BACKGROUND: Chondron is a basic microanatomical unit of articular cartilage. lncreasing evidence suggests that the pericellular matrix (PCM) is a distinct functional compartment in articular cartilage, influencing the metabolic, micromechanical environment, and degeneration of chondrocytes. But the precise functions and action mechanism need further investigation.OBJECTIVE: To review the literature pertinent to the morphology, function, isolation of the chondron in articular cartilage, and its degenerative events during the progression of osteoarthritis (OA).METHODS: This review summarized the articles published in the PubMed database before July 2009. In addition, recent data and figure of our laboratory on the morphology and biomechanics of chondron and chondrocyte were supplemented. RESULTS AND CONCLUSION: The PCM is primarily characterized by the presence of type Ⅵ collagen, and these components are widespread in the expansive extracellular matrix (ECM) in newborns, while in mature the components are mainly localized to a narrow pericellular zone. The three-dimensional morphology of chondron has been recently quantified in situ with fluorescence confocal microscopy, and the mechanical properties of the isolated individual chondrons and their PCM are measured using the micropipette technique and atomic force microscopy. More studies have shown that the presence of the PCM in chondrons has a profound influence on chondrocyte gene expression. At the same time, structural, histochemical and biomechanical studies indicate the chondron and their PCM may undergo degenerative processes with osteoarthritis, similar to those occurring in the ECM, Although the precise function of the PCM is unknown, increasing evidence in vivo or in vitro suggests that the PCM is a basic microanatomical unit in articular cartilage, influencing the metabolic and micromechanical environment of chondrocytes.Department of Orthopedics, the Second Hospital of Shanxi Medical University, Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair,Taiyuan 030001,Shanxi Province,China

Objective To observe the effects of cyclic tensile strain (CTS) on in vitro expression of glycosaminoglycans (GAG) in rabbit chondrocytes of different ages.Methods Nine male New Zealand rabbits were grouped into juvenile (2 months), adult (8 months), and senior (31 months) groups. The bilateral knee joints were harvested using sterile technique from each rabbits. In each age group, rabbit articular chondrocytes were cultivated in vitro after randomization into a control group and a CTS group, with 6 specimens in each. In the next 3 days, CTS was applied (sin10％, 0. 5 Hz, 6 h/d) in the CTS group for 6 hours per day while no CTS was applied in the control group. After the first CTS treatment, the supernatant of cell culture was collected every 12 hours from each specimen in both groups to assess the GAG levels by Alcian blue assay.Results Expressions of GAG showed significant increases in both control and CTS groups in each age group ( P ＜ 0. 05) . Different age groups showed significant differences in the GAG secretion over different time points. Compared with the younger cells, the older ones showed the most significant difference in growth between the control and CTS groups at 12 hours, though the older cells produced less GAG than the younger ones in both groups at the end of the experiment (72 hours).Conclusions CTS can stimulate rabbit chondrocytes of different ages to secrete more GAG, and younger cells tend to produce more GAG than the older ones.

Objective To explore the histological properties of isolated chondrons and chondrocytes from rabbit knee cartilage,and to determine if these properties vary with age.Methods Three groups of rabbit knees were evaluated according to different age:(1) young (2 months,n=10);(2) adult (8 months,n=10);and (3) old (31 months,n=10).The cartilage structure,proteoglycan,collagen-2,and collagen-6 content were determined by light microscopic using hematoxylin-eosin (HE),Toluidine Blue,and col-2,6 staining.The chondrons were enzymatically isolated using 0.3 g/L dispase and 0.2 g/L collagenase-2 by shaking for 3 hours.The morphology and composition of isolated chondrons were observed by HE and collagen-6 immunostaining staining after overnight coverslip monolayer culture under a microscopy.Results The chondrocytes became sparser and the total content of proteoglycans and collagen-2 were decreased in the articular cartilage with age.Compared to the chondrocytes,the surrounding rim or capsule was more obvious in the isolated chondrons,and they exhibited obvious differences in shape.The cells within one cluster from different age groups were similar to the morphology observed in cartilage in situ.The adult and old chondrons generally possessed a thicker pericellular matrix with more enclosed cells,and the chondrons contained more cells can reach 47％.Conclusion These findings further suggest that the properties of the chondrons and pericellular matrix have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with age.

