| ObjectiveOsteochondral allografts transplantation(OCT)is an effective method for the treatment of cartilage defects.Improvement of the preservation of cartilage graft in vitro directly affects the results of surgical treatment.The mechanical stimulation plays an important role in the maintenance of cartilage function.Under physiological conditions,the articular cartilage is stimulated by periodic mechanical loads.We used the rolling-sliding mechanical loading device which simulated motion of knee joint to exert mechanical stimulation on cartilage preserved in vitro,to explore the cartilage changes and its machanisim.Methods1.Periodic and aperiodic mechanical stimulation on chondrocyte viabilityThe knee joint tissues of healthy pig was used as the experimental specimen.The osteochondral disks were isolated from the knee joints of pig using a specialized Arthrex osteochondral harvester,the diameter of these explants was 8mm,the length was 10 mm with subchondral bone.We used the loading device that we devised to exert rolling-sliding mechanical stimulation on cartilage to find the result by TSMU protocols.We set the periodic group and the aperiodic group,10 specimen in each group.Cartilage in the periodic group was subjected to mechanical stimulation every three days then the culture medium was refreshed.Cartilage in the aperiodic group was subjected to single mechanical stimulation and the culture medium was refreshed every three days.The survival rate of chondrocytes was detected by FDA/EB fluorescence double staining on day 3,7,14 and 21.2.The effects of mechanical stimulation on the preservation of cartilage tissue in vitroThe cartilage was divided into the loading group and the static group,each group contained 30 specimen.Cartilage in the loading group was subjected to periodic mechanical stimulation every three days by the rolling-sliding mechanical loading device,then the DMEM culture medium was refreshed and the cartilage was preserved in vitro.In the static group,cartilage was merely preserved in culture medium during the whole process,and the culture medium was also changed every three days.The survival rate of chondrocytes was detected on day 7,14,21,28.On day 14 and 28,the morphology of cartilage was tested by HE staining,the content of proteoglycan was detected using the safranin O-fast green and toluidine blue staining method,type II collagen content was detected by immunohistochemical staining,the Young's modulus was measured by mechanical tester,to assess the viability and biomechanical property of cartilage.We also did Western Blot tests to evaluate the expression of signal pathway related protein p-MEK and p-ERK1/2,and apoptosis related protein caspase-3 and bax,to explore the related mechanisms.Result1.Periodic and aperiodic mechanical stimulation on chondrocyte viabilityChondrocyte viability decreased with time.The chondrocyte survival rate of the two groups was high on day 3,93.28±0.69% and 92.31±0.68% respectively and the difference was not statistically significant(p>0.05).The survival rate of chondrocytes in the periodic group were 86.09±0.57% and 76.38±0.95% respectively,significantly higher than those which were 83.30±0.70% and 68.53±0.78% respectively in the aperiodic group(p<0.05)on day 7 and 14.Chondrocyte survival rate in the two groups were 62.51±0.70% and 61.47±0.53% respectively on day 21,there were no significant differences in the two groups(p>0.05).2.The effects of mechanical stimulation on the preservation of cartilage tissue in vitro(1)Chondrocyte survival rate: On day 7 and 14,the survival rate of the chondrocytes in the loading group were 87.7±0.8% and 78.5±1.3% respectly,significantly higher than those which were 82.6±0.5% and 66.4±1.4% in the static group(p<0.05).On day 21,the survival rates of the two groups were 62.2±0.8% and 60.8±0.7% respectively,and on day 28 the survival rates of the two groups were 56.7±1.7% and 55.0±1.5% respectively,and the differences were not statistically significant.(2)Histology:1)Morphology: On day 14,the load group has more excellent histology than the static group,with clear level and rich matrix;On day 28,the morphological features of the two groups were poor,the matrix was pale,and the cartilage and the subch-ondral bone were loosely connected.2)Detection of content of proteoglycan: Safranin O-fast green,toluidine blue staining and Integral Optical Density(IOD)value detection showed that on day 14 the IOD value were 223.2±5.9% and 174.8±3.2% respectly in the loading group,signifi-cantly higher than those which were 166.5±3.3% and 68.53±0.78% respectively in the static group(p<0.05).There was a decrease in the content of proteoglycan in the two groups on day 28,the IOD value were 132.4±3.1% and 94.3±1.4% respectly in the loading group,126.9±2.7% and 89.9±2.1% respectly in the static group,and the differences were not statistically significant(p>0.05).3)Detection of content of type II collagen: Immunohistochemical staining and IOD value detection showed that IOD value in the loading group was 128.4±2.3%,higher than 114.3±2.4% in the static group on day 14,the difference was statistically significant(p<0.05);There was a decrease in the content of proteoglycan in the two groups on day 28,IOD values were 76.2±2.2% and 81.8±1.8% respectly,the difference was not statistically significant(p>0.05).4)Biomechanical detection: When the compression rate was 15%,the Young's modulus in the loading group was 8.58±0.23 MPa,higher than 6.87±0.12 MPa in the static group on day 14,the difference was statistically significant(p<0.05);The Young's modulus of the two groups decreased on day 28,6.34±0.10 MPa in the loading group and 6.12±0.08 MPa in the static group,there was no significant difference between the two groups(p>0.05).5)Protein expression by Western Blot analysis: After 2 hours of mechanical loading,the expression of MEK/ERK pathway related protein p-MEK and p-ERK1/2 protein increased,and the expression of apoptosis related protein caspase-3 and Bax decreased.The difference was significant(p<0.05).ConclusionsPeriodic mechanical stimulation is superior to aperiodic one in cartilage preservation.Periodic rolling-sliding mechanical stimulation can increase the survival rate of chondrocytes,maintain the content of proteoglycan and type II collagen,enhance the mechanical properties and improve the preservation effect in vitro within 14 days compared with static storage.The mechanism involved may be that mechanical stimulation activates the MEK/ERK signaling pathway to inhibit the expression of apoptotic protein and enhance the chondrocyte viability.The loading preservation scheme provides a new idea for cartilage tissue bank. |
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