The Experimental Study Of Thrombin-platelet-rich Plasma Affecting The Growth Of Human Chondrocytes Cultured In Vitro

Objective: Human chondrocytes were cultured in vitro and thendifferent concentrations of thrombin-platelet-rich plasma was put into them.The number of chondrocyte proliferation and chondrocyte total RNAsynthesis and the synthesis of type II collagen in the amount of change weredetected. According to these,we explore the affect of thrombin-platelet-richplasma to promote the growth of human chondrocytes.Methods:1. The human chondrocytes were cultured and passaged to the3rdgeneration.2. Preparation of platelet-rich plasma.3. Preparation of thrombin complexes-platelet-rich plasma. Each20ulplatelet-rich plasma contain thrombin2.2ul.Thrombin concentrations were10U/ml、20U/ml、30U/ml、40U/ml、50Uu/ml、60U/ml、70U/ml、80U/ml、90U/ml、100U/ml、200U/ml、500U/ml and1000U/ml.4. The3rdgeneration human chondrocytes were inoculated in96-wellculture plates. Each well contain1×104human chondrocytes. Grouping:Negative control group(DMEM culture medium containing only), Positivecontrol group(DMEM culture medium and PRP), Experimentalgroups(DMEM culture medium and PRP and different concentrations ofthrombin),10U/ml、20U/ml、30U/ml、40U/ml、50U/ml、60U/ml、70U/ml、80U/ml、90U/ml、100U/ml、200U/ml、500U/ml、1000U/ml. After culturedfor1day, use CCK-8(cell proliferation) to assay OD value. The optimum concentration of thrombin worth based on OD. The optimum concentration ofthrombin compare with common thrombin concentration1000U/ml bycartilage cells secreting the type II collagen gene expression which by PCRamplification, the PCR reaction product analysis. The results using the SPSSstatistical software analysis.Results:1. Human chondocyte culture: primary cells generally24-72hoursadherent,8-10days to form a monolayer cell, subculture to the3rdgeneration,cell growth is strong.2. OD value.(1)Negative control group(mean±standard deviation) andpositive control group(mean±standard deviation) was not statisticallysignificant（P＞0.05）.(2)Experimental group(mean±standard deviation) andnegative control group(mean±standard deviation) was statistically significant（P <0.05）.(3)Experimental group(mean±standard deviation) and positivecontrol group(mean±standard deviation) was statistically significant （P<0.05）.(4)There were significant differences(P <0.05)in the experimentalgroup,10U/ml、20U/ml、80U/ml-1000U/ml thrombin and60U/ml thrombin.The proliferation effect60U/ml above10U/ml,20U/ml and80U/ml-1000U/ml.30U/ml-50U/ml,70U/ml and60U/ml was not statisticallysignificant （P＞0.05）. From10U/ml-60U/ml to promote chondrocyteproliferation increased progressively and concentration dependent. Promotechondrocyte proliferation weakened from70U/ml group,80U/ml-1000U/mlwas not statistically significant（P＞0.05）.3. Chondrocyte synthesis and secretion of type II collagen results. Basedon grayscale value of the PCR, the chondrocytes of thrombin60U/mlsecretion of type II collagen gene expression is higher than the thrombin 1000U/ml（P<0.05）.Conclusion: Our results show that: in cultured human chondrocytes,thrombin platelet rich plasma composites can promote chondrocyteproliferation, but weakened in the experiment with the thrombin concentrationincreased its role in promoting chondrocyte proliferation and lowconcentrations thrombin to promote chondrocyte proliferation better.60U/mlwas optimal concentration.