Proteomic analysis of the effects of cell culture density on the metastasis of breast cancer cells.

Cancer cell progression and proliferation increase cell density, resulting in changes to the tumour site, including the microenvironment. What is not known is if increased cell density influences the aggressiveness of cancer cells, especially their proliferation, migration, and invasion capabilities. In this study, we found that dense cell culture enhances the aggressiveness of the metastatic cancer cell lines, 4T1 and ZR-75-30, by increasing their proliferation, migration, and invasion capabilities. However, a less metastatic cell line, MCF-7, did not show an increase in aggressiveness, following dense cell culture conditions. We conducted a differential proteomic analysis on 4T1 cells cultured under dense or sparse conditions and identified an increase in expression for proteins involved in migration, including focal adhesion, cytoskeletal reorganization, and transendothelial migration. In contrast, 4T1 cells grown under sparse conditions had higher expression levels for proteins involved in metabolism, including lipid and phospholipid binding, lipid and cholesterol transporter activity, and protein binding. These results suggest that the high-density tumour microenvironment can cause a change in cellular behaviour, leading towards more aggressive cancers. SIGNIFICANCE OF THE STUDY: Metastasis of cancer cells is an obstacle to the clinical treatment of cancer. We found that dense cultures made metastatic cancer cells more potent in terms of proliferation, migration, and invasion. The proteomic and bioinformatic analyses provided some valuable clues for further intensive studies about the effects of cell density on cancer cell aggressiveness, which were associated with events such as pre-mRNA splicing and RNA transport, focal adhesion and cytoskeleton reorganization, ribosome biogenesis, and transendothelial migration, or associated with proteins, such as JAM-1 and S100A11. This investigation gives us new perspectives to investigate the metastasis mechanisms related to the microenvironment of tumour sites.

Single-stage combined decompression for patients with tandem ossification in the cervical and thoracic spine.

STUDY DESIGN: Retrospective study of clinical outcomes of single-staged combined cervical and thoracic decompression for patients with tandem ossification (TO). OBJECTIVE: To describe primary clinical outcomes of this procedure. TO is introduced to described a double ossification lesion of the posterior longitudinal ligament (OPLL) or the ligament flavum (OLF) at the cervical, thoracic and lumbar spine. In clinical practice, cervical OPLL combined with thoracic OPLL or/and OLF are the most common types of TO. However, little is known about the clinical outcomes of surgical treatment and there is no consensus on the optimal treatment to this combined disorder. METHODS: Between January 2005 and December 2008, 15 patients of this complicated phenomenon were treated by single-staged combined cervical and thoracic decompression in conditions where patients' general condition allowed and individuals agreed on. Surgical intervention, perioperative complications, and clinical outcomes were reviewed in these 15 TO patients who were followed up for more than 2 years (range 2-5 years). Clinical symptoms were evaluated using the JOA scoring system and activity of daily life was evaluated by Nurick classification before surgery, at 6 months postoperatively, and at final follow-up. Patient satisfaction was determined at final follow-up. RESULTS: The mean blood loss was 1,553.3 ± 735.7 ml (range 700-2,900 ml) and the mean operation time was 280.7 ± 53.6 min (range 220-370 min). The important intraoperative and postoperative complications recorded in medical documents included CSF leakage, hematoma, C5 palsy and neurological deterioration. The JOA score was significantly higher 6 months after surgery (8.1 ± 1.8 points vs. 11.0 ± 1.6 points, p < 0.0001), and there was no significant change between 6 months after surgery and final follow-up (11.0 ± 1.6 points vs. 11.3 ± 2.1, p = 0.5894). The mean Nurick classification significantly improved from grade 3.6 ± 0.7 before surgery to grade 2.5 ± 0.9 at 6 months after surgery (p < 0.001), and well maintained as grade 2.3 ± 1.0 at final follow-up (p = 0.3343). Three patients had satisfaction scores of 3 points, 5 had scores of 2 or 1 point, and 2 had score of 0 point. Pearson correlation analysis showed a significant positive correlation between satisfaction score and JOA score (r = 0.6493, p = 0.0093), and a significant negative correlation between satisfaction score and Nurick classification (r = -0.5941, p = 0.0195). Besides, perioperative complications and progression of tandem ossification which needed revision surgery had significant adverse effect on patients' satisfaction. CONCLUSIONS: The results showed that single-staged combined decompression could provide comparable clinical outcomes, and patients' satisfaction was significantly related with postoperative neurological function. In addition, satisfaction score could be decreased by perioperative complications and progression of tandem ossification. Thus, this aggressive surgical strategy should be used more carefully with emphasis on preoperative communication with patients.