Objective To explore the relationship among the expression level of MMP?13, AGG and Col?II in the chon?drocytes caused by nutritional deficiencies in rabbits. Methods 30 New Zealand white rabbits were randomly divided into autolo?gous chondrocyte transplantation group (control group, n=10), nutrition block group (surgery group, n=10), and peripheral nutrition block group (sham surgery group, n=10). 4 weeks after treatment, the rabbits were sacrificed for undergoing the observations on general and histological level;real?time PCR assay was employed for testing the expression level of MMP?13, AGG and Col?II;cel?lular apoptosis percentage was observed through TUNEL stain. The relationship among the apoptosis level, cartilage cells histologi?cal Mankin score as well as the expression level of MMP?13, AGG and Col?II were analyzed. Results Based on the Mankin score, there was a statistic difference between surgery group and control group. On the other side, there were no statistic differenc?es between sham surgery group and control group. 4 weeks after treatment, surgery group presented a higher apoptotic percentage compared with control group;this value between sham surgery group and control showed no significant differences. There was an increased mRNA expression level of MMP?13 and a decreased mRNA expression level of AGG and Col?II in surgery group com?pared with control group;no statistic differences of all these values was found between sham surgery group and control group. His?tological Mankin score and apoptotic percentage presented positive correlation (r=0.922, P<0.001), the regression equation:Y=-0.548+0.404X, R2=0.844 (F=157.735, P<0.001); the mRNA expression level of MMP?13 and apoptotic percentage presented positive correlation (r=0.942, P<0.001), the regression equation:Y=0.951+0.116X, R2=0.883 (F=219.054, P<0.001). There was a nega?tive correlation between the mRNA expression level of MMP?13 and the mRNA expression level of AGG as well as Col?II (r=-0.956,-0.945, P<0.001). Conclusion Damage of cartilage cells causes the up?regulation of the MMP?13 expression which could ex?acerbate the degeneration of cells. It could induce the down?regulation of AGG and Col?II mRNA expression, which will cause the extracellular matrix synthesis disorder.

Objective To characterize the biomechanical behavior and properties of the chondrons enzymatically isolated from rabbit knee articular cartilage in virto. Methods Eight months old New Zealand white rabbits were randomly divided into chondroctye and chondron groups (4 rabbits in each group). In chondrocyte groups, the full articular cartilages from both knees were enzymatically isolated to chondrocytes by 0.4% pronase and 0.025% collagenase type-Ⅱ in turn. In chondron groups, chondrons were obtained from articular cartilage using the mixture of 0.3% dispase (a neutral protease) and 0.2% collagenase type-Ⅱin at 37C for 3 h. The micropipette aspiration was used to quantify changes in biomechanical properties of chondrons and chondrocytes and the viscoelastic parameters, including K1, K2, E∞ (equilibrium modulus), E0(instantaneous modulus), and μ (apparent viscosity), were calculated coupled with standard linear half-space viscoelastic solid model. Results In response to a constant negative pressure of 0.2-0.4 kPa, the chondrocytes exhibited standard linear viscoelastic solid properties. Namely, the cells showed an initial elastic response followed by a viscoelastic creep response. then cells continued to enter into the micropipette with a monotonically decreasing rate of deformation, until reaching equilibrium within about (110±18) s. Comparing with chondrocytes, the chondrons exhibited significant viscoelasticity under a greater negative pressure of 1.0-1.2 kPa. But the instantaneous length deformed into the micropipette significantly reduced, and the equilibrium time reduced to (36.5±4.5) s. The equilibrium modulus (E∞), the instantaneous modulus (E0) and the apparent viscosity (μ) of chondrons were significantly higher than the those of chondrocytes. Conclusion Comparing with chondrocytes, the chondrons exhibited significant viscoelastic properties, and viscoelastic properties of chondrons have increased in vitro.

