DAPK2 promotes autophagy to accelerate the progression of ossification of the posterior longitudinal ligament through the mTORC1 complex

Background:Ossification of the posterior longitudinal ligament(OPLL)is a prevalent condition in orthopedics.While death-associated protein kinase 2(DAPK2)is known to play roles in cellular apoptosis and autophagy,its specific contributions to the advancement of OPLL are not well understood.Methods:Ligament fibroblasts were harvested from patients diagnosed with OPLL.Techniques such as real-time reverse transcriptasepolymerase chain reaction(RT-qPCR)and Western blot analysis were employed to assess DAPK2 levels in both ligament tissues and cultured fibroblasts.The extent of osteogenic differentiation in these cells was evaluated using an alizarin red S(ARS)staining.Additionally,the expression of ossification markers and autophagy markers was quantified.The autophagic activity was further analyzed through LC3 immunofluorescence and transmission electron microscopy(TEM).An in vivo heterotopic bone formation assay was conducted in mice to assess the role of DAPK2 in ossification.Results:Elevated DAPK2 expression was confirmed in both OPLL patient tissues and derived fibroblasts,in contrast to non-OPLL controls.Silencing of DAPK2 significantly curtailed osteogenic differentiation and autophagy in these fibroblasts,evidenced by decreased levels of LC3,and Beclin1,and reduced autophagosome formation.Additionally,DAPK2 was found to inhibit the mechanistic target of the rapamycin complex 1(mTORC1)complex’s activity.In vivo studies demonstrated that DAPK2 facilitates ossification,and this effect could be counteracted by the mTORC1 inhibitor rapamycin.Conclusion:DAPK2 enhances autophagy and osteogenic processes in OPLL through modulation of the mTORC1 pathway.

Catheter-Assisted Interlaminar Approach for Cervical Epidural Steroid Injection in Patient with Cervical Stenosis Caused by Ossification of Posterior Longitudinal Ligament: A Case Report

We present the case of a 64-year-old man with cervical ossification of the posterior longitudinal ligament (OPLL) experiencing chronic neck pain and radiculopathy for 6 months. A catheter-assisted interlaminar Cervical Epidural Steroid Injection (CESI) was performed under fluoroscopic guidance, targeting the affected C2-C6 levels. Significant improvement was observed after this procedure, with decreased pain scores (visual analogue scale (VAS) 8 to 2) and improved mobility. This technique not only enhances the effectiveness of CESI but also reduces the likelihood of complications such as stroke or epidural hematoma and thus provides an alternative treatment option for patients with multiple stenotic levels who are unsuitable for surgery or are unresponsive to conservative therapy such as medication or physical therapy.

Regression of intervertebral disc calcification combined with ossification of the posterior longitudinal ligament:A case report

BACKGROUND Intervertebral disc calcification(IDC)combined with ossification of the posterior longitudinal ligament(OPLL)in cervical discs is rarely reported.This case study presents a rare case of IDC combined with OPLL in the C2-C3 segment.CASE SUMMARY Here,we present a case of a 6-year-old Asian boy with severe neck pain and stiffness.Physical examination showed no neurological or other abnormalities.Digital radiography and computed tomography(CT)revealed a calcified intervertebral disc and OPLL at the C2-C3 vertebrae.The spinal canal compromise at C2-C3 was approximately 50%on magnetic resonance imaging.The final diagnosis was IDC combined with OPLL.We applied a neck brace for the patient to protect the neck.The patient's neck pain and stiffness recovered significantly within approximately 3 wk.At the 3 mo follow-up,the follow-up CT showed resolution of the ossified intervertebral disc herniation,and a small amount of calcification and slight OPLL remained at the involved segment.CONCLUSION IDC combined with OPLL is a relatively rare condition in children.However,the majority of patients could have a favorable outcome,and the ossified mass in the canal would be spontaneously resolved with conservative therapy.

