Tsung-Ting Tsai

Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL-1β and p38 MAPK signal.

Nucleus pulposus cells (NPCs) from degenerating disks produce catabolic and inflammatory factors, including interleukin (IL)‐1, nitric oxide (NO), prostaglandin E2 (PGE‐2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disk. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time quantitative polymerase chain reaction (Q‐PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL‐1β, NO, PGE‐2, MMP‐3, and TIMP‐1 in the conditioned media were quantified by enzyme‐linked immunosorbent assay (ELISA). Our data showed that HBO treatment decreased expression of IL‐1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE‐2, and MMP‐3, and increased TIMP‐1 expression in NPCs as compared with the atmospheric treatment. These results support the hypothesis that IL‐1β and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disk degeneration, and support our proposal that HBO treatment‐induced increase of the anabolic factor (TIMP‐1)/catabolic factor (MMP‐3) ratio may provide a therapeutic approach to slow the course of intervertebral disk degeneration.

Advanced glycation end products in degenerative nucleus pulposus with diabetes

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100u2009µg/ml) for 3 days, and mRNA expressions of MMP‐2 and RAGE were measured by real‐time RT‐PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP‐2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP‐2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration.

Hyperbaric oxygen treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells

Nucleus pulposus cells (NPCs) from degenerating discs produce catabolic and inflammatory factors, including interleukin (IL)‐1 and nitric oxide (NO). Enhanced production of NO has been implicated in the apoptosis of degenerating disc cells. This study evaluates the effects of hyperbaric oxygen (HBO) on degenerated human NPCs. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA). Phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) in NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time polymerase chain reaction (PCR) analysis of aggrecan and type II collagen gene expression. The levels of IL‐1β and NO were quantified by enzyme‐linked immunosorbent assay (ELISA). To identify the HBO‐induced anti‐apoptotic pathways, expression of Bcl‐2 and Bax proteins as well as activation of cysteine‐containing aspartate‐specific proteases (caspases) 3, 8, and 9 was evaluated using Western blotting after HBO treatment. Our data showed that HBO treatment decreased the expression of IL‐1β, suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, decreased synthesis of NO, and increased the gene expression of aggrecan and type II collagen in NPCs as compared with the atmospheric treatment. HBO up‐regulated the ratio of Bcl‐2 to Bax expression and reduced the activity of caspases 9 and 3 but not of caspase 8, indicating a selective effect over the mitochondrial apoptosis pathway in degenerated NPCs. These results support our hypothesis that HBO treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells.

Increased periostin gene expression in degenerative intervertebral disc cells.

BACKGROUND CONTEXTnDisc degeneration is a multifactorial disease that may cause clinical symptoms such as chronic back pain or radiculopathy in the extremities. Periostin, an extracellular matrix protein involved in the process of fibrosis, expressed in tissues subjected to mechanical stress such as intervertebral disc. However, the expression of periostin during disc degeneration has not yet been studied.nnnPURPOSEnThe aim of this study is to elucidate the difference in gene expression profiles between degenerative and nondegenerative intervertebral discs for a better understanding of disc degeneration.nnnSTUDY DESIGNnDegenerative and nondegenerative nucleus pulposus cells were isolated from elderly patients with degenerative disc disease and younger patients with adolescent idiopathic scoliosis, respectively.nnnMETHODSnAffymetrix GeneChip Human arrays were used to derive gene expression profiles for disc degeneration, and gene expressions of periostin and other degeneration-related markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analysis. Immunohistochemical analysis of periostin and Gomori trichrome stain was performed to show the relationship of periostin, fibrosis, and disc degeneration. The mechanical stress experiment was designed to demonstrate the relationship of periostin, stress, and disc degeneration.nnnRESULTSnFourteen genes were identified to express at significantly different levels between degenerative and nondegenerative groups. An increase of periostin gene expression was observed in human degenerative nucleus pulposus cells for the messenger RNA and protein levels. Histological examination demonstrated an increased positive staining of periostin in degenerative discs from human tissues and rat needle-punctured tails and more fibrosis with architectural disorder and fragmentation in human degenerative disc as compared with nondegenerative discs. The expression of periostin was significantly induced by stress in human degenerative nucleus pulposus cells but not in nondegenerative cells.nnnCONCLUSIONSnThis study demonstrates for the first time an upregulation of periostin in addition to the expression levels of Type I collagen and matrix metalloproteinase-2 in human disc degeneration. It suggests that periostin may be a candidate gene that shows promise as a new prognostic marker and a therapeutic target that is worth further study to expand our knowledge of its role in disc degeneration.