Mechanical strain induces Cx43 expression in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that mechanical strain can lead to the development of OPLL, although the signaling mechanism is not fully understood. Connexin43 (Cx43), a gap-junction protein, has been shown to be of particular importance in bone formation. We hypothesized that Cx43 may play an important role in the signal transmission induced by mechanical strain during the development of OPLL. To explore this possibility, we cultured fibroblasts from spinal ligaments of OPLL and non-OPLL patients and preloaded mechanical stretch onto the cells via a Flexercell 4000 Tension Plus system. We evaluated expression changes in osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 via semi-quantitative RT-PCR and western blotting at 12 and 24 h after mechanical strain application in contrast to static conditions. We observed a significant gene up-regulation of OCN, ALP and COL I and Cx43 protein in OPLL cells after mechanical strain application, but no changes in non-OPLL cells. Notably, after RNA interference targeting Cx43 was performed in OPLL cells, we found that there were no significant changes in the expressions of OCN, ALP, COL I and Cx43 after the mechanical strain was applied for 24 h. Thus, we propose that the increase in Cx43 expression induced by mechanical strain in OPLL cells plays an important role in the progression of OPLL.

Choice of surgical approach for ossification of the posterior longitudinal ligament in combination with cervical disc hernia.

Ossification of the posterior longitudinal ligament (OPLL) is a common spinal disorder that presents with or without cervical myelopathy. Furthermore, there is evidence suggesting that OPLL often coexists with cervical disc hernia (CDH), and that the latter is the more important compression factor. To raise the awareness of CDH in OPLL for spinal surgeons, we performed a retrospective study on 142 patients with radiologically proven OPLL who had received surgery between January 2004 and January 2008 in our hospital. Plain radiograph, three-dimensional computed tomography construction (3D CT), and magnetic resonance imaging (MRI) of the cervical spine were all performed. Twenty-six patients with obvious CDH (15 of segmental-type, nine of mixed-type, two of continuous-type) were selected via clinical and radiographic features, and intraoperative findings. By MRI, the most commonly involved level was C5/6, followed by C3/4, C4/5, and C6/7. The areas of greatest spinal cord compression were at the disc levels because of herniated cervical discs. Eight patients were decompressed via anterior cervical discectomy and fusion (ACDF), 13 patients via anterior cervical corpectomy and fusion (ACCF), and five patients via ACDF combined with posterior laminectomy and fusion. The outcomes were all favorable. In conclusion, surgeons should consider the potential for CDH when performing spinal cord decompression and deciding the surgical approach in patients presenting with OPLL.

Consensus statement on diagnosis and treatment of cervical ossification of posterior longitudinal ligament from Asia Pacific Spine Society (APSS) 2020.

PURPOSE: The study aimed to develop an evidence-based expert consensus statement on diagnosis and treatment of cervical ossification posterior longitudinal ligament (OPLL). METHOD: Delphi method was used to perform such survey, and the panel members from Asia Pacific Spine Society (APSS) 2020 were invited to answer the open-ended questions in rounds 1 and 2. Then the results were summarized and developed into a Likert-style questionnaire for voting in round 3, and the level of agreement was defined as 80%. In the whole process, we conducted a systematic literature search on evidence for each statement. RESULTS: Cervical OPLL can cause various degrees of neurological symptoms, an it's thought to be more common in Asia population. CT reconstruction is an important imaging examination to assist diagnosis and guide surgical choice. Segmental, continuous, mixed, and focal type is the most widely used classification system. The non-surgical treatment is recommended for patients with no or mild clinical symptoms, or irreversible neurological damage, or failed surgical decompression, or condition cannot tolerant surgery, or refusing surgery. As OPLL may continue to develop gradually, surgical treatment would be considered in their course inevitably. The surgical choice should depend on various conditions, such as involved levels, thickness, and type of OPLL, skill-experiences of surgeons, which are listed and discussed in the article. CONCLUSION: In this statement, we describe the clinical features, classifications, and diagnostic criteria of cervical OPLL, and review various surgical methods (such as their indications, complications), and provide a guideline on their choice strategy.

[Anterior decompression for the treatment of cervical spondylotic myelopathy associated with ossification of posterior longitudinal ligament].