BACKGROUND:Such methods as transplanting autologous periosteum or autologous bone marrow mesenchymal stem cells (BMSCs) can promote the repair of articular cartilage defects for sure. But they all have their own limits in chondrogenic abilities,which results in an unsatisfactory curative effect.OBJECTIVE:To study the effect of transplanting BMSCs (which were induced into chondrocytes) combined with autogenous periosteum on repairing articular cartilage defects in rabbits.MATERIALS:A total of 18 New Zealand rabbits,aged 6-8 months,were divided with random digits table method into 3 groups,namely,periosteum+BMSCs group,periosteum group and blank control group,with 6 ones (12 knee joint samples) in each group. METHODS:In periosteum+BMSCs group,BMSCs were harvested and adherently cultured with trypsin digestion method. Then they were induced by transforming growth factor 81 into chondrocytes. At the same time,immunofluorescence labeling was performed to BMSCs membranes with PKH-26. Full-thickness articular cartilage defects (diameter:3mm,depth:3mm) were made to bilateral condylus medialis femoris of all rabbits. In periosteum+BMSCs group and periosteum group,defects were covered by homolateral autogenous proximal tibia periosteums,with germinal layer facing to cavitas medullaris. After that,the periosteum+BMSCs group received 3 sutures,followed by injection of 20 μL BMSCs suspension (1×109/L) into the defects,after which the last suture was taken. The periosteum group underwent coverage with periosteum on defects only. The blank control group underwent perforate only.MAIN OUTCOME MEASURES:General observation,histological observation,Wakitani's score,immunohistochemical and in situ hybridization detection of collagen type Ⅱ were performed to defects at 6 and 12 weeks following operation.RESULTS:No sutured periosteums were found desquamate. In periosteum+BMSCs group,defects were filled with hyaline cartilage-like repairing tissues at week 6 following operation;Week 12 following operation saw remodeled tissues whose cells were mainly the implanted cells labeled with PKH-26. In periosteum group,repairing tissues in defect areas were ivory white,smooth with light introcession and distinctively different from the surrounding normal cartilage tissue. In the blank control group,clearer introcession or irregular appearance,even broken surrounding cartilage tissues could be seen in the defect area. Both Wakitani's score and histological score were highest in periosteum+BMSCs group at week 6 and 12 following operation (P＜0.05),with higher ones in periosteum group than in the control group (P＜0.05). What'more,matrix around cells in the repairing tissues showed positive results to both immunohistochemical and in situ hybridization staining of collagen typeⅡ,which proved that cells in repairing tissues were the implanted ones.CONCLUSION:Transplanted BMSCs (which were induced into chondrocytes) combined with autogenous periosteum can form hyaline cartilage-like repairing tissues through which articular cartilage defects are repaired.

