The Effect Of Mechanical Forces On Cartilage: An In Vivo And In Vitro Study

PARTâ… : Effect of Mechanical Strain on Cultured Rabbit ArticularChondrocyteExperiment 1: Effects of passage on the glycosaminoglycan synthesis of the culturedrabbit articular chondrocytesObjective: To study the effect of passage on the GAG synthesis of cultured rabbit articularchondrocytes.Methods: Chondrocytes were isolated from the knee joints of five one-month rabbit knees.The morphology of the cells was detected under inverted microscope. The GAGconcentrations of supematants were measured by precipitation with alcian blue at 24, 36,48, 60h after the confluent of the Passage 0,1,2 cells.Results: The GAG concentrations of the supernatants increased with time (P＜0.001). Itdecreased with the increase of passage (P＜0.001). And post hoc tests showed the significantdifference between any two passages (P＜0.05).Conclusion: The GAG synthesis of in vitro cultured chondrocytes decrease with thepassages.Experiment 2: Effect of high-density cell culture on the synthesis ofglycosaminoglycan in rabbit articular chondrocytesObjective: To observe the performance of chondrocytes synthetizing GAG at differentinoculum densities.Methods: Five New Zealand rabbits of one month old were used in this study. Articularchondrocytes were isolated from both knees and digested using 0.4ï¼…pronase enzyme and0.025ï¼…â…¡type collagenase. The chondrocytes harvested from the same rabbit were dividedinto two sets, one was seeded at a constant density of 2×10⁴/cm₂ in primary and subculture, the other was cultured at a reduced density of 2×10³/cm² following cellular adhesion.Cellular morphology and proliferation were observed under inverted microscope. Theculture media were renewed after the primary cells and passage 1 cells were confluent.GAG concentration was determined using the modified precipitation method with Alcianblue at 12, 24, 36, 48 and 60 hours following the renewal of culture media.Results: Articular chondrocytes in the primary high-density culture group were polygonalwith clear boundaries, they have shown to form colony at 3-4 days. Cells around colonieswere more slender than those in the center of colonies, shaping as long polygon. There wasno obvious change observed in the morphology of passage 1 cells. In the low-densityculture group, cells scattered at early stage and formed colonies at 7 days, cellularmorphology showed no significant differences in comparison with high-density culturegroup. The time of primary cells becoming confluent in the low-density culture group wasprolonged compared with high-density culture group. The GAG concentration insupernatants in the primary cells of low-density culture group was significantly lower thanthat in primary cells and passage 1 cells of high-density culture group (P＜0.001, P＜0.05).The GAG concentration showed a greater difference along with the prolonging of culturetime.Conclusion: High-density culture is better then low-density culture to enhance theperformance of chondrocytes synthetizing GAG and to retard the velocity of chondrocytesdedifferentiation, which suggests high-density culture contributes to maintain thechondrocytes phenotype and can be considered as a good way of plate culture.Experiment 3: Effects of cyclic mechanical strain with varying amplication on theglycosaminoglycan synthesis of the primary cultured rabbit articular chondrocytesObjective: To study the effect of cyclic tensile strain (CTS) with varyingamplication on the GAG synthesis of primary cultured rabbit articular chondrocytes. Methods: Cyclic tensile strain (sinusoidal wave, 0.3Hz, 6h/d)of different amplication (0ï¼…,5ï¼…, 15ï¼…) was applied to monolayer cultured primary rabbit articular chondrocytes using aFlexercell 4000 strain unit. The morphology of the cells was detected under invertedmicroscope. The GAG concentrations of supernatants were measured by precipitation withalcian blue at 24, 36, 48, 60h after the beginning of the first CTS loading.Results: The cells in the middle of dishes exhibited morphologic change from a polygonalto spindle-like shape and lined perpendicularly to the radius of the dishes. The GAGconcentrations of the supernatants increase with the CTS amplication (P＜0.05).Conclusion: CTS enhance the GAG synthesis of cultured primary rabbit articularchondrocyte with an amplication-dependent manner.Partâ…¡: The Histological Compare of Articular Cartilage of the Loadingand Unloading Area in Rat Knee Joints.Objective: To observe and analysis the morphology and matrix content in articularcartilage of the loading and unloading area in rat knee joints. Methods: Wistar Knees of 5wistar rats were studied by whole-mount section technique. The sections were stained withhaematoxylin, eosin, alcian blue and safranin O to elucidate the morphological differenceof loading and unloading area. The cartilage thickness and stain difference were