| PrefaceIntervertebral disc degeneration associated with spinal degeneration disease, which include disc herniation, radiculopathy, myelopathy, spinal stenosis , instability, and low back pain, constitute the vast majority of the diagnoses treated by spine specialists. The pathogenesis of intervertebral disc degeneration is poorly understood, although it is known to be associated with a variety of cellular and biochemical changes. The most prominent of these changes include a decrease in cell density in the disc and a decrease in synthesis of cartilage specific gene products such as collagen type II and proteoglycans. As the disc degenerates, there is a change in the extracellular matrix (ECM) , and this is thought to lead to human disc disease. Recent advancements molecular biology have enabled scientists to obtain sufficient amounts of pure gene prouducts (i. e. , RNA and proteins) for experimental and clinical use. Biologic therapy to enhance the production of collagen type II and proteoglycan may be an effective method of retarding, or even reversing the disc degeneration.The discovery of growth factors and their importance in development and healing led many researchers to investigate their potential role as therapeutic a-gents in various diseases, including IVD and other muscloskeletal applications. In vitro and in vivo studies have demonstrated that exogenous application of growth factorscan up - regulate extracellular matrix synthesis by IVD cells without apparent cellular injury. Bone morphogenetic protein - 2 ( BMP - 2) is an osteoinductive cytokine that can stimulate cells anabolism in vitro and in vivo. Accordingly, the purposes of this experimental study were to demonstrate theeffect of hBMP - 2 andAd - hBMP - 2 on intertebral disc cells with difference dose in terms of expression of matrix components, aggrecan and collagen type II synthesis.Materials and methodsDisc tissue acquisition and cell cultureIntervertebral disc tissue was obtained from 18 patients (age ranged from 27 to 45 years) with lumbar disc herniation during operations. The nucleus pulp-ous carefully were obtained from the central aspect of the disc, and all tissues of the disc specimen except nucleus pulpous were discarded. The specimens were minced into fragments of approximately 1 mm x 1 mm x 1 mm in volume. The tissues were digested at 37CC with 0. 2% pronase and 0. 02% type H collagenase . And then the cells were plated in tissue culture flasks. Primary cultures were sustained for about 2-3 weeks in DMEM/ F12 medium containing 20% heat -inactivated FBS at 37°C in a humidified atmosphere with 5% CO2. The medium was changed twice a week. Detection of Collagen type IIHuman intervertebral disc cells were cultured in vitro and crept slice, the SP immunobistochemical method was used to examine the expression of collagen type II. Detection of Type H collagen and aggrecanAt confluency (60% ~ 70% fusion) , the degenerative cells were treated with hBMP -2(0ng/mll10ng/ml and lOOng/ml). A total of 4 ml cell supernatant was collected from each experimental group of lumbar disc cells cultured in monolayer of 25 cm2 culture flask at third and sixth days. Enzyme - linked im-munosorbent assay ( ELISA) were subsequently performed on each sample to quantify Type II collagen and aggrecan. Detection of Type II collagen and aggrecan mRNAAt about 60% -70% confluence, the degenerative cells were treated with hBMP - 2 (Ong/ml, lOng/rnl and lOOng/ml). The cells were collected from each experimental group of lumbar disc cells cultured in monolayer of 25 cm2culture flask at sixth day. A reverse transcription polymerase chain reaction (RT - PCR) technique was used to determine the mRNA level of Type II collagen and aggrecan. (3 - actin primer was used as an internal control. Ad -hBMP -2 transfect lumbar intervertebral disc cellsThe cultured cells were plated about 70 ~ 80% confluence in cell culture flaks with 1 x 10 cells and organized into four groups. After washing the mono-layer cells three times with EBS to remove any remaining culture medium, DMEM/ Fl 2 medium in 1 ml aliquots for Ad - hBMP -2 were added to get 0MOI, 50MOI, 100MOI and 150MOI. After 4 hours .of incubation at 37T:, 3 ml of culture medium was added to each group, the cells were continued to culture for detection. Identify the intervertebral disc cells after transfectionHuman intervertebral disc cells were cultured in vitro and crept slice, the SP immunobistbchemical method was used to examine the expression of collagen type II after transfection. When the cultured cells were plated about 70 -80% confluence, Ad ^ hBMP - 2 were added to get 150MOI. , then the cells were continued to culture, for 3 days. The cells were treated with cblchicines before confluence 12 hours'and identified by chromosome analysis. Adenoviral Transduction RateTo quantify the percent of successfully transduced intervertebral disc cells for a given MOI,. Human intervertebral disc cells were cultured in vitro and crept slice. When the cultured cells were plated about 70. ~ 80% confluence, Ad-LacZ were, added. to get 50MOI. , then the cells were continued to culture for3 days. The sections were finally