Increased sulfatase 1 gene expression in degenerative intervertebral disc cells

Sulfatase 1 (SULF1) plays a key role in cell signaling involving in cell growth, differentiation, proliferation, and migration. Abnormal SULF1 expression has been implicated in the development of various cancers and diseases of the skeletal and nervous systems. The present study aims to examine the difference in SULF1 expression between degenerative and non‐degenerative intervertebral discs (IVDs) to provide an enhanced understanding of disc degeneration. Degenerative and non‐degenerative disc tissues were surgically harvested from patients and experimental rats. Disc degeneration‐specific genes were identified by microarray analysis. The gene expression of SULF1 was measured by sulfatase assay, reverse transcription‐polymerase chain reaction (RT‐PCR), real‐time RT‐PCR, and western blotting. Also, the presence of SULF1 in human and rat discs was confirmed by immunohistochemistry. More specifically in human cells, an increase of SULF1 gene expression was observed in degenerative cells at both mRNA and protein levels, as well as in time‐ and dose‐dependent manner in response to TNF‐α treatment. Increased staining of SULF1 was detected in degenerative discs compared to non‐degenerative discs for humans and rats. These findings show an upregulation of SULF1 in degenerative discs for the first time, and suggest that there is a link between SULF1 and disc degeneration.

Unplanned revision spinal surgery within a week: a retrospective analysis of surgical causes

BackgroundThe need for revision surgery after a spinal surgery can cause a variety of problems, including reduced quality of life for the patient, additional medical expenses, and patient-physician conflicts. The purpose of this study was to evaluate the causes of unplanned revision spinal surgery within a week after the initial surgery in order to identify the surgical issues most commonly associated with unplanned revision surgery.MethodsWe retrospectively reviewed the medical records of all patients at who received a spinal surgery at a regional medical center from July 2004 to April 2011 in order to identify those who required a revision surgery within one week of their initial surgery. Patients were excluded if they received a vertebroplasty, kyphoplasty, or nerve block surgery, because those surgeries are one-day surgeries that do not require hospital admission. In addition, patients with a primary diagnosis of wound infection were also excluded since reoperations for infection control can be expected.ResultsThe overall incidence of unplanned revision spinal surgery during the time period covered by this review was 1.12xa0% (116/10,350 patients). The most common surgical causes of reoperation were screw malposition (41 patients), symptomatic epidural hematoma (27 patients), and inadequate decompression (37 patients). Screw malposition was the most common complication, with an incidence rate of 0.82xa0%. Screw instrumentation was significantly associated with revision surgery (pu2009=u20090.023), which suggests that this procedure carried a greater risk of requiring revision. The mean time interval to reoperation for epidural hematomas was significantly shorter than the intervals for other causes of revision spinal surgery (pu2009<u20090.001), which suggests that epidural hematoma was more emergent than other complications. Also, 25.93xa0% of patients who underwent hematoma removal experienced residual sequelae; this percentage was significantly higher than for other surgical causes of revision spinal surgery (pu2009=u20090.013).ConclusionsThe results suggest that to avoid the need for reoperation, screw malposition, inadequate decompression, and epidural hematoma are the key surgical complications to be guarded against. Accordingly, adequate decompression, epidural hematoma prevention, and proper pedicle screw placement may help reduce the incidence of revision surgery.

Correlation of blood bone turnover biomarkers and Wnt signaling antagonists with AS, DISH, OPLL, and OYL

BackgroundWnt signaling plays an important role in development and maintenance of many organs and tissues. The most-studied secreted Wnt inhibitors are sclerostin (SOST), Dickkopf-related protein 1 (DKK-1), and secreted frizzled related protein 1 (SFRP-1) which play important roles in bone turnover. The present study investigated the relationship between serum Wnt inhibitors and diseases with excessive ossification structures, such as ossification of posterior longitudinal ligament (OPLL), ankylosing spondylitis (AS), diffuse idiopathic skeletal hyperostosis (DISH), and ossification of yellow ligament (OYL).MethodsTwenty-five patients with AS, DISH, OPLL, or OYL were recruited in this study. Fasting peripheral blood samples were collected from all patients and nine controls. Various biomarkers of bone turnover including osteocalcin (OSC), osteoprotegerin (OPG), SFRP-1, DKK-1, and SOST were investigated.ResultsOur data showed that serum levels of OSC were higher, but Dkk-1 levels were lower in AS, DISH, OPLL, and OYL patients than those in the controls. Serum levels of SFRP-1 were significantly higher in DISH patients than those in the controls. Serum levels of SOST were significantly higher in DISH and OPLL patients than both levels in the controls. Serum levels of OPG were lower in AS patients than those in the controls. Serum levels of OSC were higher in the OPLL patients than those in the AS patients. Serum levels of DKK-1, SFRP-1, SOST, and OPG were not significantly different between the different disease groups.ConclusionsIn this exploratory study, both OSC and DKK-1 levels are correlated with the clinical conditions associated with excessive ossification, indicating that blood OSC and DKK-1 levels may serve as diagnostic biomarkers for AS, DISH, OPLL, and OYL. These findings may also help discover potential drug therapies for management of these diseases in the future.

