Changes of death receptor regulator expression in the articular cartilage of patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in the expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the articular cartilage of patients with Kashin-Beck disease (KBD) and the role of these proteins in the pathogenesis of KBD.</p><p><b>METHODS</b>The cartilage samples were collected from patients with established diagnosis of KBD and osteoarthritis and from healthy control subjects undergoing amputation due to traffic accidents. The expressions of Fas-associated death domain protein (FADD) and cellular-FLICE inhibitory protein (c-FLIP) in the cartilage were detected by immunohistochemistry, and the positive chondrocytes were counted in different layers of the articular cartilage under microscope.</p><p><b>RESULTS</b>The positivity rates of FADD in the middle layer of articular cartilage from patients with KBD [(28.68∓2.19)%] and osteoarthritis [(35.40∓2.34)%] were significantly higher than that in normal cartilage [(10.51∓5.02)%, F=16.245, P=0.000], but the rates in the upper and deeper layers were comparable among the 3 groups (P=0.206-0.761). In KBD cartilage, FADD expression was the highest in the middle layer [(28.68∓5.38)%] followed by the deeper layer [(17.94∓8.38)%]. Compared with the healthy controls, KBD and osteoarthritis patients showed significantly higher FLIP expression in the upper layer of the cartilage (F=5.929, P=0.018) but similar expressions in middle and deeper layers.</p><p><b>CONCLUSIONS</b>KBD patients have significant increased FADD expression in the middle layer but decreased FLIP expression in the upper layer of the cartilage, suggesting that the death receptor pathway and its regulators play important roles in the pathogenesis of KBD.</p>

Expression of Caspase-8 and Bcl-2 in the cartilage loose bodies in patients with Kashin-Beck disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of Caspase-8 and Bcl-2 in the formation of loose bodies in Kashin-Beck disease (KBD).</p><p><b>METHODS</b>Specimens of cartilage loose bodies were collected from 50 adult patients with KBD, and the samples of articular cartilage were collected from 10 healthy adults to serve as control. Avidin-biotin alkaline phosphatase immunohistochemistry was employed to examine Bcl-2 and Caspase-8 positivities in the chondrocytes in the loose bodies.</p><p><b>RESULTS</b>In KBD loose bodies, the percentage of chondrocytes positive for Bcl-2 and Caspase-8 [(18.40∓8.78)% and (67.54∓12.29)%, respectively] were significantly higher than those of the control group [(12.25∓1.58)% and (24.70∓4.35)%, respectively]. Caspase-8 was found to promote chondrocyte apoptosis in the loose bodies, and this effect overrode the apoptosis-suppressing effect of Bcl-2. Bcl-2 and Caspase-8 positivities were found mainly in the deep hypertrophic chondrocytes in the cartilage or in cells adjacent to the bone tissues.</p><p><b>CONCLUSION</b>KBD loose bodies contain an increased percentage of apoptotic chondrocytes positive for Bcl-2 and Caspase-8. The apoptosis-inducing effect of Caspase-8 was a dominant feature in the cartilage pathology of KBD compared to the apoptosis-suppressing effect of Bcl-2.</p>

Effects of selenium,iodine deficiency and their combination on bone and cartilage growth in parental and first filial generation rats / 中国地方病学杂志

