respectively. But there was a significantly difference between groups A and B, compared with groups C and D, in tibia length (P = 0.013 and 0.037), 95% CI were (3.7439, 5.1581) and (5.2550, 7.2070) respectively.

      Pathohistological changes in the growth plate

      Pathological changes over time to epiphyseal plates are shown in Fig. 3. There was no chondrocyte necrosis of the epiphyseal plate in the resting zone, proliferative zone, or the hypertrophic zone in group A at weeks 4, 8 or 12. Epiphyseal cartilage cells in group A were rich, cell columns were neatly arranged in the proliferative zone, nuclei were deeply stained, the cytoplasm was clear, and cell outlines were clear. Chondrocytes in the hypertrophic zone were arranged closely. There was no chondrocyte necrosis in group B in week 4. In week 8, however, two of 10 cases had FN in the hypertrophic zone in group B, and in week 12, five of eight cases had FN. The arrangement of proliferative cell columns was irregular, and cell columns were shorter and sparser than in the control group. In group C, normal chondrocyte morphology was not observed, but a fused extracellular matrix was apparent by week 12. Two cases in group C showed LN in the hypertrophic or proliferative zones, and the proliferative cell columns were irregular and sparse. There was no chondrocyte necrosis in group D by week 4, but there was FN in four of 10 cases by week 8. At week 12, five cases in group D showed LN in the hypertrophic or proliferative zones, and three cases showed only FN. The arrangement of proliferative cell columns in group D was irregular, and cell columns were of varying lengths and were sparse.
      Figure thumbnail gr3
      Fig. 3Histological examination of the tibial epiphyseal plate at week 12 (hematoxylin and eosin, original magnification 20×). (a) In group A, cell columns were arranged regularly, the nuclei were clear, and the cytoplasm was transparent. (b) In group B, cell columns were disordered, condensed nuclei with pyknosis and lysis can be seen in the mast cell layer, and cells appear to have vacuole changes. There was some LN in the hypertrophic zone. (c) In group C, cell columns were disordered and sparse, there is a patchy cell-free zone, and LN can be seen in the hypertrophic zone. (d) In group D, there is obvious chondrocyte necrosis, normal chondrocyte morphology has disappeared, and the chondrocytes are fused with the extracellular matrix.

      Pathohistological changes in the metaphysis

      In group A, trabecular bone in the tibial metaphysis showed a longitudinal arrangement, like a section of a stalactite. Two to three chondrocyte columns extended downwards to become one trabecular bone. In group C, the primary trabecular bone was sparse and interrupted in the metaphysis; some transverse bone appeared in the metaphysis. In the group D, metaphyseal trabecular bone was more obviously sparse and interrupted compared with group C. In rats with serious disease, massive transverse trabecular bone appeared in the metaphysis, and transverse trabecular bone sealed the inferior part of the growth plate (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Histological examination of the tibial metaphysis at week 12 (hematoxylin and eosin, original magnification 20×). (a) In group A, the tibial metaphysis was full of regular trabecular bone in a longitudinal arrangement. (b) In group B, some trabecular bone was sparse and interrupted, but most of it was normal. (c) In group C, primary trabecular bone was obviously sparse and interrupted. Transverse trabecular bone was present in the metaphysis in some rats. (d) In group D, trabecular bone was obviously interrupted, and the inferior part of the growth plate was sealed by massive transverse trabecular bone in the metaphysis.
      In group B, a cartilage matrix filled in the gap between proliferative cell columns, which is the longitudinal arrangement of collagen fibers, and Masson trichrome staining of the collagen was blue. In group A, staining was deep. In groups C and D, however, staining was pale, relatively sparse, and disappeared in the necrosis zone. T-2 toxin may affect epiphyseal cartilage cell synthesis and the secretion of collagen components.