OBJECTIVE: To study the technique and effect of anterior decompression for the treatment of cervical spondylotic myelopathy associated with ossification of posterior longitudinal ligament (OPLL). METHOD: Sixty-one patients (42 male and 19 female, 45 - 74 years with mean age of 57 years old) underwent anterior decompression for the treatment of cervical spondylotic myelopathy associated with OPLL. Among them, OPLL was definitely diagnosed in 49 patients preoperatively, and was found during the operation in the other 12 patients. The occupying rate of OPLL ranged 32%-70% with an average of 52%. The preoperative JOA scores ranged 4 - 14 points with an average of 9.6 points. In additional to conventional decompression, the ossification was removed completely after discectomy and corpectomy. RESULTS: Corpectomy was performed in 41 cases, discectomy in 6 cases and combination of corpectomy and discectomy in 14 cases. The follow-up of all patients ranged from 6 to 36 months (mean 16 months). The postoperative JOA scores ranged 8-16 points with an average of 12.8 points. The neurological improvement rate ranged from 25.0% to 87.5% with an average of 65.2%. The transient leakage of cerebrospinal fluid (CSF) occurred in 5 cases, and stopped after conservational treatment. No neurological deterioration developed. CONCLUSIONS: The difficulty and risk of anterior decompression are significantly increased in the patients with cervical spondylotic myelopathy associated with OPLL. Remove of ossification after corpectomy and discectomy could provide complete decompression and better results.

MiR-499a-5p suppresses apoptosis of human nucleus pulposus cells and degradation of their extracellular matrix by targeting SOX4.

Emerging evidence suggests that microRNAs (miRNAs, miRs) play important roles in the development of intervertebral disc degeneration (IVDD). Nonetheless, the expression level and biological function of miR-499a-5p in IVDD are still unclear. In this study, we found that miR-499a-5p was significantly downregulated in degenerative tissues of the human nucleus pulposus (NP) compared with healthy tissues. Knockdown of miR-499a-5p promoted NP cell (NPC) apoptosis, stimulated caspase activation, enhanced MMP3 and MMP13 expression, and downregulated aggrecan and type II collagen. Furthermore, TNF-α-treated NPCs showed increased apoptosis and induced an imbalance between anabolism and catabolism of the extracellular matrix (ECM); these changes were attenuated by miR-499a-5p overexpression. Research into possible mechanisms revealed that miR-499a-5p suppressed the expression of SOX4 both at mRNA and protein levels and directly bound to the 3' untranslated region of SOX4 mRNA. Ectopic expression of SOX4 attenuated the negative effect of miR-499a-5p on NPC apoptosis and the positive effect on ECM synthesis. Taken together, these results indicate that miR-499a-5p may attenuate TNF-α-induced NPC apoptosis and an imbalance between anabolism and catabolism of the ECM by downregulating SOX4.

Computer-assisted virtual operation planning in anterior controllable anterior-displacement and fusion surgery for ossification of the posterior longitudinal ligament based on actual computed tomography data.

OBJECTIVES: To investigate the effect of computer-assisted virtual operation planning (CAVOP) on anterior controllable anterior-displacement and fusion (ACAF) surgery for ossification of the posterior longitudinal ligament (OPLL). PATIENTS AND METHODS: A total of 25 patients with OPLL were enrolled in the study from September 2017 to December 2017. Preoperative Computed tomography (CT) scanning data were input into Mimics software to reconstruct three-dimensional (3D) models of actual cervical OPLL.Preoperative simulation of each surgical procedure and measurement of main parameters for intraoperative decision were conducted. Postoperative CT were used to test the clinical value of the preoperative planning. Width of vertebrae-OPLL complex (VOC), thickness of resected vertebral body (VB), height of intervertebral spacer, and length of screws and anterior plate were analyzed. RESULTS: There were no significant differences between the length of screws, width of VOC, and thickness of anterior resection of vertebrae in preoperative CT and postoperative CT. Statistical differences were found between preoperative and postoperative height of intervertebral space and length of anterior plate. CONCLUSION: A virtual ACAF surgical procedure for OPLL is feasible and useful clinically in surgical planning. It may provide a valuable tool for surgeons in formulating an appropriate surgical plan.

Morphometric Analysis of the Uncinate Process as a Landmark for Anterior Controllable Antedisplacement and Fusion Surgery: A Study of Radiologic Anatomy.

OBJECTIVE: This study used the uncinate process (UP) base as the landmark to measure the various distances of the interested anatomic structures to improve the practicality of anterior controllable antedisplacement and fusion (ACAF) for ossification of the posterior longitudinal ligament (OPLL). METHODS: Computed tomographic (CT) scan data of 20 OPLL patients were studied. We investigated the base distance of the UP, transverse foramen (TF) to UP base, pedicle to UP base, posterior to anterior UP, maximal width of OPLL, and width of the vertebrae-OPLL complex (VOC). RESULTS: Base distance of the UP shows an increasing trend from C3 to C7. The average base distance of the UP ranges from 14.6 mm at C3 to 22.7 mm at C7. The TF to UP distance ranges from 4.6 to 7.2 mm. The pedicle to UP distance is significantly shorter than upper levels at C7 and C6. The posterior to anterior UP ranges from -3.7 to -5.7 mm with an increasing trend from C3 to C7. The maximal width of OPLL is 13.2 mm on average. The mean width of the VOC in the ACAF cases is 16.8 mm on average. CONCLUSIONS: The results show that the UP can serve as a landmark for the location of longitudinal osteotomies in ACAF. However, preoperative measurement of CT images should be conducted for an individual dependent surgical planning of ACAF.