Enlarged laminectomy for ossification of posterior longitudinal ligament in cervical spine

Objective To introduce surgical strategy of enlarged laminectomy (with partial facet joint dissection to expose nerve root) ,and to discuss its benefit for cervicalossification of posterior longitudinal ligament (OPLL)

The clinical features and treatment strategies of thoracic ossification of posterior longitudinal ligament

Objective To study the clinical features and surgical strategies of thoracic spinal stenosis caused by ossification of posterior longitudinal ligament(OPLL).Methods From January 2004 to March 2009,21 cases of tho-racic spinal stenosis

"Cave-in"technique: 360° circumferential decompression for thoracic spinal stenosis with ossification of posterior longitudinal ligament

Objective To investigate the surgical techniqueand efficiency of the "Cave-in" 360° circumferential decompression for thoracic spinal stenosis(TSS)with ossification of posterior longitudinal ligament(OPLL).Methods From October 2005 to

Association between two polymorphisms of the bone morphogenetic protein-2 gene with genetic susceptibility to ossification of the posterior longitudinal ligament of the cervical spine and its severity

Background Ossification of the posterior longitudinal ligament （OPLL） has a strong genetic background. Previous studies have shown that bone morphogenetic protein-2 （BMP2） and BMP2 mRNA are expressed in ossifying matrix and chondrocytes adjacent to cartilaginous areas of OPLL tissues and mesenchymal cells with fibroblastic features in the immediate vicinity of the cartilaginous areas. It is suggested that BMP2 plays different roles in the different stages of development of OPLL. However, it remains unknown which factors induce ligament cells to produce BMP2. Methods OPLL patients （n=-192） and non-OPLL controls （n=304） were studied. Radiographs of the cervical spine were analyzed for extent of OPLL. We investigated whether single nucleotide polymorphisms of exons 3（-726） T/C and 3（-583） NG in the BMP2 gene are statistically associated with genetic susceptibility to OPLL in Chinese Han subjects. Results There was no statistical difference between the occurrence of exons 3（-726） T/C and 3（-583） NG and the occurrence of OPLL in the cervical spine. However, there was a significant association between occurrence of exon 3（-726） T/C polymorphism and occurrence of OPLL in males of cases and controls in the cervical spine. In addition, no significant association was found between the exons 3（-726） T/C and 3（-583） A/G with number of ossified cervical vertebrae in OPLL patients. Conclusions Exon 3（-583） A/G polymorphism in BMP2 gene is not associated with the occurrence and the extent of OPLL in the cervical spine. Chinese Han male patients with TC and CC genotypes in exon 3（-726） T/C have genetic susceptibility to OPLL but not to more extensive OPLL in the cervical spine.

Linkage of three polymorphisms on chromosome 20p12 to ossification of the posterior longitudinal ligament of spine and its severity in Han Chinese patients

Background Ossification of the posterior longitudinal ligament （OPLL） is characterized by the replacement of ligamentous tissue with new ectopic bone formation, and has a strong genetic background. Because of the abnormal bone metabolic features and the strong genetic component, osteoporosis is a related disorder with OPLL. Three polymorphisms on chromosome 20p12 were identified associated with the risk of osteoporosis and osteoporotic fracture.The rs996544 （C/T） ＂TT＂ and rs965291 （G/A） ＂AA＂ genotypes conferred higher risks for vertebral and hip fractures. The osteoporosis haplotype is defined by two polymorphisms, rs1116867 （A） and D35548 （T）. However, it remains unknown whether these three polymorphisms predispose to an increased frequency and severity of OPLL in Han Chinese patients.Methods A total of 420 OPLL patients and 506 age- and sex-matched controls were studied. Three single nucleotide polymorphisms （SNPs）, rs996544 （C/T）, rs965291 （G/A） and rs1116867 （A/G）, were analyzed by direct sequencing.Associations between these SNPs with the occurrence and extent of OPLL were statistically evaluated.Results There was no significant association between the rs996544 （C/T） polymorphism and the prevalence of OPLL.The rs1116867 （A/G） polymorphism ＂AG＂ genotype was associated with the occurrence of OPLL. The rs1116867 （A/G） polymorphism ＂G＂ allele was associated with the occurrence of OPLL, but not with the extent of OPLL. The rs965291 （G/A） polymorphism in female patients was statistically different between cases and controls （P 〈0.05）. The rs965291 （G/A） polymorphism ＂A＂ allele was associated with the occurrence of OPLL in female patients. For the rs965291 （G/A）polymorphism, patients with the ＂A＂ allele （genotype, ＂AG＂ or ＂AA＂） showed a significantly greater number of ossified cervical vertebrae than those without the ＂A＂ allele （genotype, ＂GG＂, P 〈0.05）, particularly in female patients.Conclusions The rs1116867 （A/G） and rs965291 （G/A） polymorphisms on chromosome 20p12 are associated with the occurrence and the extent of OPLL, at least in Han Chinese subjects. Our data should advance our understanding of the molecular etiology of OPLL and may guide approaches to prevent the onset of OPLL.

