| Objective Ossification of the posterior longitudinal ligament (OPLL) is a commoncervical disorder, which can cause severe nerve compression symptoms. Even minortrauma may lead to incomplete or complete paralysis. The disease has great impact onpatients’ lives. Since1970s, a number of fundamental and clinical researches on cervicalOPLL had been done at home and abroad. These researches have proven that thepathological process of OPLL is the posterior longitudinal ligament fibroblastsdifferentiate into osteoblasts and causing ectopic bone formation ultimately. The process ofossification was affected by varieties factors, such as genes and mechanical stress. For theOPLL patient with severe symptoms, surgery remains the maine treadtment. The surgicalapproach is divided into posterior, anterior, or anterior combined with posterior. ThePosterior surgery can expand the canal volume and decompress the spinal cord and nerveroots indirectly, but it can not remove the ossification foci in front of the spinal cord andinterrupt the progress of ossification. Anterior surgery is thought to be a risky procession,although the ossification foci could be removed. The incidence of cerebrospinal fluidleakage and spinal cord injury was relatively high. Most surgeons choose to retain theossification foci which called "floating" method, but the residues of ossification matter wascontinued growth, which impact on clinical efficacy in turn. In the follow-up, lots ofpatients’ symptoms increased gradually with progress of ossification, or the symptomsrecurrence after surgery. Some patients even need re-operation. Mechanical stress wasthought to be an iUmportannt faRctor foer theg progiresssiont oef ossrification. It is necessary toexplore a method to inhibit or slow down the sustained growth of the ossification foci.Chitosan was a deacetylated product of chitin, which has good biological compatibility. Itwas mainly used to prevent post-operative adhesions and scarring currently and hadachieved exact effect. There are experiments have proven that chitosan could inhibithuman skin fibroblast autocrine transforming growth factor-β1(TGF-β1), reduce theextracellular matrix deposition and collagen protein synthesis, and suppress theproliferation and differentiation of fibroblasts. The deposition of the extracellular matrix,as well as the synthesis of collagen is the performance of ossification in posteriorlongitudinal ligament fibroblasts. Moreover, it has been verified that high glucose couldpromote collagen synthesis in the posterior longitudinal ligament via endogenous TGF-β1, resulting in hypertrophy of the ligament. Therefore, we hypothesize that chitosan mayaffect the secretion of TGF-β1in fibroblasts derived from cervical posterior longitudinalligament, and inhibit or delay the progression of osteogenic differentiation. This study wasto confirm that mechanical stretch stress can promote the TGF-β1expression in posteriorlongitudinal ligament fibroblasts derived from OPLL patients, and accelerate the process ofossification. Chitosan would be added to the culture solution in order to analyze the effecton the ossification progress of posterior longitudinal ligament fibroblasts.Methods Fourteen specimens of posterior longitudinal ligament derived from OPLLpatients were obtained via anterior approach surgery including12anterior cervicalcorpectomy and fusion (ACCF) and2anterior cervical discectomy and fusion (ACDF). Allthe specimens were placed in sterile test tube containing culturee meddium and sent to thecell culture chamber under37°C immediately. Fibroblasts culture in vitro was taken bytissue culture method. The tissue and cell were identified by immunostaining andhistochemistry technique. Fibroblasts from OPLL patients were preloaded with mechanicalstress using the Flexercell4000system. The mRNA expression of type I collagen (COL I),alkaline phosphatase (ALP), and TGF-β1was detected at pre-stress and12h,24h afterstress loading by real-time RT-PCR.0.1ng/ml,1ng/ml and10ng/ml concentration ofexogenous TGF-β1was added to culture solution of statically cultured fibroblasts, while2ug/ml TGF-β1antibody was added to culture solution of Loading stress fibroblasts, thenthe mRNA expressions of COL I, ALP, and TGF-β1were detected after24h in order toresearch the role of TGF-β1in the progress of ossification.0mg/ml,0.1mg/ml,1mg/mlconcentration of chUitosan nwas aRdded teo culgture siolsutiont aned aprplying mechanical stress for12hours and24hours, the fibroblasts’ mRNA expression of COL I, ALP and TGF-β1wasdetected by real-time RT-PCR. Supernatant was also reserved. The type I collagenC-telopeptide (CTX-1) was detected by enzyme-linked immunoassay (ELISA) and theALP activity unit was detected by biochemical technology. Then the inhibitory effect ofchitosan on the progress of OPLL would be analyzed.Results After Masson trichromatic staining and Von kossa H&E staining, ossificationphenomenon can be seen clearly around the tissue samples of posterior longitudinalligament from OPLL patients. All of14specimens of posterior longitudinal ligament wereused for cell culture in vitro by tissue culture method and12cases of them are successful.After treating by HE and immune fluorescent staining method, it can be observed under the microscope that cells are fusiform, spindle and polygonal-star, while the nucleus is largeand oval with unclear boundary of cells, which are the typical morphologicalcharacteristics of fibroblast cells. After applying mechanical stress for12hours and24hours seperately, the mRNA expression of COLI, ALP and TGF-β1was higher thancontrol group. The result in24hours were statistical significance (P<0.05). It verifies thatmechanical stress can promote fibroblast cells of posterior longitudinal ligamentosteogenic differentiation. The mRNA expression of COL I and ALP was increased whenexogenous TGF-β1was added to the culture solution for24hours, and it showed aconcentration-dependent phenomenon. The results in10ng/ml were statistical significance(P<0.05). However, the increased range was significantly lower than that of the controlgroup after applying mechanical stress for24hours when2ug/ml TGF-β1antibody wasadded to culture solution (P<0.05). It verifies that TGF-β1plays an important role in theprocedure of ossification. Affected by0.1mg/ml Chitosan, the increased range of mRNAexpression of COL I, ALP and TGF-β1was no significant difeferendce with the controlgroup after applying mechanical stress for12hours (P>0.05), but the increased range ofCOL I was significantly decreased after24hours (P<0.05). Affected by1.0mg/mlchitosan, the increased range of mRNA expression of ALP was significantly lower than thecontrol group after applying mechanical stress for12hours (P<0.05), while the increasedrange of COL I, ALP and TGF-β1were all significantly decreased after24hours (P<0.05).The CTX-1content in supernatant of1.0mg/ml chitosan group was significantly lowerthan the0.1mg/ml group and control group after applying mechanical stress for24hours(P<0.05), while the ALP content of1.0mg/ml Chitosan group was significantly lower thanthe0.1mg/ml ChitUosan gnroup Rand coentrolg grouips aftetr aepplyirng mechanical stress for12hours and24hours (P<0.05).Conclusions The success rate for posterior longitudinal ligament fibroblasts cell culturein vitro by tissue culture method was high. Mechanical stretch stress stimulation canaccelerate the process of ossification and promote osteogenic differentiation for fibroblastsof posterior longitudinal ligament. TGF-β1plays an important role in the procedure.Chitosan can affect the secretion of TGF-β1in posterior longitudinal ligament fibroblastsderived from OPLL patients, and inhibit the progression of osteogenic differentiation. |
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