PDGF-BB Expression Is Related To Fibrosis Of Hypertrophied Ligamentum Flavum Of Lumbar Vertebra

BackgroudLumbar spinal canal stenosis (LSS) occurs as a result of degenerative changes in the posterior structures of the Lumbar spine, including hypertrophy of the ligamentum flavum (LF) as well as the bony proliferation of the facet joints. LSS is a well-recognized disease that causes severe disability by compressing the cauda eguina and nerve roots which need to be resolved by operation. The patients not only suffer a lot from disease but also cost lost of money. AS to the pathological mechanism of the LF hypertrophy which is main caused LSS is not clear, the aim of research is to prevent LF develop into hypertrophy and reduce risk of disease of LSS.In the ligament from junior patients, elastic fibers consisted of 75% in extracellular matrix. However, the hypertrophied LF and showed loss of elastic fibers, increased collagen fibers and ossificated LF showed calcification, ossification, chondrometaplasia which is related to collagenⅡ. An increase in collagen content was the main cause of LF hypertrophy. The LF sample was divided into 3 sections and evaluated respectively:dural, middle, and dorsal layer. LF consists of a superficial and a deep component. Superficial part is called dorsal layer and deep part near to spinal dura mater is called dural layer. The part locates between superficial and deep component is called middle layer. It is continuous in the midline.Causes of LF hypertrophy are multi-factorial including spinal levels, age, mechanical stress and growth factors. Thickness of LF increased with age and LF degenerated with aging. LF showed loss of elastic fibers, increased collagen fibers, caused LF hypertrophy. The thickness of LF hypertrophy increased with lumbar spinal levels but decreased in L5S1 spinal levels. Normal LF undergo the traction during spine have flexion and extension. With age increased the spine grew into instable and caused LF scar. Some grouth factors were related to fibrosis such as transforming growth factor (TGF-β) is related to early stage of hypertrophied LF. AS to the pathological mechanism of the LF hypertrophy, TGF-0 which increasing collagen synthesis in a variety of soft tissues caused LF hypertrophy especially during the early phase and inflammatory cytokines which also make some contribution to hypertrophy. Higher expression of TIMP-2 was found in LSS compared to lumbar disc herniation group (LDH) and MMP-3 was also found in hypertrophied LF. Bone morphogenetic protein-2 (BMP-2) is key to ossification of LF in LSS. However, these factors can not fully explain the occurrence of hypertrophy.LF hypertrophy is a process of injury-repair-reinjury-rerepair, endding in scar formation, ossification, et al. LF is divided into three layers such as dural layer(yellow deep), dorsal layer (white superficial) and middle layer according to anatomical Plane. The dorsal layer is subjected to maximum load than another two layers and is susceptibility to scar. LF hypertrophy is due to accumulation of inflammation-related scar tissue. Upon injury enzyme COX-2 that belongs to cyclooxygenase is found in hypertrophied LF. Patients who take NSAIDS medicine-or COX-2 inhibitor can relieve the symptom from lumbar spinal canal stenosis caused by hypertrophy of the LF in clinical trial. The results suggested that inflammatory response is one reasons of brought out scar formation which is deposed of collagen.Platelet-derived growth factor (PDGF) produced by platelets, macrophages, vascular endothelium, fibroblasts, and keratinocytes. PDGF is a potent mitogenic factor and is thus believed to be capable of enhancing wound Healing. Framework of collagen and elastin fibers replaced by the granulation tissue, which followed by tissue remodeling involving the synthesis of new collagen mediated by PDGF, and the breakdown of old collagen by PDGF. The final product of this process is scar tissue. PDGF comprises a family of homo or heterodimeric growth factors including PDGF-BB. PDGF-BB is a isoform of PDGF, that have function through PDGF-βacceptor. Experimental evidence for a role of PDGF-BB in lung fibrosis was obtained by the demonstration that intratracheal injection of PDGF-BB causes transient proliferation of pulmonary mesenchymal and epithelial cells accompanied by collagen deposition. PDGF is thus tight related to the tissue scar and collagen construction which form fibrosis.The purpose of this study was to prove this hypothesis. Normal LF undergo the over flexion and extension caused the LF scar, which produced by platelets and released PDGF to repaired scar in the LF. Scar formed caused elastic fibers decreased and collagen increased which develop into fibrosis that is tightly related to hypertrophied LF lead to Lumbar spinal canal stenosis.Objectives1.To study correlation among LF thickness,fibrosis and the expression of PDGF-BB.2.To explore the method of the isolation and culture of HLF cells in vitro, and establish this cell lines.3.To investigate the biological effect of PDGF-BB in LF cells in vitro.Methods1.To identify PDGF-BB expression in hypertrophied ligamentum flavum of patients with lumbar spinal canal stenosis and relate it to fibrosis.