Classification and management of the tandem ossification of the posterior longitudinal ligament and flaval ligament

Objective To provide appropriate guidelines for treatment of tandem ossification of the posterior longitudinal ligament （OPLL） and flaval ligament （OFL）. Data sources Published articles about OPLL and OFL were selected using Medline and Embase electronic databases. Study selection An English literature search from January 1980 to December 2006 was conducted. Because many reported cases were incorporated in OFL studies, the key words for search were OFL or OFL and OPLL. The first step revealed 93 studies of which 13 reports of tandem OPLL and OFL （tandem ossification） were selected. Results All studies were case series or case report and advocated that the primary therapy for tandem ossification should be operative. The clinical outcomes of surgery were evaluated in most reports, predominantly using the JOA scores. Gender is the only factor which has prognostic value. A higher proportion of women was found in the failure group A two- stage classification of tandem ossification was developed to relate diagnosis to outcome. Conclusions All patients with suspected ossification of the spinal ligaments should undergo routine MRI screening of the whole spine. The correlation of the classification with surgical treatments needs further studies to validate its usefulness. Chin Med J 2009; 122（2）：219-224

A single nucleotide polymorphism in the human bone morphogenetic protein-2 gene （109T〉G） affects the Smad signaling pathway and the predisposition to ossification of the posterior longitudinal ligament of the spine

Background Although various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging, and hormonal disturbances have been suggested as causes of ossification of the posterior longitudinal ligament （OPLL）, the etiology of OPLL is not fully understood. The purpose of this study was to investigate whether bone morphogenetic protein （BMP）-2 is a candidate gene to modify the susceptibility of OPLL and the mechanism of signal transduction in ossification. Methods A total of 420 OPLL patients and 506 age- and sex-matched controls were studied. The complete coding sequence of the human BMP-2 gene was analyzed using polymerase chain reaction （PCR） and direct sequencing. All single nucleotide polymorphisms （SNPs） were detected and genotyped. BMP-2 expression vectors containing positive polymorphisms were constructed and transfected into the C3H10T1/2 cells. The expression of BMP-2 and the Smad signal pathway in positive cell clones were detected by Western blotting. The alkaline phosphatase （ALP） activity was determined using quantitative detection kits. Results The frequencies for the 109T〉G and 570A〉T polymorphisms were different between the case and control groups. The ＂TG＂ genotype in 109T〉G polymorphism is associated with the occurrence of OPLL, the frequency of the ＂G＂ allele is significantly higher in patients with OPLL than in control subjects （P 〈0.001）. The ＂AT＂ genotype in 570A〉T polymorphism is associated with the occurrence of OPLL, the frequency of the ＂T＂ allele is significantly higher in patients with OPLL than in control subjects （P=0.005）. Western blotting analysis revealed that the expression of P-Smadl/5/8 protein transfected by wild-type or mutant expression vectors were significantly higher than control groups （P 〈0.05）, but there was no statistical difference in each experimental group （P 〉0.05）. The expression of Smad4 protein transfected by wild-type or mutant expression vectors was significantly higher than control groups （P 〈0.05）. The expression of Smad4 protein transfected by pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） was significantly higher than the other experimental groups （P 〈0.05）. The increase in ALP activity has been detected in pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） transfected cells up to 4 weeks after stable transfection. Activity of ALP was （30.56±0.46） nmol.min^-1.mg^-1 protein and （29.62±0.68） nmol.min^-1.mg^-1 protein, respectively. This was statistically different compared with the other experimental groups （P 〈0.05）. Conclusions BMP-2 is the predisposing gene of OPLL. The ＂TG＂ genotype in the 109T〉G and the ＂AT＂ genotype in the 570A〉T polymorphisms are associated with the occurrence of OPLL. The 109T〉G polymorphism in exon-2 of the BMP-2 gene is positively associated with the level of Smad4 protein expression and the activity of ALP. The Smad mediated sicjnaling pathway plays an important role durincl the Datholoqical process of OPLL induced by SNPs of BMP-2 aene.