Objective:To investigate the short-term effects of articular injection of hyaluronic acid combined with glucocorticoid in patients with knee osteoarthritis.Methods:From October 2017 to June 2018, a total of 188 patients diagnosed with knee osteoarthritis received parallel articular injection. There were 60 cases with mild knee osteoarthritis, 72 with moderate and 56 with severe according to the WOMAC knee functional score. There patients were divided into group rank Ⅰ48 cases, Ⅱ 49 cases, Ⅲ 45 cases, Ⅳ 46 cases according to the knee joint X-ray Kellgren-Lawrence classification. The unified treatment regimen was 2.5 ml Sodium Hyaluronate (SHA) injection for the first time, SHA 2.5 ml and compound betamethasone injection (CBI) 1 ml for the second week, and 2.5 ml of SHA for the third week. WOMAC score and Lequesne index were used to evaluate joint function before the first injection and after SHA and SHA+CBI injection. The improvement rate of Lequesne index ≥30% or improvement rate of WOMAC score ≥25% was regarded as effective treatment.Results:Lequesne index and WOMAC score decreased gradually in the mild, moderate and severe groups after 3 weeks of injection. Among these patients, the improvement rates of Lequesne index after SHA injection and SHA+CBI injection were 36.44%±8.46% and 49.26%±13.75% in the mild group, 23.09%±12.61% and 30.66%±14.95% in the moderate group, and 10.50%±8.78% and 11.07%±6.52% in the severe group. The improvement rate of WOMAC score in the mild group after SHA injection and after SHA+CBI injection was greater than 25%. After SHA injection, the improvement rate of WOMAC score was 13.06%±10.21% in the moderate group, and 27.49%±13.61% after SHA+CBI injection. Those in severe group were all less than 25%. Kendall's staub correlation analysis results showed that there was a strong positive correlation between WOMAC function score and X-ray Kellgren-Lawrence classification ( r=0.744, P<0.001). The Lequesne index and WOMAC scores of the Kellgren-Lawrence X-ray classification decreased gradually after 3 weeks of injection. The improvement rate of Lequesne index period in group rank Ⅰ after SHA and SHA+CBI injection was 36.64%±10.05% and 52.00%±8.19%, respectively. That for group rank Ⅱ was 32.05%±8.09% and 41.95%±10.53%, group rank Ⅲ 16.93%±10.34% and 27.77%±10.25%, group rank Ⅳ 7.52%±5.53% and 7.60%±6.66%. The improvement rate of WOMAC score period in group rank Ⅰ after SHA and SHA+CBI injection was 29.48%±11.77% and 42.59%±13.55%, respectively. That for group rank Ⅱ was 26.72%±10.21% and 30.49%±16.90%, group rank Ⅲ 13.78%±5.96% and 23.05%±9.52%, group rank Ⅳ 4.77%±3.80% and 4.27%±4.23%. Conclusion:For mild or X-ray classification Ⅰ, Ⅱ knee osteoarthritis patients, articular injection SHA or SHA+CBI are effective. Further, SHA+CBI is better than single injection of SHA. SHA+CBI injection was effective for moderate knee osteoarthritis patients. For severe or X-ray classification Ⅲ, Ⅳ patients, SHA or SHA+CBI injection at interval are invalid.

Objective:To investigate the differences in gene expression levels in knee chondrocytes and chondrons in vitro.Methods:The chondrocytes and chondrons were isolated from full thickness of the 8-months ( n=5) rabbit knees cartilage. Chondrons from right knee were enzymatically isolated using 0.3% dispase and 0.2% collagenase-2 with shaking for 3 hours. Chondrocytes were isolated by 0.4% Pronase and 0.025% collagenase-2 from left knee. The mRNA levels in chondrocytes and chondrons were analyzed by quantitative real-time PCR, including matrix proteins [aggrecan(Agg), collagen(Col-2), Col-6A6, Col-10, Col-11], MMPs and inhibitors (MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, TIMP-3), cytoskeletal proteins (Sox-9, vinculin, tubulin, actin), cytokines (IL-β, TNF-α). Analysis was performed using SPSS 16.0 statistical software, and the two-group comparisons were considered as significant by t-test at P<0.05. Results:Compared to the chondrocytes, the Agg [(5.78±0.90) vs (1.89±0.27), t=9.26, P<0.001], Col-2 [(6.29±0.76) vs (3.06±0.60), t=7.46, P<0.001], Col-6A6 [(0.89±0.18) vs (0.22±0.06), t=7.90, P<0.001], Col-10 [(3.83±0.76) vs (1.00±0.26), t=7.88, P<0.001] and TIMP-1 [(1.98±0.85) vs (1.03±0.34), t=2.32, P=0.049], TIMP-2[(3.46±1.50) vs (1.52±1.06), t=2.36, P=0.046], TIMP-3 [(3.96±0.50) vs (1.36±0.18), t=10.94, P<0.001], Sox-9 [(7.09±2.93) vs (3.24±0.77), t=2.84, P=0.022], vinculin [(3.42±1.69) vs (1.46±0.68), t=2.41, P=0.043], tubulin[(9.34±0.71) vs (2.35±0.80), t=14.61, P<0.001] showed higher expression in the chondrons. Compared to the chondrocytes, the MMP-1 [(1.02±0.30) vs (2.67±0.45), t=6.91, P<0.001], MMP-3 [(1.21±0.32) vs (2.52±0.79), t=3.44, P=0.009], MMP-13 [(1.23±0.34) vs (3.42±0.86), t=5.30, P=0.007], IL-1β [(1.02±0.14) vs (2.70±0.49), t=7.37, P<0.001], TNF-α [(0.99±0.08) vs (3.15±0.54), t=8.85, P<0.001] showed lower expression in the chondrons. There were no difference between chondrons and chondrocytes for Col-11, MMP-9, actin ( P>0.05). Conclusion:The gene expression of extracellular matrix components are higher and the gene expression levels of inflammatory factors and MMPs are decreased in chondrons compared with the chondrocytes, suggesting the chondrons have more multiplication potential as seeding cells for tissue-engineered cartilage.