Objective To study the effects of selenium deficiency,iodine deficiency and combined selenium and iodine deficiency on bone and cartilage growth in the parental and the first filial generation rats. Methods Forty-eight weanling healthy SD rats were randomly divided into selenium deficieney, iodine deficiency, combined selenium and iodine deficiency and control groups according to their body mass. These rats were fed with selenium deficiency, iodine deficiency, combined selenium and iodine deficiency, and normal fodder, respectively. The parental rats (about 3 months old) were mated in each group 8 weeks after the beginning of the experiment. Right tibias and left knee joints were collected when the parental generation rats were about 6 months and the first filial generation rats were about 3 months old. Tibial length, mid-shaft tibial diameter, and articular cartilage diameters of the right tibias were measured by vernier caliper. Left knee joints were embedded and cut into sections and the thickness of the growth plate cartilage, layers of proliferative and hypertrophic chondrocytes in growth plate cartilage were observed under the light microscope. Results The selenium deficiency had significant effect on serum selenium level of the parental and the first filial generation rats(F value were 239.56,232.68, P< 0.01), and also on serum T4 level of the first filial generation rats(F value were 6.95, P < 0.05). The iodine deficiency had significant effect on serum T3 and T4 level in the two generations rats(F value were 14.11,14.05,30.29,34.53, P < 0.01 ). There were interactions between selenium deficiency and iodine deficiency on serum T4 level in the first filial generation rats (F= 5.99, P< 0.05). The serum selenium of selenium deficiency group[ (30.28 ± 6.34), (43.95 ± 9.75)μg/L],combined selenium and iodine deficiency group[ (30.33 ± 5.18), (35.40 ± 3.16)μg/L] were significantly lower than iodine deficiency group[(345.83 ± 29.55), (245.24 ± 9.95)μg/L] and the controls[ (358.64 ± 30.50), (236.50 ±9.75) μg/L] in the two generations. The serum T3 of combined selenium and iodine deficiency group [(0.55 ± 0.05 ),(0.88 ± 0.14)nmol/L] were significantly lower than the controls[(0.75 ± 0.08), (1.26 ± 0.26)nmol/L] in the two generations. The serum T4 of iodine deficiency [ (24.11 ± 2.29), (42.10 ± 8.92) nmol/L ] and combined selenium and iodine deficiency group[ (20.66 ± 1.93), (26.55 ± 5.98)nmol/L] were significantly lower than the controls[ (36.15 ±2.74), (52.79 ± 8.84)nmol/L] and selenium deficiency group[ (28.12 ± 3.33), (52.02 ± ll.99)nmol/L] in the two generations. The selenium deficiency and iodine deficiency had significant effect on tibial length, thickness of the growth plate cartilage, layers of proliferative and hypertrophic chondrocytes in first filial generation rats(F values were 24.31,6.98,40.76,56.15,25.24,82.82, 10.07,5.57, P <0.05 or <0.01). There were interactions between selenium deficiency and iodine deficiency on tibial length, thickness of the growth plate cartilage, layers of proliferative and hypertrophic chondrocytes (F values were 5.68,24.86,41.82,9.12, P <0.05 or <0.01 ). The tibial length of the selenium deficiency group[ (33.17 ± 0.34)mm] and combined selenium and iodine deficiency group[ (31.30 ± 0.87)mm] were significantly lower than the controls[ (34.12 ± 0.32)mm, P< 0.05]. Thickness of the growth plate cartilage [ (1.60 ± 0.18)mm ], layers of proliferative chondrocyte (8.54 ± 0.81), and hypertrophic chondrocyte (4.95 ± 0.37)of the combined selenium and iodine deficiency group were significantly decreased when compared to the selenium deficiency group[ (3.03 ± 0.10)mm, 14.68 ± 0.84,6.60 ± 0.31], iodine deficiency group[ (2.90 ± 0.09)mm, 13.75 ±0.33,6.61 ± 0.84 ] and the controls [ (3.19 ± 0.09) mm, 14.94 ± 0.36, 6.64 ± 0.26, P <0.05]. Thickness of the growth plate cartilage, layers of proliferative chondrocyte of the iodine deficiency group were lower than the controls(P<0.05). Conclusions Selenium deficiency impair tibial growth in first filial generation rats, iodine deficiency retarded the chondroncyte proliferation and decreases the thickness of growth plate cartilage in first filial generation rats, and combined selenium and iodine deficiency significantly impair the growth of bone and cartilage in first filial generation rats.

Effects of selenium and/or iodine deficiency on chondrocyte apoptosis in rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effects of selenium and/or iodine deficiency on chondrocyte apoptosis in articular cartilage in rats.</p><p><b>METHODS</b>Forty-eight Sprague-Dawley rats were randomly divided into selenium deficiency group, iodine deficiency group, combined selenium and iodine deficiency group, and control group. Chondrocyte apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method, and Bcl-2 and Bax in articular cartilage were stained by immunohistochemistry in F3 generation of rats.</p><p><b>RESULTS</b>In articular cartilage, the positive rate of apoptotic chondrocytes stained by TUNEL in the upper and middle zones in selenium deficiency group, iodine deficiency group, and combined selenium and iodine deficiency group (all P < 0.05) were significantly higher than that in control group. The apoptotic chondrocytes were prominent in the middle zone. The positive percentage of chondrocytes apoptosis was not significantly different among these three groups (P > 0.05). Compared with the control group, the expressions of both Bcl-2 and Bax were significantly higher in the upper and middle zone in the selenium deficiency group, iodine deficiency group, and combined selenium and iodine deficiency group (all P < 0.05); however, the expressions of Bcl-2 and Bax were not significantly different among these three groups (P > 0.05).</p><p><b>CONCLUSION</b>Selenium and/or iodine deficiency may induce chondrocyte apoptosis.</p>