      Histological cartilage evaluation

      According to the OARSI scores for the osteochondral tissue samples, all compartments in group C and group D showed slight cartilage degeneration (intact surface with hypertrophy and/or edema) at 12 weeks. In general, the lower scores in groups C and D were associated with significantly more cartilage degeneration compared with groups A and B (P < 0.05). OARSI scores were not significantly different between groups A and B or between groups C and D (respectively P values: 0.12 and 0.06). However, scores were significantly different between groups D and A and between groups D and B (P = 0.012 and 0.037, respectively) at 12 weeks (Fig. 5).
      Figure thumbnail gr5
      Fig. 5OARSI cartilage histopathology assessment system score for the osteochondral tissue samples. A score of 0 represents normal cartilage, whereas a score of 24 represents a severely degenerated joint surface. Groups C and D had significantly more degeneration than groups A and B. An asterisk indicates a significant difference (P < 0.05); compared with group A, the 95% CI of groups B, C, and D are (−0.6531, 0.1198), (−2.3298, −1.6369) and (−2.5348, −1.8852); compared with group C, the 95% CI of groups A, B, D are (1.6369, 2.3298), (1.3032, 2.1301) and (−0.5846, 0.1313). Vertical lines atop each bar indicate the standard deviation.

      Discussion

      An animal model of KBD is the best way to study its pathogenesis, prevention, and the treatment. Many researchers have tried unsuccessfully to establish several KBD animal models using different single risk factors
      • Yao Y.F.
      • Kang P.D.
      • Li X.B.
      • Yang J.
      • Shen Bin
      • Zhou Z.K.
      • et al.
      Study on the effect of T-2 toxin combined with low nutrition diet on rat epiphyseal plate growth and development.
      • Yang S.
      Chondronecrosis induced in rhesus monkeys fed with grains and water of Kashin–Beck's disease endemic area.
      • Pasteels J.L.
      • Liu Fu-De
      • Hinsenkamp M.
      • Rooze M.
      • Mathieu F.
      • Perlmutter N.
      Histology of Kashin–Beck lesions.
      • Yang J.B.
      • Sun D.J.
      • Wang G.
      Observation on the culture of Fusarium isolated from the cereal produced in KBD areas causing cartilage necrosis of growth plate in broiler chickens.
      • Wang L.H.
      • Fu Y.
      • Shi Y.X.
      • Wang W.G.
      T-2 toxin induces degenerative articular changes in rodents: link to Kashin–Beck disease.
      . Therefore, we investigated the possibility that KBD-like symptoms could be induced in a rat model through the combination of exposure to a potential biological mycotoxin (T-2 toxin) and environmental deficiency (selenium and iodine deficiency) in the setting of malnutrition (low-protein diet). The rats we used were 1-month-old when our study began, and they are of a type that grows quickly until 4–14 weeks of age, which is similar to the growth pattern seen in humans between 5 and 15 years of age found in KBD-endemic areas. The pathological and radiographic changes we found were very similar to those seen in patients with KBD. After the rats were fed a low-nutrition diet (low in protein, selenium, and iodine) and given T-2 toxin, they demonstrated growth plate alteration and bone deformation. At week 4, their epiphyseal plates revealed different necrosis changes, including LN in the hypertrophic and proliferative zones. Furthermore, the plates had short cell columns that were sparse and interrupted in the proliferative zone. Metaphyseal trabecular bone was obviously sparse and interrupted, and massive transverse trabecular bone appeared in the metaphysis and sealed the inferior part of the growth plate. We speculate that the transverse trabecular bone sealed the inferior part of the growth plate, which may result in bone growth retardation and deformity. Yang et al. used Fusarium-contaminated grain to feed chickens
      • Wang L.H.
      • Fu Y.
      • Shi Y.X.
      • Wang W.G.
      T-2 toxin induces degenerative articular changes in rodents: link to Kashin–Beck disease.
      . In the fifth week of their study, the proliferative and transition zone cells developed a large area of cell necrosis. This type of organization was similar to the changes of deep cartilage necrosis in KBD; however, this view has not widely been accepted. Xiong
      • Xiong Y.
      Toxic effect of T-2 toxin on articular cartilage in Chinese experimental mini-pig.
      found that T-2 toxin also can induce deep zone necrosis or patchy necrosis of the articular cartilage in miniature pigs, but the epiphyseal plates in those animals did not show as great a change as we found in rats.
      The basic pathological feature of KBD is a FN of chondrocytes in the hypertrophic zone of the growth plate cartilage and in the deep zone of articular cartilage, which can result in growth retardation, secondary OA, and disability in advanced stages