Clinical results and intramedullary signal changes of posterior decompression with transforaminal interbody fusion for thoracic myelopathy caused by combined ossification of the posterior longitudinal ligament and ligamentum flavum

Background Surgical treatment of thoracic myelopathy caused by ossification of the posterior longitudinal ligament （OPLL） and ossification of the ligamentum flavum （OLF） is technically demanding, and the results tend to be unfavorable. Various operative approaches and treatment strategies have been attempted, and posterior decompression with transforaminal thoracic interbody fusion （PTTIF） may be the optimal method with which the anterior-posterior compression was removed in one step. It is comparatively less traumatic with fewer serious complications.

T allele at site 6007 of bone morphogenetic protein-4 gene increases genetic susceptibility to ossification of the posterior longitudinal ligament in male Chinese Han population

Background Several candidate genes of ossification of the posterior longitudinal ligament （OPLL） susceptibility have been identified, but their polymorphisms account for only a small percent of the total variance. Bone morphogenetic protein-4 （BMP4） is a potent ectopic ossification inducing factor. BMP4 protein and mRNA are present in cells from OPLL patients, but not non-OPLL controls. A single nucleotide polymorphism of 6007C〉T（rs17563） of BMP4 has been reported to affect bone density in postmenopausal women. Thus, BMP4 may function in OPLL development. Appropriately, the relationship between BMP4 polymorphisms and OPLL was investigated. Methods A case-control association study investigated the genetic etiology in 179 OPLL patients and 298 non-OPLL controls. Extent of OPLL was analyzed by radiologic examinations. Whether single nucleotide polymorphism （SNP） of -5826G〉A（rs1957860） 5＇ of the transcription start site and 6007C〉T（rs17563） in exon 4 of the BMP4 gene were statistically associated with genetic susceptibility to OPLL in Chinese Han subjects was assessed. Results A significant statistical difference in genotype of 6007C〉T polymorphism between male OPLL patients and male controls was evident, and the frequency of ＂TT＂ genotype in male OPLL patients was significantly higher than in male controls （P=0.039）. The frequency of the ＂T＂ allele was also significantly higher in male OPLL subjects than in male controls （P=0.014, OR=1.57）. A significant difference was also observed between the 6007C〉T polymorphism and the number of ossified cervical vertebrae in OPLL patients, while no statistical difference was apparent between the -5826G〉A polymorphism and OPLL occurrence. Conclusions The T allele in the 6007C〉T polymorphism may be a risk factor for male Han Chinese with ossification of the posterior longitudinal ligament in the cervical spine. Chinese Han male patients with CT and TT 6007C〉T genotypes have a genetic susceptibility to OPLL and more extensive OPLL in the cervical spine.

Circumferential decompression via the posterior approach for the surgical treatment of multilevel thoracic ossification of the posterior longitudinal ligaments： a single institution comparative study