Construction of tissue-engineered cartilage by seeding chondrocytes on allogeneic cancellous bone matrix gelatin / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the use of cancellous bone matrix gelatin (BMG) combined with chondrocytes in constructing tissue-engineered cartilage by observing the growth, proliferation and differentiation of chondrocytes on allogeneic cancellous BMG.</p><p><b>METHODS</b>The articular chondrocytes isolated from a 1-month-old rabbit were multiplied to a monolayer and seeded onto cancellous BMG to construct tissue-engineered cartilage in vitro during a period of 6 weeks. Samples were taken from the construct after 1, 2, 4, and 6 weeks of culture and evaluated by histology, immunohistochemistry and transmission electron microscopy (TEM).</p><p><b>RESULTS</b>The chondrocytes excreted matrix proteoglycan and collagen on cancellous BMG. With the prolongation of the culture time, the cells proliferated in the construct and the cells in the lacunae increased. Numerous chondrocytes were present the central region of the cancellous BMG and surrounded by extracellular matrix. By 6 weeks of culture, the BMG was covered with 15-20 layers of chondrocytes and cartilaginous tissue occurred in the pores throughout the cancellous BMG. Immunohistochemical staining showed rich and evenly distributed type II collagen around the chondrocytes, and TEM revealed an ultrastructure of the chondrocyte similar to that of native chondroctyes, with abundant extracellular matrix produced around the cells.</p><p><b>CONCLUSION</b>Tissue-engineered cartilage can be constructed in vitro using allogeneic cancellous BMG combined with chondrocytes. Allogeneic cancellous BMG serves as a good scaffold material for tissue-engineered cartilage to promote the growth and proliferation of the seeded chondrocytes and allows maintenance of the differentiation phenotype of the cells.</p>

Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro.</p><p><b>METHODS</b>Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro.</p><p><b>RESULTS</b>T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above.</p><p><b>CONCLUSION</b>T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.</p>

Butenolide induces apoptosis of cultured chondrocytes: study of its mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe cell apoptosis and Bcl-2 and Bax expression changes of chondrocytes induced by butenolide (BUT) and the inhibitory effect of selenium against BUT-induced chondrcyte apoptosis, to gain insights into the mechanism by which BUT induces chondrcyte apoptosis.</p><p><b>METHODS</b>Cartilage tissue reestablished from human fetal articular chondrocytes in vitro were treated with BUT at the concentrations of 0.1, 1.0 and 5.0 microg/ml and with the protective factor selenium. TUNEL method was used to detect chondrocyte apoptosis, which was quantified by flow cytometry. Immunohitochemistry was performed to analyze the expression of Bcl-2 and Bax in the reestablished cartilage tissue.</p><p><b>RESULTS</b>BUT exposure induced chondrocyte apoptosis, and the apoptosis rate increased with the concentration increment of BUT from 0 to 1.0 mg/ml, resulting also increased positive expression rate of Bcl-2 and Bax(P<0.05). The apoptosis rate of chondrocytes in BUT+ selenium group was significantly lower than that of BUT groups (P<0.05), as was the positivity rate of Bcl-2 and Bax expression (P<0.05).</p><p><b>CONCLUSION</b>BUT induces chondrocyte apoptosis in positive relation with BUT concentration (from 0 to 1.0 mg/ml) and causes increased expressions of Bcl-2 and Bax. Selenium can inhibit the chondrocyte apoptosis induced by BUT.</p>

Comparison of apoptosis of articular chondrocytes in the pathogenesis of Kashin-beck disease and primary osteoarthritis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate chondrocyte apoptosis and expression of Fas and inducible nitric oxide synthase (iNOS) in articular cartilage in the pathogenesis of Kashin-beck disease (KBD) and primary osteoarthritis (OA).</p><p><b>METHODS</b>The collected samples of articular cartilage were divided into three groups: normal control (15 cases), KBD adults (15 cases) and OA (15 cases). Chondrocyte apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling method, and Fas and iNOS in articular cartilage were stained by immunohistochemistry.</p><p><b>RESULTS</b>The positive percentages of chondrocyte apoptosis stained in articular cartilage of KBD and OA were significantly higher than that of the control (P < 0.01), and the positive percentage of chondrocytes apoptosis in the eroded areas of articular cartilage were significantly higher than in the non-eroded areas in articular cartilage of the same patient with KBD and OA (P < 0.05). There was no significant difference in positive percentage of chondrocytes apoptosis between KBD and OA. The positive percentages of Fas and iNOS in chondrocytes were significantly higher in KBD and OA than in control (P < 0.01). Significant differences in Fas and iNOS expression between the eroded areas and non-eroded areas were seen in articular cartilage of patients with KBD and OA (P < 0.05), but such difference did not exist between KBD and OA.</p><p><b>CONCLUSION</b>Cell apoptosis seems to be associated with the pathogenesis of both KBD and OA. Fas and iNOS might mediate chondrocyte apoptosis.</p>

