Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint. The pericellular matrix, cellular membrane of the chondrocytes, and their...Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint. The pericellular matrix, cellular membrane of the chondrocytes, and their cytoskeletal structures are key elements in the mechanical environment. The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties. Methods Three age groups of rabbit knees were evaluated： （1） young （2 months, n=10）; （2） adult （8 months, n=10）; and （3） old （31 months, n=10）. Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods. Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation. The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining. Results In response to an applied constant 0.3-0.4 kPa of negative pressure, all chondrocytes exhibited standard linear viscoelastic solid properties. Model predictions of the creep data showed that the average equilibrium modulus （E~）, instantaneous modulus （E0）, and apparent viscosity （~） of old chondrocytes was significantly lower than the young and adult chondrocytes （P 〈0.001）; however, no difference was found between young and adult chondrocytes （P 〉0.05）. The adult and old chondrons generally possessed a thicker pericellular matrix （PCM） with more enclosed cells. The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure （1.0-1.1 kPa） without the deformation seen in the old chondrons. The viscoelastic properties （E,, E0, and/~） of young and adult chondrons were significantly greater than that observed in young and adult cells, respectively （P 〈0.001）. The adult chondrons were stiffer than the young chondrons under micropipette aspiration （P 〈0.001）. Conclusions Our findings provide a theoretical model to measure the viscoelastic properties of the chondrons as a whole unit by micropipette aspiration, and further suggest that the properties of the chondrocytes and PCM have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with aging.展开更多
Background Chondrocytes＇ phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure.Microfilaments,microtubules,and intermediate filaments are three important components of the cyto...Background Chondrocytes＇ phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure.Microfilaments,microtubules,and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes.The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.Methods Chondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods （n=24）.The isolated cells were stabilized for three days and then exposed to low,medium,and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest：colchicine for microtubules,acrylamide for intermediate filaments,and cytochalasin D for actin microfilaments.A group of control cells were treated with carrier.Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents.The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy （LSCM） with immunofluorescence staining on day 3.The mRNA levels of aggrecan （AGG） and type Ⅱ collagen （Col-2） and their levels in culture medium were analyzed using real-time PCR and enzymelinked immunosorbent serologic assay （ELISA） on days 3,6,and 9.Results In the initial drug-dose-response study,there was no significant difference in the vitality of cells treated with 0.1 μmol/L colchicine,2.5 mmol/L acrylamide,and 10 μg/L cytochalasin D for two days when compared with the control group of cells.The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei.Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three,six,and nine days,and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells.Cellular proliferation,monolayer morphology,and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.Conclusions The disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis,increased matrix metalloprotease expression,and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures.Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.展开更多
基金This work was supported in part by 973 Program （No. 2009CB526514） and National Natural Science Foundation （No. 30872616 and No. 81071495） of China.
文摘Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint. The pericellular matrix, cellular membrane of the chondrocytes, and their cytoskeletal structures are key elements in the mechanical environment. The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties. Methods Three age groups of rabbit knees were evaluated： （1） young （2 months, n=10）; （2） adult （8 months, n=10）; and （3） old （31 months, n=10）. Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods. Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation. The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining. Results In response to an applied constant 0.3-0.4 kPa of negative pressure, all chondrocytes exhibited standard linear viscoelastic solid properties. Model predictions of the creep data showed that the average equilibrium modulus （E~）, instantaneous modulus （E0）, and apparent viscosity （~） of old chondrocytes was significantly lower than the young and adult chondrocytes （P 〈0.001）; however, no difference was found between young and adult chondrocytes （P 〉0.05）. The adult and old chondrons generally possessed a thicker pericellular matrix （PCM） with more enclosed cells. The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure （1.0-1.1 kPa） without the deformation seen in the old chondrons. The viscoelastic properties （E,, E0, and/~） of young and adult chondrons were significantly greater than that observed in young and adult cells, respectively （P 〈0.001）. The adult chondrons were stiffer than the young chondrons under micropipette aspiration （P 〈0.001）. Conclusions Our findings provide a theoretical model to measure the viscoelastic properties of the chondrons as a whole unit by micropipette aspiration, and further suggest that the properties of the chondrocytes and PCM have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with aging.
基金This work was supported in part by grants from National Natural Science Foundation of China （No. 31340010 and No. 31271033）, Science Foundation of Ministry of Education of China （No. 20121417120004）, and Natural Science Foundation of Shanxi Province （No. 2013021036-3）.
文摘Background Chondrocytes＇ phenotype and biosynthesis of matrix are dependent on having an intact cytoskeletal structure.Microfilaments,microtubules,and intermediate filaments are three important components of the cytoskeletal structure of chondrocytes.The aims of this study were to determine and compare the effects of the disruption of these three cytoskeletal elements on the apoptosis and matrix synthesis by rabbit knee chondrocytes in vitro.Methods Chondrocytes were isolated from full-thickness knee cartilage of two-month-old rabbits using enzymatic methods （n=24）.The isolated cells were stabilized for three days and then exposed to low,medium,and high doses of chemical agents that disrupt the three principal cytoskeletal elements of interest：colchicine for microtubules,acrylamide for intermediate filaments,and cytochalasin D for actin microfilaments.A group of control cells were treated with carrier.Early apoptosis was assessed using the Annexin-FITC binding assay by flow cytometry on days 1 and 2 after exposure to the disrupting chemical agents.The components and distribution of the cytoskeleton within the cells were analyzed by laser scanning confocal microscopy （LSCM） with immunofluorescence staining on day 3.The mRNA levels of aggrecan （AGG） and type Ⅱ collagen （Col-2） and their levels in culture medium were analyzed using real-time PCR and enzymelinked immunosorbent serologic assay （ELISA） on days 3,6,and 9.Results In the initial drug-dose-response study,there was no significant difference in the vitality of cells treated with 0.1 μmol/L colchicine,2.5 mmol/L acrylamide,and 10 μg/L cytochalasin D for two days when compared with the control group of cells.The concentrations of colchicine and acrylamide treatment selected above significantly decreased the number of viable cells over the nine-day culture and disrupted significantly more cell nuclei.Real-time PCR and ELISA results showed that the mRNA levels and medium concentrations of AGG and Col-2 were significantly decreased for cultures treated with colchicine and acrylamide when compared with untreated cells at three,six,and nine days,and this inhibition was correlated with higher matrix metalloprotease-13 expression in these cells.Cellular proliferation,monolayer morphology,and matrix metabolism were unaffected in cytochalasin D-treated cells when compared with control cells over the nine-day culture period.Conclusions The disruption of the microtubulin and intermediate filaments induced chondrocyte apoptosis,increased matrix metalloprotease expression,and decreased AGG and Col-2 expression in rabbit knee chondrocyte cultures.Our findings suggest that microtubulin and intermediate filaments play a critical role in the synthesis of cartilage matrix by rabbit knee chondrocytes.