[Clinical characteristics and results of cervical spinal cord injury in the patients with ossification of the posterior longitudinal ligament].

OBJECTIVE: To investigate the clinical characteristics and results of cervical spinal cord injury (SCI) in the patients with ossification of the posterior longitudinal ligament (OPLL). METHODS: Nineteen patients with cervical SCI associated with OPLL were retrospectively analyzed. Data collection included: pre- and postoperative neurological function, OPLL-type, MRI signal changes and surgical approaches. RESULTS: Spinal cord associated with OPLL was injured severely by mild trauma. Methylprednisolone sodium succinate was used within 8 h after trauma in 12 cases. Two of them died of complications. The neurological functions were markedly improved in the other 10 cases. Seventeen cases had surgical treatment. The neurological functions (Frankel grade) were improved significantly in the operated patients except for one, who died 27 d after operation. CONCLUSIONS: The patients with OPLL are prone to have severe SCI, which directly associates with the preexisting OPLL-type and hyper-intensity signal change in the spinal cord on MRI. Both of using methylprednisolone sodium succinate administration within 8 h after trauma and surgical decompression may improve the neurological outcomes.

Upregulated expression of connexin43 in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.

STUDY DESIGN: A case-control study was conducted. OBJECTIVE: To investigate different expressions of connexin43 (Cx43) between spinal ligament fibroblasts from patients with ossification of the posterior longitudinal ligament (OPLL) and non-OPLL patients and demonstrate knockdown of Cx43 protein expression by RNA interference inhibiting expression of osteoblast-specific genes in OPLL cells. SUMMARY OF BACKGROUND DATA: The OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant cellular signaling pathways remain unclear. METHODS: Twenty patients presenting with OPLL and 18 non-OPLL patients underwent anterior decompression between January 2008 and June 2009. Specimens of the posterior longitudinal ligament were collected intraoperatively. Tissue fragment cell culture was performed. Inverted phase contrast microscopy and hematoxylin-eosin staining were used to observe cell morphology. The mouse antivimentin antibody was used to identify the cultured cells via immunocytochemistry and immunofluorescence. The messenger RNA expression of osteoblast-specific genes of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of Cx43 was detected via Western blotting. And then, after 72 hours, when RNA interference against Cx43 was performed in OPLL cells, expression of the indexes mentioned earlier was compared again between the transfection group and the nontransfection group. RESULTS: Cultivated cells were observed 7 to 10 days after cell culture. Hematoxylin-eosin staining showed fusiform and multiangular star morphologies, large and elliptical cell nuclei, and ill-defined cell appearances. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The messenger RNA expressions of OCN, ALP, and COL I and protein expressions of Cx43 from OPLL fibroblasts were greater than those from non-OPLL cells, and the difference was significant. Furthermore, knockdown of Cx43 protein expression inhibited the messenger RNA expressions of OCN, ALP, and COL I remarkably in the transfection group compared with the nontransfection group, 72 hours after RNA interference targeting Cx43 was performed in OPLL cells. CONCLUSION: Tissue fragment culture of the cervical posterior longitudinal ligament provided a successful fibroblast culture, showing good adherence and subculture. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, in which Cx43 played an important role.

Paresis associated with aconuresis caused by intervertebral disc calcification at c7-t1: a case report and review of the literature.

STUDY DESIGN: Case report. OBJECTIVE: To report a case of a 19-year-old boy with intervertebral disc calcification (IDC) at C7-T1, who presented with paresis and aconuresis. Surgical outcome was assessed. SUMMARY OF BACKGROUND DATA: IDC, commonly seen in the cervical spine region in children, is well-known as a self-limiting disorder with no or little symptoms. Surgical intervention is usually not required. METHODS: A 19-year-old boy presented with acute back pain, progressive numbness, and weakness of both lower extremities and aconuresis for 1 week. There was no traumatic history or signs of infection. Radiograph, computed tomography with reconstruction, and magnetic resonance imaging revealed C7-T1 IDC with severe spinal cord compression. Decompression with anterior cervical corpectomy and fusion was performed. RESULTS: Follow-up showed complete resolution of the back pain and complete recovery of motor power and sensory function in both lower extremities and return of normal micturition function. The patient had full recovery with no complications. CONCLUSION: Serious neurologic deficit, especially a bladder dysfunction, caused by calcified intervertebral disc is rare. However, favorable outcome can be achieved in those cases where rapid diagnosis is made and followed by spinal cord decompression.