(1) Specimens collection.The LF were collected during the patient's surgery after written informed consent was obtained. This protocol was approved by the institutional ethic boards of hospital. The mean age of the LDH (and standard deviation) was 50.50±5.0years (range,45.0 to 59.0 years) and 56.00±7.50years (range,40.0 to 65.0 years) in those with LSS. there was no significant difference (p=0.069). Twenty patients who were undergoing decompression because of spinal canal stenosis due to LSS and nerve root compression caused LDH that had been unresponsive to conservative measures for at least three months. None of these patients received epidural or selective nerve-root blocks. The patients who had isthmic and degenerative spondylolisthesis, scoliosis, or fractures were excluded from this study. We followed the method proposed by Fukuyama et al and CT or MRI was performed for further examination to measure the thickness of LF samples from 20 patients. On the T1-weighted axial images through the facet joint, the LF was clearly seen as a low-signal intensity mass just ventral side of the facet joint. Thickness at the middle portion of LF was measured. The LF thickness at L3/4,L4/5, and L5/S1 was measured.(2). To evaluated the degree of fibrosis using Masson trichrome stains.Observation under LPF low power field (×10).The degree of fibrosis were evaluated and graded as following. Grade 0 indicates normal tissue showing no fibrotic region; grade 1, fibrosis at≤25% of the entire area; grade 2, between 25% and 50%; grade 3, between 50% and 75%; and grade 4,≥75%. Analysis the degree of the fibrosis between LDH and LSS in dural layer, middle layer, dorsal layer respectively.(3) Picric-sirius red polarized light to identify the type of collagen.Slice was baked in 60℃incubator for 20minutes Before dewaxing. Slice soaked in Xylene I for 10 minutes, and xyleneⅡfor 10 minutes;alcohol rehydration gradual deoxidizing; Soaked in ddH2O for 3 minutes; Slice was washing for 10 minutes and rinsing by distilled water; Deal with 0.2% phosphorus molybdenum acid for 1～5minutes; 0.1% sirius scarlet picric acid solution dyeing for 90 minutes; 0.01N hydrochloric acid rinsing for 2 minutes; 70% alcohol rinsing for 45sec; rehydration gradual deoxidizing transparency; Slice soaked in Xylene I for 5 minutes, and xyleneⅡfor 5 minutes; Slice was sealed by Neutral gum. Each picrosirius red-stained section was imaged with an Olympus BX51 microscope (Olympus, Center Valley, Pennsylvania), a 20x objective, and circularly polarized light.(4) Determination of the location expression of PDGF p and PDGF-BB.The LF specimens were fixed in 10% neutral formalin and embedded in paraffin. There were 5um thick sections created and were dewaxed in xylene and rehydrated in graded ethanol solutions. Sections were then incubated with rabbit polyclonal anti-PDGF p and anti-PDGF-BB antibody (Millipore, San Antonio, TX, USA) followed dilution of 1:100. A routine immunoperoxidase staining technique using 3,3-diaminobenzidine tetrahydrochloride (DAB) was performed. A human dermis tissue sample used as a positive control.(5) Biologic study expression of PDGF-BB.6 samples from LDH group and 6 from dural layers,6 from dorsal layers in LSS groups were used. Immediately after removal, the sample was rinsed with saline and bone, blood, fat were separated. LF histologically consisted of dural layer(yellow deep), dorsal layer (white superficial) and middle layer. Each samples cut anatomically into three layers. The wet weight of each was determined and the tissue was freeze-clamped in liquid nitrogen and stored at-80℃until use. A total of 100 mg of the LF tissue was homogenized for protein and mRNA test respectively.2. Isolation and culture of HLF cells in vitro.Tissue samples of ligamentum flavum were harvested during surgery from patients with thoracolumbar burst fractures or from patients with lumbar disc herniation. The samples were washed with phosphate-buffered saline (PBS), after which any surrounding tissues attached to the specimen were carefully removed. The collected ligaments were minced into pieces of approximately 0.2 mm3 and washed twice with PBS. The minced tissue was digested at 37℃for 90 min with 0.2% collagenase type