Background The treatment strategies for multilevel thoracic ossification of the posterior longitudinal ligaments （T-OPLL） were rarely reported. The aim of this study was to investigate the clinical outcomes and complications of circumferential decompression for multilevel T-OPLL and compare two different methods in the management of the OPLL （resection or floating）. Methods Data of sequentially treated patients who received surgical treatment for thoracic spinal stenosis caused by multilevel T-OPLL from January 2005 to February 2012 were retrospectively reviewed. Based on the surgical approaches applied, the patients were divided into two groups. Group A consisted the patients who received posterior decompression and group B consisted the patients who received circumferential decompression via the posterior approach. Group B was further divided into two subgroups： subgroup 1 （the resection group） where the OPLL was completely resected and subgroup 2 （the floating group） where the OPLL was floated. Results A total of 49 patients were included in the study. Fourteen patients with single posterior decompression were included in group A and 35 patients who received circumferential decompression were included in group B. In group B, 29 patients had complete resection of the ossified posterior longitudinal ligaments, while the other six underwent a flotation procedure. The follow-up data were available in 39 patients. Mean JOA scores improved from 5.4 ± 1.8 to 7.5 ± 2.8 in group A and from 3.7 ± 1.8 to 7.9 ± 2.4 in group B. The main complications included cerebrospinal fluid （CSF） leakage and postoperative neurelogic deterioration （ND）. Twenty-three of the 25 cases with postoperative CSF leakage achieved a complete recovery at the last follow-up and 12 of the 15 cases with ND achieved some neurological improvement at the last follow-up. Conclusions Circumferential decompression via the posterior approach is an effective surgical method for thoracic spinal stenosis caused by multilevel OPLL of the thoracic spine. Patients who receive complete resection of the ossified posterior longitudinal ligaments may have better recovery rate than the ＂floating＂ group.

Quantitative analysis of cyclooxygenase 2 in the posterior longitudinal ligament of cervical spondylotic myelopathy

Background Cervical spondylotic myelopathy （CSM）, in part, results from degeneration of the posterior longitudinal ligament （PLL）, which mechanically compresses the spinal cord. Much research was done on the ossification of PLL, but not concerning the non-ossifying degeneration of cervical PLL. The degeneration of cervical PLL may be related to inflammation. The aim of this study was to elucidate the pathological features of the PLL and the role of cyclooxygenase 2 （COX-2） in the degeneration of the PLL in CSM. Methods A total of 23 PLL specimens were collected during surgery from patients with CSM for the histological and immunohistochemical （type II collagen and Ki-67） study. For the control group 14 cervical PLL autopsy specimens were investigated in the same manner, mRNA expression of COX-2 was quantitatively measured by real-time reverse transcription-polymerase chain reaction （RT-PCR） from 18 PLL specimens of patients with CSM and 18 PLL specimens of autopsy cases. Immunohistochemistry was used to evaluate the cellular location of COX-2 in PLL. Results A distinct amount of fibrotic area, chondrometaplastic tissue and calcification were found in the PLL of the patient group, compared with the control group. Type II collagen was apparent around chondrometaplastic cells. Ki-67 positive reaction was less than 5%. A COX-2 positive reaction was found in 9 of the patient specimens （39.1%） in which the COX-2 was released from vascular endothelial cells in the PLL. However, such reactions were not found in the control group. Real-time PCR showed that the mRNA expression level of COX-2 in the patient group was significantly higher than that in the control group （P〈0.01）. Conclusions Chondrometaplastic tissue producing type II collagen was identified as the most predominant pathological feature in the degenerative PLL. The higher expression of COX-2 might be related to degeneration of the PLL in CSM.

A 92-year-old man with retropharyngeal hematoma caused by an injury of the anterior longitudinal ligament

Traumatic retropharyngeal hematoma is a rare condition and may be lethal in some cases. In patients with this condition, the absence of a vertebral fracture or a major vascular injury is extremely rare. We present the case of a 92-year-old man who hit his forehead by slipping on the floor in his house. He had no symptoms at the time; however, he experienced throat pain and dyspnea at 6 hours after the injury. On arrival, he complained of severe dyspnea; therefore, an emergency endotracheal intubation was performed. A lateral neck roentgenogram after intubation showed dilatation of the retropharyngeal and retrotracheal space and no evidence of a cervical vertebral fracture. Cervical computed tomography （CT） with contrast medium revealed a massive hematoma extending from the retropharyngeal to the superior mediastinal space but no evidence of contrast medium extravasation or a vertebral fracture. However, sagittal magnetic resonance imaging （MRI） revealed an anterior longitudinal ligament （C4-5 levels） injury. We determined that the cause of the hematoma was an anterior longitudinal ligament injury and a minor vascular injury around the injured ligament. Therefore, we recommend that patients with retropharyngeal hematoma undergo sagittal cervical MRI when roentgenography and CT reveal no evidenee of injury.