      • Hinsenkamp M.
      • Mathieu F.
      • Claus W.
      • Collard J.F.
      • de Maertelaere V.
      Effects of physical environment on the evolution of Kashin–Beck disease in Tibet.
      • Ren F.L.
      • Guo X.
      • Zhang R.J.
      • Wang Sh.J.
      • Zuo H.
      • Zhang Z.T.
      • et al.
      Effects of selenium and iodine deficiency on bone, cartilage growth plate and chondrocyte differentiation in two generations of rats.
      • Yao Y.F.
      • Kang P.D.
      • Li X.B.
      • Yang J.
      • Shen Bin
      • Zhou Z.K.
      • et al.
      Study on the effect of T-2 toxin combined with low nutrition diet on rat epiphyseal plate growth and development.
      . The developmental deformities are most likely a result of impaired chondrocyte differentiation and endochondral ossification, caused by the impairment of the growth plate cartilage of the bone growth center.
      KDB is endemic to the Aba region of Sichuan Province. Most people in areas affected with KBD live at an altitude of 2500–3500 m. Our investigation revealed that grain storage in the area is easily contaminated by mildew. The natural environment and the local customs are similar to those in Tibet. The high altitude and extremely severe weather conditions affect the species of grain planted but also the physical environment of the patients
      • Hinsenkamp M.
      • Mathieu F.
      • Claus W.
      • Collard J.F.
      • de Maertelaere V.
      Effects of physical environment on the evolution of Kashin–Beck disease in Tibet.
      . The local residents' diet tends to consist mostly of barley (>90%) and is low in protein, fat, and vitamin and minerals, especially selenium and iodine, minerals that are deficient in the region
      • Yao Y.F.
      • Pei F.X.
      • Li X.
      • Yang J.
      • Shen B.
      • Zhou Z.K.
      • et al.
      Preventive effects of supplemental selenium and selenium plus iodine on bone and cartilage development in rats fed with diet from Kashin–Beck disease endemic area.
      • Zhao Q.
      • Shen Q.B.
      • Zeng X.G.
      Geochemical characteristics of Kashin–Beck disease districts in the Aba area, Sichuan.
      • Chen K.H.
      • Fang Sh.M.
      The survey report of iodine deficiency in Sichuan Aba region.
      . This dietary pattern can easily induce malnutrition
      • Dang S.
      • Yan H.
      • Yamamoto S.
      • Wang X.
      • Zeng L.
      Poor nutritional status of younger Tibetan children living at high altitudes.
      . Therefore, we speculated that a low-nutrition diet (low in protein, selenium, and iodine) and the intake of mycotoxins play a role in the occurrence of KBD in the Aba area.
      We found that the histological and the radiographic changes at different times in group D were similar to the pathological changes seen in KBD. According to the OARSI scores of the osteochondral tissue samples, all compartments in groups C and D showed slight cartilage degeneration (intact surface with hypertrophy and/or edema) at 12 weeks. Although the pathogenesis of KBD is not clear, it is believed that chondrocyte necrosis is associated with the pathological changes caused by free radicals or other pathological factors
      • Mo D.X.
      KBD cartilage necrosis histopathology and clinical significance.
      • Zhang B.D.
      • Guo X.
      • Bai G.L.
      • Ping Z.G.
      • Zuo H.
      • Ren F.L.
      • et al.
      The changes of nitric oxide, No synthase and sFas/APo-1 in serum among the patients with Kashin–Beck disease.
      • Shi Z.
      • Cao J.
      • Chen J.
      • Li S.
      • Zhang Z.
      • Yang B.
      • et al.
      Butenolide induced cytotoxicity by disturbing the prooxidant-antioxidant balance, and antioxidants partly quench in human chondrocytes.
      . As a cofactor of glutathione peroxidase, selenium participates in the lipoxygenase pathway in the organic antioxidant system. Selenium deficiency can damage cellular and mitochondrial membranes
      • Tapiero H.
      • Townsend D.M.
      • Tew K.D.
      The antioxidant role of selenium and seleno-compounds.
      • Behne D.
      • Kyriakopoulos A.
      Mammalian selenium-containing proteins.
      . Zhao et al. reported that polysaccharide extracted from selenium-enriched Ganoderma lucidum had a DNA-protective effect against oxidative damage by hydroxyl radicals and that a high selenium concentration had a much stronger effect against hydroxyl and superoxide radicals
      • Zhao Q.
      • Shen Q.B.
      • Zeng X.G.
      Geochemical characteristics of Kashin–Beck disease districts in the Aba area, Sichuan.
      . In addition, Zhang et al. reported that moniliformin toxin promoted the catabolism of aggrecan and type II collagen in cultured human chondrocytes and that selenium could partially alleviate the damage of aggrecan induced by moniliformin toxin