I in serumless containing DMEM. Collagenase-treated ligament-chips were washed with DMEM and then placed in 25cm2culture flasks in DMEM supplemented with 10% fetal bovine serum (FBS),100 U/ml penicillin and 100 pg/ml streptomycin, and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37℃.The medium was firstly changed at day 5-7 after explant culture and then replaced at three-day intervals. The outgrown cells grown to confluence were subcultured using trypsinization with 0.2% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA).Under an inverted phase microscope, the cultures were examined for cell outgrowth and Cell morphology and growth status. Immunofluorescence staining was used to detect the expression of vimentin and collagen typeⅠ.3. Application rhPDGF-BB on LF cells in vitro.(1) Proliferation of LF cells.Proliferation of LF cells were evaluated using the MTT. Cells were plated at 5000 cells/cm2 in 96-well plate and cultivated in MEM/FBS for 24 hrs. After the cells were adherent, they were exposed to 200ng/ml recombinant human PDGF-BB (rhPDGF-BB) for 0h,6h,12h,24h,48h,72h or 0,10,50,100,200ng/ml rhPDGF-BB for 12h. LF cells unexposed to rhPDGF-BB was taken as control group. The absorbance (OD) was then measured at a wavelength of 490 nm by a Microplate Autoreader (Bio-Tek Instruments, VT, USA). The growth inhibitory rate was calculated by the following formula:growth inhibitory rate=(average OD value in the control group-average OD value in the treatment group)/average OD value in the control group×100%.(2) Determination of expression of collagen I mRNA in LF cells.LF cells were seeded into 6-well plates at 1×105 cells/ml in a volume of 2 ml medium. LF cells were exposed to 200ng/ml recombinant human PDGF-BB (rhPDGF-BB) for 0h,6h,12h,24h,48h,72h or 0,10,50,100,200ng/ml rhPDGF-BB for 12h. Cells were collected and Total RNA was extracted using the TRI Reagent according to the manufacture's instruction. Relative expression of collagen I was calculated using GAPDH mRNA expression as an internal control.(3) Determination of expression of collagenⅠprotein in LF cells.LF cells were seeded into 6-well plates at 1×105 cells/ml in a volume of 2 ml medium and were exposed to 200ng/ml rhPDGF-BB for 0h,6h,12h,24h,48h,72h or 0,10,50,100,200ng/ml rhPDGF-BB for 12h. Supernatants of cells were collected and their total protein was then prepared with RIPA lysis buffer. The expression of collagen I protein were determined by Elisa.(4) Determination of expression of collagenⅢmRNA in LF cells.LF cells were seeded into 6-well plates at 1×105 cells/ml in a volume of 2 ml medium. LF cells were exposed to 200ng/ml recombinant human PDGF-BB (rhPDGF-BB) for 0h,6h,12h,24h,48h,72h or 0,10,50,100,200ng/ml rhPDGF-BB for 12h. Cells were collected and Total RNA was extracted using the TRI Reagent according to the manufacture's instruction. Relative expression of collagenⅢwas calculated using GAPDH mRNA expression as an internal control.(5) Determination of expression of collagenⅢmRNA in LF cells.LF cells were seeded into 6-well plates at 1×105 cells/ml in a volume of 2 ml medium and were exposed to 200ng/ml rhPDGF-BB for 0h,6h,12h,24h,48h,72h or 0,10,50,100,200ng/ml rhPDGF-BB for 12h. Supernatants of cells were collected and their total protein was then prepared with RIPA lysis buffer. The expression of collagen I protein were determined by Elisa.Results1.To investigating the difference of fibrosis and expression of PDGF-BB between LSS and LDH, to sudy correlation among LF thickness,fibrosis and the expression of PDGF-BB.(1) The features of specimens.The mean age of the LDH (and standard deviation) was 50.50±5.0years (range, 45.0 to 59.0 years) and 56.00±7.50years (range,40.0 to 65.0 years) in those with LSS. there was no significant difference (p=0.069).The mean thickness of the LF (and standard deviation) was 2.8±0.7mm (range,1.63 to 3.87 mm) from LDH group and 5.3±1.0mm (range,3.95 to 7.48 mm) from LSS group. The difference was significant (p=0.000).(2) The fibrosis of dural, middle, dorsal layer between LSS and LDH group.The fibrosis of LSS was significantly higher than LDH group (P=0.001).The fibrosis of dural, middle, dorsal layer between LSS and LDH group have significantly difference. Value of P were 0.025,0.001,0.002,respectively. Value of P between dorsal and dural of LSS was 0.000.(3) Identify the type of collagenThe expression of collagen were identified collagen type I and collagen type III both in LDH and LSS group.(4) Determination of the location expression of PDGFβand PDGF-BB.10 samples of LSS group showed positive staining of PDGF-p and PDGF-BB. They were stained in the fibroblast in hypertrophied LF especially in dorsal layer in LSS group.