compared.Results: There is great different in the loading and unloading area in the chondrocyte arrayand cartilage thickness. The cartilage of the unloading area has less chondroitin sulfate(CS)and keratan sulfate(KS) but more hyaluronic acid (HA).Conclusion: The unloading area of the articular cartilage is different from loading area histologically. It elucidated that the graft of unloading area has no structure suitable to themechanic environment of the loading area in mosaicplasty.Partâ…¢: The Effect of Sports on Articular Cartilage of RatExperiemnt 1: The Design and Application of Rat TreadmillObject: To develop a rat treadmill.Method: The treadmill machine has been designed to compulsively make animals exerciseby manual controlled electrical shock. The shock is delivered to the animals by theelectrode at the end of the machine. The fore part of the treadmill was dark and the middleof the treadmill was strongly lighted. The rats would see the strong light before beingshocked. After 45 days intensive running exercise (1km/d,20m/min), rat knee cartilagestudied by whole-mount section and safranin O stain technique and compared with control.Result: The rats soon learned to run back to the dark part of the treadmill after severaltimes of shock stimulation. And in 2-3 days, they can learn to run in the treadmill withoutshock stimulation. The safranin O staining of uncalcified cartilage was reduced in therunning rats group (P＜0.05).Conclusion: These designs make full use of the instinct to dark place and learning abilityof the rats, so it greatly reduced the shock. The it provide a good tools to study the effect ofsports on muscle-skeletal system. Experiment 2: Articular Cartilage Glycosaminoglycan Distribution in the rat KneeJoint After Strenuous Running Exercise.Object: To study the influences of the strenuous running training program on the knee jointarticular cartilage.Method: At the age of 16 weeks, 5 male wistar rats started running on a horizontaltreadmill. Thereafter, the rats were trained for 45 days continuously. The rats ran 1km/d atthe speed of 1.2km/h. 5 matched male rats served as controls. Knees of the rats werestudied by whole-mounted section, HE stain, safranin O stain.Result: The cartilage surfaces were intact after the running exercise. Theglycosaminoglycan concentration was decreased an average of 66ï¼…in the superficialzone, 56ï¼…on the middle zone, but no difference was found in the deep zone. And theglycosaminoglycan concentration was increased 38ï¼…in the calcified cartilage.Conclusion: Strenuous running induced marked decrease of proteoglycan in theuncalcified cartilage, but induced no osteoarthritis in this rat strenuous running model.Experiment 3: The Effect of Strenuous Uphill Running on the Articular Cartilage ofthe KneeObject: To study the influences of the strenuous uphill running training program on thearticular cartilage of the knee.Method: At the age of 16 weeks, 5 male wistar rats started running on a nearly horizontaltreadmill. Thereafter, the rats were trained for 45 days continuously. The rats ran 1km/d atthe speed of 1.2km/h in the first 15 days and 20°uphill in the last 30 days. 5 matched malerats served as controls. The knees of the rats were studied by whole-mounted section, HEstain, safranin O stain, immunochemistry staining for colleganâ…¡and OARSI score.Result: The cartilage surfaces strained with india ink became irregular and OARSI scoresignificantly wordened after 45 days training. Conclusion: Strenuous uphill running induced osteoarthritis in the knee joints of rats. Theincreased loading of the cartilage may be the reason.PARTâ…£: Correlation between radiographically diagnosed osteophytes and varusangle in the knees of osteoarthritis patients.Objective: To assess the correlation between the presence of radiographically diagnosedosteophytes and varus angle in the knees of osteoarthritis patients.Methods: A total of 18 knees in 13 patients with knee OA was studied. The length ofmarginal osteophytes that appeared in radiogram was measured. A special scale methodcalled the "spur index" was devised for the study to determine a corrected measurement ofthe osteophyte length, varus angle were measured with Limb X Film Measuring Plate(LXMP). The correlation between varus angle and osteophytes formation was evaluated byPearson correlation.Results: No correlation between the osteophytes of lateral tibiofemoral compartments andvarus angle was found. In medial tibiofemoral compartment the osteophytes' lengthcorrelates positively with varus angle (P＜0.05).Conclusion: This study demonstrate a strong positive association between the varus angleand osteophytes' size in medial tibiofemoral compartment. The secretion of cytokines afterabnormal mechanical stimulation may be the reason of this finding. More studies about themechanobiology of chondrocyte will be needed to confirm this finding and test ourhypothesis.