Transforaminal interbody debridement and fusion to manage post-discectomy discitis in lumbar spine

BACKGROUNDnTraditionally, nonoperative management with long-term antibiotics and bed rest has been recommended as first-line treatment for most patients with postoperative discitis. A recent trend in treatment under a limited range of indications has been to perform surgical débridement followed by long-term administration of antibiotics. This descriptive study investigated whether transforaminal lumbar interbody débridement and fusion (TLIDF) combined with intravenous antibiotics is appropriate to manage postdiscectomy discitis.nnnMETHODSnThis study retrospectively analyzed demographic data, laboratory data, and radiography and magnetic resonance imaging of 10 patients with postoperative discitis who underwent surgical TLIDF followed by antibiotic treatment. Preoperative and postoperative spine sagittal alignment, visual analog scale scores, and Kirkaldy-Willis criteria for functional outcomes were evaluated.nnnRESULTSnAn infection clearance rate of 100% was ultimately achieved for the patients who underwent TLIDF with short posterior instrumentation. TLIDF yielded better outcomes than traditional conservative treatment in terms of spine alignment correction, functional outcomes, and quality of life.nnnCONCLUSIONSnBased on previously reported data and the findings of this study, we suggest that surgical intervention should be used in certain cases, as it can achieve better outcomes than conservative treatment. We recommend a novel single posterior approach with TLIDF and posterior pedicle screw instrumentation for management of postdiscectomy discitis.

BMP-2 gene transfection of bone marrow stromal cells to induce osteoblastic differentiation in a rat calvarial defect model

Gene therapy for bone tissue engineering has been widely developed. Recently, non-viral DNA-based gene therapy has been reported to be a safer and more efficient method of delivering DNA into target cells. We used a non-viral gene transfection reagent to delivery bone morphogenetic protein-2 (BMP-2) gene into bone marrow stromal cells (BMSCs). Primary BMSCs were isolated from rat femurs and transfected with BMP-2 plasmids. The transfection rate was analyzed using flow cytometry. The concentration of BMP-2 protein was quantified using an enzyme-linked immunosorbent assay. Levels of osteopontin and osteocalcin were measured to evaluate osteogenic differentiation. In vivo, we designed a critical-size calvarial defect rat model to study new bone regeneration, using Matrigel as a scaffold to carry BMP-2-transfected bone marrow stromal cells into the defect site. New bone formation was assessed by micro-computed tomography, X-ray, immunohistochemical staining and histomophometry. The transfection rate after 72u202fh was 31.5%. The BMP-2 protein level as well as osteopontin and osteocalcin expressions were higher in the experimental group (transfected with BMP-2) than the control group (transfected with green fluorescent protein, GFP). The in vivo study suggested that bone healing occurred 12u202fweeks after scaffold implantation. In addition, BMP-2-transfected bone marrow stromal cells provided better osteogenic differentiation than primary bone marrow stromal cells. Our findings suggest that non-viral gene therapy may be useful in bone tissue engineering.nnnSIGNIFICANCEnThe study has clinical implications for the wider use of BMP-2-transfected BMSCs for cell-based transplantation therapy in bone regeneration.

Comparison of the efficacy of hyperbaric oxygen therapy in patients with postoperative spinal and extremity wound lnfections

Background: Hyperbaric oxygen (HBO) has been shown to promote the healing of ischemic and infected tissue; therefore, it may be useful for treating non-healing postoperative wound infection. The purpose of this study was to compare the efficacy of HBO therapy in the treatment of postoperative spinal and extremity wound infections. Methods: From 1995 to 2011, 284 patients with postoperative wound infection were enrolled in this study and divided into 2 groups: groups I, 26 patients (spinal wound infection) and group II, 235 patients (upper and lower extremities wound infection). HBO therapy was indicated when infection showed no improvement of clinical symptoms/signs and laboratory testing following appropriate antibiotic therapy for at least 2 weeks. All patients received HBO therapy one cycle (20 times) as an adjunctive treatment, and the following data were collected retrospectively: age, sex, comorbidity (diabetes mellitus, liver cirrhosis, and end-stage renal disease), and type of bacteria (gram-positive, gram-negative, and anaerobic). These clinical results and outcomes after HBO therapy were analyzed Results: Sex and diabetes mellitus incidence were significantly different between the 2 groups; the extremities infection group had more male and fewer diabetic patients. No significant difference between the groups was found in terms of other factors, comorbidity, or type of bacteria. Although the overall clinical outcome after HBO treatment was not significantly different between the groups, the spinal group showed a higher percentage of excellent results after infection with a gram-positive pathogen. Conclusion: Patients with post-operative spinal wound infection under HBO therapy have similar treatment responses to patients with the extremities wound infection. Clinical outcomes are not affected by comorbidities such as diabetes mellitus, liver cirrhosis, or end-stage renal disease. Therefore, HBO therapy is recommended as an adjunctive treatment for postoperative spinal wound infection.