(5) Biologic study expression of PDGF-BB.Western blot assays showed that the expression of protein PDGF-BB is highest in dorsal layer in LSS compare to dural layer in LSS (P=0.023) and entire part in LDH (P=0.023); There is no significant difference between dural layer of the Lss and LDH group (P=0;096). The mRNA expressions of PDGF-BB,20 fold over-expressed were significantly higher in the dorsal layer of LSS than dural layer of LSS group (P=0.019) and entire LDH group (P=0.013). There is no significant difference between dural layer of the Lss and LDH group (P=0.865).(6).Correlation between LF thickness and fibrosis.The fibrotic score increased with thickening of the LF at all 3 sites from dural, middle to dorsal layer, showing a positive and significant linear correlation. The regression coefficients were 0.81 (P<0.05),0.77 (P<0.05), and 0.41 (P<0.05), respectively.(7).Correlation between LF thickness and expression of PDGF-BB.There showed a relatively strong.correlation 0.41(P=0.000) between PDGF-BB mRNA and thickness of LF.(8).Correlation between fibrosis and expression of PDGF-BB.PDGF-BB mRNA showed a very strong correlation (r=0.69) with fibrosis, and the association was shown to be statistically significant (P=0.001). The data indicated that with fibrosis aggravated, hypertrophied LF deposited much more PDGF-BB when compared with the LDH patients.2. Isolation and culture of HLF cells in vitro.(1) Inverted phase microscope LF cells were successfully isolated using a collagenase-pretreated explant method. Within 10-14 days after explants, outgrowth of cells was observed from ligament tissue explants and became monolayer. Morphologically, LF cell lines varied widely in appearance, and ranged from thin, spindle-shaped cells to polygonal cells or oval-shaped cells. The primary cells showed radial growth at sub-confluence. The passage cells arranged mainly spindle-shaped, some were fan-shaped or polygonal. They showed vortex-like growth at sub-confluence. When LF cells subculture continuously to the fifth passage, they still maintain a good morphology.(2) Immunofluorescence staining.Immunofluorescence staining showed that the vimentin and collagen typeⅠexpressed in the all LF cells.3. Application rhPDGF-BB on LF cells in vitro.(1) Proliferation of LF cells.Shows proliferation of LF cells which were exposed to PDGF-BB at concentrations ranging from 0 to 200 ng/ml for 0h,6h,12h,24h,48h,72h. The cell numbers at the concentrations of 100 ng/ml were significantly higher than control group at 6h,12h,24h,48h (P<0.05), showed no significantly difference at 72h (P> 0.05); The cell numbers at the concentrations of 200 ng/ml showed no significantly difference than control group at 6h,12h,24h,72h (P>0.05), showed significantly difference at 48h (P<0.05).(2) Determination of expression of collagen I mRNA in LF cells.The mRNA expression of collagen I of LF cells exposed to PDGF-BB of 200ng/ml for 12h have significantly difference than control group (P<0.01). Real-Time-PCR measurement showed that PDGF-BB treatment results in a time-dependent (F=435.865, P=0.000) and dose-dependent (F=103.817, P= 0.000) increasing expression of collagen I mRNA.(3) Determination of expression of collagen I protein in LF cells.The protein expression of collagen I of LF cells exposed to PDGF-BB of 200ng/ml for 24h have significantly difference than control group (P<0.01).Elisa measurement showed that PDGF-BB treatment results in a time-dependent (F= 1478.703,P=0.000) and dose-dependent (F=127.424, P=0.000) increasing expression of collagen I.(4) Determination of expression of collagenⅢmRNA in LF cells.The mRNA expression of collagenⅡof LF cells exposed to PDGF-BB of 100ng/ml for 12h have significantly difference than control group (P<0.01). Real-Time-PCR measurement showed that PDGF-BB treatment results in a time-dependent (F=914.894,P=0.000) and dose-dependent (F=136.842,P=0.000) increasing expression of collagenⅢmRNA.(5) Determination of expression of collagenⅢprotein in LF cells.The protein expression of collagenⅢof LF cells exposed to PDGF-BB of 200ng/ml for 72h have significantly difference than control group (P<0.01).Elisa measurement showed that PDGF-BB treatment results in a time-dependent (F=848.137,P=0.000) and dose-dependent (F=10184.74,P=0.000) increasing expression of collagenⅢ.Conclusions1. A higher PDGF-BB expression existed in the hypertrophied ligamentum flavum in lumbar spinal canal stenosis patient and could be a risk factor for fibrosis.2. Cells identified by the vimentin and collagen typeⅠwere LF Cells after primary culture.3. PDGF-BB increase expression of collagenⅠandⅢthat were related to fibrosis which caused the development of hypertrophy of the of the ligamentum flavum in lumbar spinal canal stenosis.