      • Zhang A.
      • Cao J.L.
      • Yang B.
      • Chen J.H.
      • Zhang Z.T.
      • Li S.Y.
      • et al.
      Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin–Beck disease.
      . Moreover, in China, endemic selenium deficiency is often accompanied by endemic iodine deficiency; all selenium-deficient areas of China are also areas of iodine deficiency disease
      • Ma T.
      • Guo J.
      • Wang F.
      The epidemiology of iodine deficiency diseases in China.
      . Thus, KBD was once considered a complication of iodine deficiency disease. Low levels of selenium and iodine were thought to be the most important environmental factors causing the diseases. A cross-sectional study showed that especially in Tibetan KBD-affected areas, low urinary iodine, high thyrotropin, and low serum thyroxine-binding globulin values were associated with an increased risk of KBD, whereas a low serum selenium concentration was not
      • Moreno-Reyes R.
      • Suetens C.
      • Mathieu F.
      • Begaux F.
      • Zhu D.
      • Rivera M.T.
      • et al.
      Kashin–Beck osteoarthropathy in rural Tibet in relation to selenium and iodine status.
      . Iodine is an essential element for the synthesis and metabolism of thyroid hormones. Deficiency of iodine leads to decreased thyroid hormone production, which induces dysfunction in bone growth and developmental growth
      • Guo R.L.
      • Wang B.L.
      • Zheng H.
      • Zuo A.J.
      • Liang D.C.H.
      • Zhang J.Y.
      Pathological investigation of bone development retardation caused by iodine deficiency.
      • Guo R.L.
      • Wang B.L.
      • Zheng H.
      • Zuo A.J.
      • Liang D.C.H.
      • Zhang J.Y.
      Impairment of bone development in iodine-deficient rats.
      • Wexler J.A.
      • Sharretts J.
      Thyroid and bone.
      • Winkler R.
      • Griebenow S.
      • Wonisch W.
      Effect of iodide on total antioxidant status of human serum.
      • Mo D.X.
      Pathology of selenium deficiency in Kashin–Beck disease.
      . The deficiency of iodine can also decrease antioxidation, which has been reported in the literature
      • Ma T.
      • Guo J.
      • Wang F.
      The epidemiology of iodine deficiency diseases in China.
      . Depending on the mechanism for radical damage in KBD, antioxidation of iodine may play an important role in decreasing the lesions in KBD.
      Exposure to T-2 toxin, produced by mycotoxins in the stored grains in KBD-endemic areas where the weather is damp and cold, is widely considered another risk factor for the development of KBD
      • Mo D.X.
      Pathology of selenium deficiency in Kashin–Beck disease.
      . Transient exposure to T-2 toxin is a biological factor, which may explain the temporal wave-like or “sporadic attack” incidence of KBD. In group D of our study, metaphyseal trabecular bone was noticeably sparse and interrupted and massively transverse, similar to the pathological changes seen in KBD. Therefore, we believe that T-2 toxin contamination of food supplies, in combination with selenium and iodine deficiency and a low-protein diet, may be one of the most important causes of KBD.
      Our study has shown that an animal model of KBD can be approached by feeding rats a low-nutrition diet (low levels of selenium, iodine, and protein) and exposing them to T-2 toxin. The pathological and radiographic changes that we observed were very similar to those in patients with KBD. This animal model can be very helpful for research on the etiology, pathogenesis, and prevention of KBD. The combination of a low-nutrition diet and T-2 toxin exposure may play an important role in the etiology and pathogenesis of KBD.

      Authors' contributions

      PK conceived the research question, coordinated development of the study design, completed the first draft of the manuscript, and edited the manuscript. FP secured funding, advised and coordinated manuscript development, wrote and edited a portion of the manuscript, approved the final version of the manuscript prior to submission, and is also a guarantor. YY and JY developed and edited the manuscript and provided intellectual contributions to the study. BS and ZZ contributed to the development of the research question, edited the manuscript, and provided intellectual contributions during manuscript revisions.

      Conflict of interest

      We declare that we have no conflicts of interest.

      Acknowledgments

      This research was funded by China National Science & Technology pillar program during the 11th 5-year-plan period (2007BA125B04) and by National Natural Science Fund of China (81271976/H0605-81171763). We thank Professor X. Guo from the Medical College of Xi'an Jiaotong University (Xi'an, China) for excellent technical assistance.

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