incubated in X - gal substreat for 4 hours at 31X.. the sections were observed with light microscopy. Transgene ExpressionHuman intervertebral disc cells were cultured in vitro, the degenerative cells were treated with Ad -hBMP -2(0MOI, 50MOI, 100MOI and 150MOI) and Ad - Lac - Z. The expression of hBMP -2 in these cells after infection with different doses was determined by immunofluorescence and western blot. Detection of Type II collagen and aggrecan after transfectionAt about 60% ~70% confluence, the degenerative cells were treated withAd-hBMP-2(0MOI,50MOI, 100MOI and 150MOI). A total of 4 ml cell supernatant was collected from each experimental group of lumbar disc cells cultured in monolayer of 25 cm culture flask at third and sixth days. ELISA were subsequently performed on each sample to quantify Type II collagen and aggre-can. Detection of Type II collagen and aggrecan mRNA after transfectionAt about 60 -70% confluence, the degenerative cells were treated with hBMP-2(0MOI, 50MOI, 100MOI and 150MOI). The'cells were coUected from each experimental group of lumbar disc cells cultured in monolayer of 25 cm culture flask at sixth day. RT - PCR technique was used to determine the mRNA level of Type II collagen and aggrecan. (3 - actin primer was used as an internal control. Statistical analysisSPSS12. 0 software was used to analyze the data. All data were expressed as values of mean and standard deviation. One - way - ANOVA was used in each group. A P value of < 0.05 was considered statistically significant.ResultsIdentify lumbar intervertebral cellsThe lumbar intervertebral disc cells were polygonal or asteroidal with the round nucleus centrally and secretive granules can be observed in plasma. The secondary passages of human nucleus pulposus cells can express type H collagen by immunocytochemistry. The expression of aggrecan and collagen type II by hBMP - 2At third and sixth day, the OD values of the nucleus pulpous groups treated with hBMP - 2 from the dose of lOng/ml to lOOng/ml were higher than those in control groups (P < 0. 05 ) , the expression of aggrecan and collagen type II in these cells was gradually increased after treated with different dose hBMP - 2 in dose dependent manner. The expression of aggrecan and collagen type II mRNA by hBMP - 2At sixth day, the OD values of the nucleus pulpous groups treated withhBMP -2 from the dose of lOng/ml to lOOng/ml were higher than those in control groups, aggrecan and collagen type H mRNA expression were up - regulated with increasing hBMP - 2 dose in dose dependent manner. Identify lumbar intervertebral cells after transfectionAfter transfection, the disc cells were presented normal morpha The second generation human intervertebral disc cells could grow in bloom and hold their shape. Chromosome analysis showed that there were 23 pairs chromosome in a disc cell and had not appeared polyploid and heteroploid. Transgene ExpressionApproximately 100% of cells exposed to the Ad - LacZ virus successfully expressed transgene at the 50 MOI evaluated. The cells created |3 - galactosi-dase that reacted with X - gal and made substrate specific blue. The expression of hBMP - 2 in these cells was gradually increased after transfection with different dose. The expression of hBMP - 2 transfected with Ad - hBMP - 2 from the dose of 50 MOI to 150MOI were lighter than those in control groups by fluorescence. The result of Western Blot showed the expression of hBMP - 2 in these cells was gradually increased after transfection from 50 MOI to 150 MOI, but the expression of hBMP - 2 was negative in control and Ad - LacZ samples. Aggrecan and collagen type II synthesis after transfectionAt third and sixth days, the OD values of the nucleus pulpous groups transfected with Ad - hBMP -2 from the dose of 50 MOI to 150MOI were higher than those in control groups (P <0.01) , The aggrecan and Collagen type II values of experimental groups were increased gradually in dose dependent manner after being infected. Expression of aggrecan and collagen type II mRNAIn human invertebral disc cell culture, difference was observed between control and culture treated with Ad - hBMP - 2 from the dose of 50 MOI to 150MOI in the expression of aggrecan and collagen type II mRNA. Aggrecan and collagen type II expressions were up - regulated by Ad - hBMP - 2 after transfection with increasing viral concentrations.Conclusions1. hBMP-2 is efficient to accelerates aggrecan and collagen type E synthesis in dose dependent manner.2. hBMP - 2 is efficient to accelerates aggrecan and collagen type II mRNA synthesis in dose dependent manner.3. Ad - BMP - 2 can infect human intervertebral disc cells with high efficiency and not interfere with the disc cells structure and function.4. The expression of hBMP - 2 in these cells was gradually increased after transfection with different dose.5. Expression of hBMF' - 2 gene is efficient to accelerates aggrecan and collagen type H synthesis in dose dependent manner.6. Ad - hBMP - 2 after transfection up - regulate the expression of aggrecan and collagen type II mRNA with increasing viral concentrations. |
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