The Experimental Study On The Effect Of Low Intensity Pulsed Ultrasound On Degenerative Human Nucleus Pulposus Cells In Three-dimensional Culture

PART ⅠISOLATION, CULTURE AND IDENTIFICATION OFNUCLEUS PULPOSUS CELLS FROM DEGENERATIVEHUMAN INTERVERTEBRAL DISCObjective To get enough nucleus pulposus cells for further studyfrom degenerated disc tissue through the primary culture and subculture ofnucleus pulposus cells.Methods The harvested nucleus pulposus tissue samples fromdegenerative discs were minced into small fragments of approximately1mm3, digested in0.25%trypsin solution at37°C for20minutes and then in0.2%type II collagenase solution for2-4hours. Then the isolated cellswere collected and suspended in DMEM/F12medium with20%fetalbovine serum, penicillin (100U/ml) and streptomycin (100ug/ml) at37°C,5%CO2atmosphere for monolayer culture. Then, the identification ofhuman nucleus pulposus cells was performed by COL2α1antibody immunocytochemistry staining with and the cell surface maker of CD24detected by flow cytometry. Besides, the senescent nucleus pulposus cellwas detected by SA-β-gal staining.Results The primary culture and subculture of degenerative humannucleus pulposus cells derived from degenerative discs were successfullyperformed. The degenerative human NP cells were identificated accordingto its characterization under microscope, immunocytochemistry stainingusing COL2α1antibody and CD24-PE maker. The degree of cellsenescence in degenerative human nucleus pulposus cells is relatively high(mean percentage is23%).Conclusion To get enough cells for further study through the primaryculture and subculture of degenerative human nucleus pulposus cells fromdegenerative discs is possible and easy. PART ⅡTHE EXPERIMENTAL STUDY ON THE EFFECT OFLOW-INTENSITY PULSED ULTRASOUND ONDEGENERATIVE HUMAN NUCLEUS PULPOSUS CELLSIN THREE-DIMENSIONAL CULTUREObjective To investigate the effect of low-intensity pulsedultrasound(LIPUS) with a multiple range of intensity on degenerativehuman nucleus pulposus cells in three-dimensional culture.Methods The nucleus pulposus cells acquired from degenerativelumbar intervertebral discs were cultured in monolayer culture andidentified. After two passages, the cells were cultured in calcium alginatebeads and stimulated by LIPUS at a multiple range of intensity(0,15,30,60,120mW/cm~2) for1week (20min/d). The effect of LIPUSwere evaluated by the ratio of cell apoptosis, mRNA and protein expressionlevels of COL2α1and aggrecan, which were detected by flow cytometry,RT-PCR and western blot respectively.Results The ratio of cell apoptosis in groups of30,60and120mW/cm~2was significantly lower than the control group (P<0.05)，exceptfor group of15mW/cm~2(P>0.05). There was significant difference betweengroups of30,60and120mW/cm~2and control group both in mRNA andprotein expression level of COL2α1and aggrecan (P<0.05), but no difference between each other group (30,60,120mW/cm~2).Conclusion The LIPUS can inhibit the apoptosis of degenerativehuman nucleus pulposus cell cultured in calcium alginate beads andupregulate the synthesis of COL2α1and aggrecan. It may be a usefultreatment for delaying the progression of disc degeneration. PART ⅢTHE STUDY ON THE EFFECT AND MOLECULARMECHANISM OF LOW-INTENSITY PULSEDULTRASOUND ON DEGENERATIVE HUMAN NUCLEUSPULPOSUS CELLS IN THREE-DIMENSIONAL CULTUREObjective To investigate the effect of low-intensity pulsedultrasound(LIPUS) on degenerative human nucleus pulposus cells in three-dimensional culture and the underlying molecular mechanism.Methods The nucleus pulposus cells acquired from degenerativelumbar intervertebral discs were cultured in monolayer culture and identified.After two passages, the cells were cultured in calcium alginate beads. TheLIPUS group was stimulated by LIPUS for1week (20min/d); the control group was cultured in the same circumstance without LUPUS stimulation；LIPUS+LY294002group was stimulated by LIPUS for1week and treatedwith LY294002simultaneously. The ratio of cell apoptosis was detected byflow cytometry. The concentration of aggrecan, COL2α1, MMP-3andTIMP-1in culture supernatant was detected by ELISA.The mRNAexpression levels of aggrecan，COL2α1and Sox9were detected by RT-PCR.The protein expression levels of aggrecan, COL2α1, Sox9, FAK, p-FAK,Akt, p-Akt, Bcl-2and active caspase-3were detected by western blot.Results The concentration of aggrecan, COL2α1and TIMP-1inLIPUS group was obviously higher than control group (P <0.05), but theconcentration of MMP-3in LIPUS group was significantly lower thancontrol group (P <0.05). The mRNA expression levels of aggrecan，COL2α1and Sox9in LIPUS group were significantly higher than controlgroup(P<0.05). The protein expression levels of aggrecan, COL2α1, Sox9,Bcl-2, p-FAK and p-Akt in LIPUS group were significantly higher thancontrol group; and there was no significant difference in the protein level ofFAK and Akt compared with control group; but the protein expression levelof active caspase-3in LIPUS group was lower than control group. When thecells were stimulated by LIPUS and treated with LY294002simultaneously,the ratio of cell apoptosis in LIPUS+LY294002group was significanthigher than both in LIPUS group and control group(P <0.05), and themRNA and protein expression levels of aggrecan and COL2α1in LIPUS+LY294002group were significantly lower than both in LIPUSgroup and control group (P <0.05).Conclusion The LIPUS can inhibit the apoptosis of degenerativehuman nucleus pulposus cell and stimulate the extracellular matrix synthesisof degenerative human nucleus pulposus cells cultured in calcium alginatebeads via activating integrin-FAK-PI3K/Akt pathway.