BACKGROUNDChondrocytes' 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.

METHODSChondrocytes 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 enzyme-linked immunosorbent serologic assay (ELISA) on days 3, 6, and 9.

RESULTSIn 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.

CONCLUSIONSThe 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.

Animals ; Cartilage, Articular ; cytology ; metabolism ; Chondrocytes ; cytology ; Collagen ; metabolism ; Cytoskeleton ; metabolism ; Knee Joint ; cytology ; metabolism ; Microscopy, Confocal ; Rabbits

BACKGROUNDOsteoporosis (OP) is a common bone disease, which adversely affects life quality. Effective treatments are necessary to combat both the loss and fracture of bone. Recent studies indicated that caffeic acid phenethyl ester (CAPE) is a natural chemical compound from honeybee propolis which is capable of attenuating osteoclastogenesis and bone resorption. Therefore, this study aimed to investigate the effect of CAPE on bone loss in OP mice using micro-computed tomography (CT) and histology.

METHODSEighteen mice were prepared and evenly divided into three groups. The six mice in the sham+PBS group did not undergo ovariectomy and were intraperitoneally injected with PBS during the curing period. Twelve mice were ovariectomized (OVX) to induce OP. Six of them in the OVX+CAPE group were intraperitoneally injected with 0.5 mg/kg CAPE twice per week for 4 weeks after ovariectomy. The other six OVX mice in OVX+PBS group were treated with PBS. All the mice were sacrificed 4 weeks after ovariectomy. The tibias were bilaterally excised for micro-CT scan and histological analysis. The Mann-Whitney U test was used to test the statistical differences among groups.

RESULTSBone loss occurred in OVX mice. Compared with the sham+PBS group, mice in the OVX+PBS group exhibited a significant decrease in bone mineral density (BMD, P < 0.05), bone volume fraction (BV/TV, P < 0.01), trabecular thickness (Tb.Th, P < 0.05), and trabecular number (Tb.N, P < 0.01), as well as a non-insignificant increase in the number of osteoclasts (N.Oc/B.Pm). With CAPE treatment, the microarchitecture of the tibial metaphyses was significantly improved with a reduction of osteoclast formation. Compared with the OVX+PBS group, BV/TV in the OVX+CAPE group was significantly increased by 33.9% (P < 0.05).

CONCLUSIONCAPE therapy results in the protection of bone loss induced by OVX.

Animals ; Bone Density ; drug effects ; Caffeic Acids ; pharmacology ; Female ; Metabolism ; drug effects ; Mice ; Mice, Inbred C57BL ; Ovariectomy ; Phenylethyl Alcohol ; analogs & derivatives ; pharmacology ; Propolis ; chemistry ; Tomography, X-Ray Computed