The Effect Of Dynamic Compressive Force On The Chondrogenic Differention Of Precartilaginous Stem Cells Induced By IGF-1and The Role Of Erk In The Mechanical Siganal Transduction

Part1The effect of dynamic compressive force on the expression of Sox9and extracellular matrix of rat precartilaginous stem cellsObjectiveTo investigate the impact of intermittent dynamic compressive force on the expression of Sox9and extracellular matrix in rat precartilaginous stem cells(PSCs).Methods1. The primary PSCs were harvested from La Croix ring of growth plate in the rat and cultured in the medium.2. The PSCs were isolated and purified using magnetic activated cell sorting3. The PSCs were randomly divided into4groups:control group, the pressure group of one day, the pressure group of3days and the pressure group of7days; the pressure groups were intermittently subjected to the compressive pressure of90mmHg for1d,3d, and7d.4. The mRNA expression levels of Sox9, type Ⅱ collagen and aggrecan were measured by real-time quantitative polymerase chain reaction (qPCR)5. The protein expression levels of Sox9and type Ⅱ collagen were measured by western-blot.Results1. After being isolated and purified, the PSCs attached the culture flask stably and exhibited polygon or triangular shape under light microscope, with good growth condition and luminosity.2. Under dynamic compressive pressure of90mmHg, the mRNA expression levels of Sox9, type Ⅱ collagen and aggrecan increased significantly compared with the control groups at all endpoints (P<0.05);3. The expression levels of Sox9and type Ⅱ collagen protein at1d didn’t change obviously(P>0.05), but increased significantly at3d and7d (P<0.05).4. The expression levels of aggrecan mRNA, Sox9and type Ⅱ collagen mRNA and protein increased with the time of stimulation.ConclusionThe intermittent dynamic compressive force promotes the expression levels of Sox9, type Ⅱ collagen and aggrecan mRNA and protein in rat PSCs in vitro, indicating which could facilitate induction of PSCs into chondrogenic differentiation. Part2The effect of dynamic compressive force on the expression of Sox9and extracellular matrix induced by IGF-1in PSCs ObjectiveTo investigate the effect of dynamic compressive force on the expression of Sox9and extracellular matrix induced by IGF-1in PSCs.Methods1. The PSCs were isolated, purified and cultured in the medium containing10%fetal bovine serum as mentioned above.2. The PSCs were randomly divided into4groups:control group, the pressure group, the IGF-1group and the pressure and IGF-1group; the cells in the pressure group were intermittently subjected to the compressive pressure of90mmHg for14d; the concentration of IGF-1in the medium is100ng/ml.3. The morphologies of PSCs are observed in the light microscope and the effect was confirmed by Alcian blue staining and immunohistochemistry staining.4. The mRNA expression levels of Sox9, type II collagen and aggrecan were measured by real-time quantitative polymerase chain reactionResults1. After being induced by mechanical stress and/or IGF-1, the PSCs exhibited polygon shape under light microscope, with good luminosity.2. The alcian blue and immunohistochemistry staining showed positive results in the IGF-1group, the pressure group and the pressure with IGF-1group.3. Compared with the control group, the mRNA expression levels of Sox9, type II collagen and aggrecan increased significantly in three other groups (P<0.05); However, the mRNA expression levels of Sox9, type Ⅱ collagen and aggrecan decreased significantly in the pressure group compared with IGF-1group (P<0.05).ConclusionThe dynamic compressive force decreases the expression levels of Sox9, type II collagen and aggrecan mRNA induced by IGF-1in rat PSCs in vitro, indicating which could inhibit induction of PSCs into chondrogenic differentiation Part3The role of extracellular signal regulated kinase in the mechanical signal transduction of PSCsObjectiveTo investigate the role of extracellular signal regulated kinase in the mechanical signal transduction of PSCs.Methods1. The PSCs were isolated, purified and cultured in the medium containing10%fetal bovine serum as mentioned above.2. The PSCs were divided into2groups randomly:control group and the pressure group, the pressure group were subjected to the compressive pressure of90mmHg; each contains two subgroups:control group and the PD98059group, the concentration of PD98059in the medium is20μmol/L.3. The mRNA expression levels of Sox9and type Ⅱ collagen were measured by real-time quantitative polymerase chain reaction;4. The protein expression levels of Sox9and type Ⅱ collagen were measured by western-blot.Results1. Under the compressive pressure of90mmHg, the protein expression levels of pERK increased significantly compared with the control group (P<0.05).2. Compared with the control groups, the mRNA and protein expression levels of Sox9and type Ⅱ collagen increased in pressure group (P<0.05); the expression levels of Sox9and type Ⅱ collagen decreased significantly in both groups after (P<0.05).Conclusion ERK inhibitor can reduce the expression levels of Sox9and type II collagen induced by the dynamic compressive stress, indicating that the ERK signaling pathway may be involved in the process of cell response to mechanical signal transduction.