Removal versus preservation of the posterior longitudinal ligament in Bryan cervical disc arthroplasty

Background Bryan cervical disc arthroplasty can be used to restore and maintain the mobility and function of the involved cervical spinal segments. The efficiency of posterior longitudinal ligament （PLL） resection in anterior cervical decompression and fusion has been demonstrated. However, no clinical reports have compared PLL removal with preservation in Bryan cervical disc arthroplasty. This study aimed to assess the role of removal of PLL in Bryan cervical disc arthroplasty at an 18-month follow-up.

Intraoperative ultrasonography in ＂cave-in＂ 360° circumferential decompression for thoracic spinal stenosis

The surgical outcomes of decompression for thoracic spinal stenosis （TSS） are unfavorable. The purpose of this study was to determine the efficacy of intraoperative ultrasonography during ＂cave-in＂ 360~ circumferential decompression surgery in patients with TSS. Methods Thirteen patients with TSS underwent ＂cave-in＂ 360° circumferential decompression surgery between May 2010 and November 2010. Intraoperative ultrasonography was used after removal of the posterior wall of thoracic spinal canal to assess the morphologic restoration of the spinal cord and the anterior surface of the spinal canal. In seven patients, ultrasonography was used again after circumferential decompression to compare the cross-sectional area of the spinal cord before and after circumferential decompression. Results The average period of follow-up was （12±2） months （range 9-15 months）. The Japanese Orthopedic Association score was significantly higher at the final follow-up （8.5±2.1, range 3-10） than preoperatively （5.2±1.1, range 3-7; P 〈0.01）. The cross-sectional area of the spinal cord was （30.8±6.6） mm2 before and （53.6±19.1） mm2 after circumferential decompression （P 〈0.01）. For five patients with TSS caused by thoracic disc herniation, the levels of circumferential decompression performed corresponded to those expected preoperatively. In contrast, for eight patients with TSS caused by ossification of the posterior longitudinal ligament, on average 1.6±0.9 fewer levels of circumferential decompression were performed than expected preoperatively. Conclusions ＂Cave-in＂ 360° circumferential decompression is an effective therapeutic option for TSS. Intraoperative ultrasonographic evaluation may reduce the levels of circumferential decompression and ensure sufficient decomoression, and increase the efficacy of this surqical technique.

Effect of decompression range on decompression limit of cervical laminoplasty

Backgrounds:Cervical posterior decompression surgery is used to relieve ventral compression indirectly by incorporating a backward shift of the spinal cord, and this indirect decompression is bound to be limited. This study aimed to determine the decompression limit of posterior surgery and the effect of the decompression range.Methods:We retrospectively reviewed the data of 129 patients who underwent cervical open-door laminoplasty through 2008 to 2012 and were grouped as follows: C4-C7 ( n = 11), C3-C6 ( n = 61), C3-C7 ( n = 32), and C2-C7 ( n = 25). According to the relative location of spinal levels within a decompression range, the type of decompression at a given level was categorized as external decompression (ED;achieved at the levels located immediately external to the decompression range margin), internal decompression (ID;achieved at the levels located immediately internal to the decompression range margin), and central decompression (CD;achieved at the levels located in the center, far from the decompression range margin). The vertebral-cord distance (VCD) was used to evaluate the decompression limit. The C2-C7 angle and VCD on post-operative magnetic resonance images were analyzed and compared between groups. The relationship between VCD and decompression type was analyzed. Moreover, the relationship between the magnitude of the ventral compressive factor and the probability of post-operative residual compression at each level for different decompression ranges was studied. Results:There was no significant kyphosis in cervical curvature (>-5°), and there was no significant difference among the groups ( F = 2.091, P = 0.105). The VCD of a specific level depended on the decompression type of the level and followed this pattern: ED < ID < CD ( P < 0.05). The decompression type of a level was sometimes affected by the decompression range. For a given magnitude of the ventral compressive factor, the probability of residual compression was lower for the group with the larger VCD at this level. Conclusions:Our study suggests that the decompression range affected the decompression limit by changing the decompression type of a particular level. For a given cervical spinal level, the decompression limit significantly varied with decompression type as follows: ED < ID < CD. CD provided maximal decompression limit for a given level. A reasonable range of decompression could be determined based on the relationship between the magnitude of the ventral compressive factor and the decompression limits achieved by different decompression ranges.