Toxic effect of butenolide on chondrocyte differentiation and the protective effect of selenium / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of butenolide (BUT) on cultured chondrocytes differentiation and the possible protective effects of selenium (Se).</p><p><b>METHODS</b>Ex-vivo cultured chondrocytes were divided into six groups: (1) Control group (without BUT and Se); (2) Se 0.1 microg/ml control group; (3) BUT 0.1 microg/ml group; (4) BUT 1.0 microg/ml group; (5) BUT 5.0 microg/ml group; and (6) BUT 1.0 microg/ml + Se 0.1 microg/ml group. The expression of collagen II (Col II), collagen X (ColX), basic fibroblast growth factor (bFGF), and parathyroid hormone-related peptide (PTHrP) in (or around) chondrocytes in all groups were analyzed by immunohistochemistry.</p><p><b>RESULTS</b>The expressions of Col II in 1.0 microg/ml BUT group and 5.0 microg/ml BUT group were significantly lower than those in the control group (P < 0.05). The expression of Col II in 1.0 microg/ml BUT + Se group were significantly higher than those in the 1.0 microg/ml BUT group and 5.0 microg/ml BUT group (P < 0.05). The expressions of bFGF and PTHrP of BUT groups were significantly higher than those in the Se and control groups (P < 0.05). No expression of ColX was observed in all groups.</p><p><b>CONCLUSION</b>BUT can affect the collagen II synthesis of the chondrocytes. Selenium supplementation may play a protective role.</p>

Mechanism of chondrocyte apoptosis in Kashin-Beck disease and primary osteoarthritis: a comparative study / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate chondrocyte apoptosis and the expressions of Bcl-2, Bax, Fas and iNOS in the articular cartilage between Kashin-Beck disease (KBD) and primary osteoarthritis (OA) and explore the difference in pathogenesis between the two diseases.</p><p><b>METHODS</b>The articular cartilage specimens were collected from 15 normal human subjects, 15 adult patients with KBD and 15 with OA. Chondrocyte apoptosis was detected by TUNEL method, and the expressions of Bcl-2, Bax, Fas and iNOS in articular cartilage were examined with B-SA immunohistochemistry.</p><p><b>RESULTS</b>The percentages of apoptotic chondrocytes positive for TUNEL staining in the articular cartilage were significantly higher in patients with KBD and OA than in normal control subjects (F=20.90-53.16, df=42, P<0.01), and the erosive areas of the articular cartilage contained greater percentage of apoptotic chondrocytes than the non-erosive areas in the same patient with KBD (t=4.154, df=28, P<0.01) or OA (t=6.004, df=28, P<0.01). No significant difference was noted in the positive apoptotic chondrocytes between KBD and OA (t=1.329-1.362, df=28, P>0.05). The percentage of chondrocytes positive for Bcl-2, Bax, Fas and iNOS were significantly higher in KBD and OA patients than in the control subjects (F=25.46-215.31, df=42, P<0.01), and significant differences were observed in Bcl-2, Bax, Fas and iNOS expressions between the erosed areas and non-erosed areas in articular cartilage in patients with KBD (t=2.608-7.77, df=28, P<0.05) and OA (t=2.278-5.413, df=28, P<0.05), but their expressions showed no significant difference between the two diseases (t=0.284-1.590, df=28, P>0.05).</p><p><b>CONCLUSION</b>There was no significant difference in apoptotic chondrocytes and Bcl-2, Bax, Fas and iNOS expressions in the cartilage between adult patients with KBD and OA.</p>

Migration of intravenously grafted mesenchymal stem cells to injured heart in rats / 生理学报

The present study aimed to determine the role of tissue injury in migration of mesenchymal stem cells (MSCs) intravenously transplanted into heart and to establish experimental basis for improving stem cell therapy in its targeting and effectiveness. MSCs were isolated from bone marrow of male Sprague-Dawley rats and purified by density centrifuge and adhered to the culture plate in vitro. Female rats were divided randomly into four groups. Myocardial ischemia (MI) transplanted group received MSCs infusion through tail vein 3 h after MI and compared with sham-operated group or normal group with MSCs infusion, or control group received culture medium infusion. MI was created in female rats by ligating the left anterior descending coronary artery. The heart was harvested 1 week and 8 weeks after transplantation. The characteristics of migration of MSCs to heart were detected with expression of sry gene of Y chromosome by using fluorescence in situ hybridization (FISH). Ultrastructural changes of the ischemic myocardium of the recipient rats were observed by transmission electron microscope (TEM). One week or 8 weeks after transplantation, sry positive cells were observed in the cardiac tissue in both of MI transplanted group and sham-operated group, the number of sry positive cells being significantly higher in MI transplanted group (P<0.01). No significant difference was found in the number of sry positive cells between 1 week and 8 weeks after transplantation. No sry positive cells were observed in the hearts of control and normal group. In addition, the ultrastructure of some cells located in the peri-infarct area of MI rats with MSCs transplantation was similar to that of MSCs cultured in vitro. These results indicate that MSCs are capable of migrating towards ischemic myocardium in vivo and the fastigium of migration might appear around 1 week after MI. The tissue injury and its degree play an important role in the migration of MSCs.