Research on the Biological Characteristics of Human Chondrocytes.cultured Influence Effect by TypeⅠor TypeⅡCollagen
|North Sichuan Medical College
|typeⅠcollagen culture plate typeⅡcollagen culture plate human articular chondrocytes culture in vitro
Objective: To investigate the effect of type I and type II collagen on theinfluence of the biological characteristics of human chondrocytes.Throughobserve the Biological characteristics of human chondrocytes seed in threekinds of Culture plates in vitro.Methods: Cartilage tissue samples from the total hip arthroplasty ofpatients with femoral neck fracture in the Affiliated Hosptial of NorthSichuan Medical orthopedics .Chondrocytes has harvested from articularcartilage was monolayer culture in culture plaste in the lab. After 3 months ofcartilage cell culture passage to the 7th generation, recorded as P0-P7,observation of cell morphology,selected the third-general ofchondrocytes ,respectively ,to send in the ordinary culture plates(ordinaryplate),type I collagen-coated culture plates(type I plate),type IIcollagen-coated culture plates (type II plate) continuing culture for 10days.Through evaluation of cell activity、doubling time and observation thecell morphology under light microscope to analyze the results of cell culture.By ELISA technology ,PCR quantitative detection technology,toluidine bluestaining,GAG quantitative detection of cartilage cells to understanddifferentiation and proliferation capacity of chondrocytes in three kindsculture plates .Results:（1）Collection 20 cases of articular cartilage specimens which match the criteria,select the normal morphology, strong activity cattilage cellsof 10 cases specimens for research.（2）Cell morphology was observed undera microscope,the cartilage cell jues to digest were round,adherent cells werepolygonal.With cell passaging,the cells gradually elongate,fusiform,similar tofibroblasts,the former three generations of cartilage cells with significantproliferative activity and cell morphology. from the 4th generation graduallyinto a long spindle, the 7th generation cartilage cells chang into fibroblastscells.(3) the 3rd generation cartilage cells seed in the ordinary cultureplate(ordinary plate)、type I collagen-coated culture plates(type I plate)、typeII collagen-coated culture plate (type II plate) continuing culture for 10days .we found the most number of cartilage cell in type II plate,for type Iplate 2 times,for ordinary plates 5 times,the result suggest that theproliferation is fast.Detection the cartilage cells secrete collagen type I,II theamount of protein in three different culture plates,found that type II boardcartilage cells secrete the least amount of type I collagen, and with theordinary board test result has statistically significant difference (P < 0.01), butwith type I board test results has no statistically significantdifference(P >0.01).Cartilage cells in type II plate secrete the most amount oftype II collagen and with the other plate test result has statistically significantdifference(P >0.01). Toluidine blue staining of cartilage cells and DMMBcolorimetric quantitative detection of the amount of cartilage cells to secreteGAG found that cartilage cells on the type II plates secrete the mostGAG ,and with the other plates test results has statistically significantdifference (P < 0.01).The above results suggest cartilage cells on the type IIplate has strong proliferation.Conclusion: 1.The study found that use DMEM dissolved collagenase ,use typeⅡcollagenase digestion step method and times to collect the cartilage cells ,canreduce the toxicity of digestive enzymes on chondrocytes to maintain cellviability.2.Collagen and cartilage cell interaction,collagen-coated cultute platesused in the primary chondrocytes in vitro can makes cell adherent as soon aspossible , reduce the amout of suspension cell death.3.Cultured cartilage cells cultured in collagen plate is better thanordinary culture plate,typeⅡcollagen in culture caritilage cells cultured inpackages are more able to maintain cell shape,extend the differentiationpattern of time ,have the ability to cause cells to differentiate.