Bacteria detected after instrumentation surgery for pyogenic vertebral osteomyelitis in a canine model.

OBJECTIVE: This study was designed to identify the presence, type and origin of bacteria adjacent to the metal implant in the infected region in a canine model of pyogenic vertebral osteomyelitis treated with single-stage anterior autogenous bone grafting and instrumentation. METHODS: Dogs with pyogenic spondylodiscitis underwent one-stage debridement, autogenous bone grafting and titanium plate instrumentation and perioperative antibiotic therapy. The implants and adjacent vertebral bones were removed surgically at various postoperative time points (4, 8, 12 and 24 weeks) for bacteria detection. Bacteria were detected from retrieved spinal implants as well as surrounding bones by culture and/or pyrosequencing methods in 17 (85%) of all 20 animals. The positive rate for bacteria presence was 45% by culture and 80% by pyrosequencing method. RESULTS: Radiological or macroscopic examination showed no signs for infection recurrence in any animal regardless of bacteria presence at the surgical site. However, organism identical with the causative bacterium for spinal infection was found in only two of nine culture-positive animals. CONCLUSION: Within the confines of the study, the use of metallic implants in an infected area did not lead to a clinically relevant infection although bacteria may exist at the surgical site. The use of metallic implants in an infected area of the spine is safe. The metallic implants may not be the "culprit" for the persistence or recurrence of infection.

AO spine injury classification system: a revision proposal for the thoracic and lumbar spine.

PURPOSE: The AO Spine Classification Group was established to propose a revised AO spine injury classification system. This paper provides details on the rationale, methodology, and results of the initial stage of the revision process for injuries of the thoracic and lumbar (TL) spine. METHODS: In a structured, iterative process involving five experienced spine trauma surgeons from various parts of the world, consecutive cases with TL injuries were classified independently by members of the classification group, and analyzed for classification reliability using the Kappa coefficient (κ) and for accuracy using latent class analysis. The reasons for disagreements were examined systematically during review meetings. In four successive sessions, the system was revised until consensus and sufficient reproducibility were achieved. RESULTS: The TL spine injury system is based on three main injury categories adapted from the original Magerl AO concept: A (compression), B (tension band), and C (displacement) type injuries. Type-A injuries include four subtypes (wedge-impaction/split-pincer/incomplete burst/complete burst); B-type injuries are divided between purely osseous and osseo-ligamentous disruptions; and C-type injuries are further categorized into three subtypes (hyperextension/translation/separation). There is no subgroup division. The reliability of injury types (A, B, C) was good (κ = 0.77). The surgeons' pairwise Kappa ranged from 0.69 to 0.90. Kappa coefficients κ for reliability of injury subtypes ranged from 0.26 to 0.78. CONCLUSIONS: The proposed TL spine injury system is based on clinically relevant parameters. Final evaluation data showed reasonable reliability and accuracy. Further validation of the proposed revised AO Classification requires follow-up evaluation sessions and documentation by more surgeons from different countries and backgrounds and is subject to modification based on clinical parameters during subsequent phases.

Stage specific effect of leptin on the expressions of estrogen receptor and extracellular matrix in a model of chondrocyte differentiation.

Endochondral ossification is a dynamic process. The interaction between leptin and estrogen in this process is complicated. Whether there is a stage specific crosstalk between leptin and estrogen in the differentiation process of the chondrocytes in the growth plate remains unknown. The aim of our study was to investigate the effect of leptin on the expression of estrogen receptors and extracellular matrix in ATDC5 cells, an in vitro model of endochondral ossification. First, we quantified the physiological expressions of estrogen receptors α, ß (ERα, ERß), leptin receptor (Ob-Rb), type II and type X collagens in definite stages of endochondral ossification in ATDC5 cells using real-time PCR. Dynamic and stage specific expression characteristics of these target genes were observed. Simultaneous expressions of Ob-Rb with ERα or ERß in ATDC5 cells were also found with dual-label confocal immunofluorescency. Then using Western blotting analysis and/or real-time PCR, we detected that, leptin treatment up-regulated the expressions of ERα, ERß and type II collagen, but down-regulated type X collagen expression and the ERα/ERß ratio in the chondrogenic differentiation stage. Meanwhile, leptin down-regulated the expressions of ERα, type II and type X collagens, and the ERα/ERß ratio, but up-regulated the expression of ERß in the hypertrophic differentiation stage. Significant positive correlation existed between ERα and type II collagen expression, and between the ratio of ERα/ERß and type X collagen production. In summary, the crosstalk between leptin and estrogen receptor might be differentiation stage specific in ATDC5 cells.

Difference in subchondral cancellous bone between postmenopausal women with hip osteoarthritis and osteoporotic fracture: implication for fatigue microdamage, bone microarchitecture, and biomechanical properties.

OBJECTIVE: Osteoarthritis (OA) and osteoporosis (OP) of the hip rarely occur in the same patient. The purpose of this study was to determine whether this difference might be attributable to the different quantity and quality of subchondral cancellous bone in the two conditions. METHODS: Subchondral cancellous bone from the femoral head was obtained at the time of hip arthroplasty from 60 postmenopausal women, 30 with OA and 30 with OP. In each group, 10 specimens were subjected to compressive fatigue loading and 20 were left nonloaded. Specimens were examined by compressive mechanical testing, micro-computed tomography scanning, fluorescence microscopy, and nanoindentation techniques. RESULTS: Both the ultimate stress and the elastic modulus of cancellous bone from OA patients were significantly higher than those of cancellous bone from OP patients (P < 0.05). Compared to cancellous bone from OP patients, the bone volume fraction and trabecular thickness were significantly increased, but bone matrix mineralization was significantly decreased, in cancellous bone from OA patients (P < 0.05 for each comparison). The microcrack density was significantly higher in OP cancellous bone than in OA cancellous bone (P < 0.001), irrespective of fatigue loading. In addition, fatigue loading resulted in a significant increase in microcrack density in both OA and OP cancellous bone (P < 0.001). There was no significant difference in nanoindentation elastic modulus and hardness between cancellous bone from OA and OP patients, as well as between bones with and without fatigue loading. CONCLUSION: The difference in mechanical properties between OA and OP cancellous bone is attributed to different bone mass and bone structure. OP cancellous bone is susceptible to fatigue damage due to insufficient structure. However, increased bone volume and plate-like structure provide OA cancellous bone a superior capacity to resist fatigue damage.

Primary Aspergillus spondylodiscitis in a liver transplant recipient.

Solid organ transplant recipients are at increased risk for Aspergillus infections. However, the cases of Aspergillus spondylodiscitis are rare and mostly resulted from the hematogenous spread of invasive pulmonary Aspergillosis. Here, we report a case of primary spondylodiscitis in a liver transplant recipient. Six months after transplantation, a chronic and progressive lumbar back pain was presented. The patient had no fever and the white blood cell count was normal. High plasma (1→3)-beta-d-glucan (BDG) level was detected at the time of back pain. The pathogen was Aspergillus flavus. Clinical and radiological healing was achieved through posterior only debridement and voriconazole therapy.

Principles of management of thoracolumbar fractures.

There is little consensus on treatment of thoracolumbar fractures, which are one of the most controversial areas in spine surgery. The great variations in clinical decision making may come from differences in evaluation of spine stability with these fractures. Few high-quality studies concerning optimal treatment of thoracolumbar fractures have been conducted. This article reviews the conflicting results and recommendations for management of thoracolumbar fractures of currently published reports. Specifically, it addresses issues regarding evaluation of stability, indications for operative treatment, timing of surgery, surgical approach, and fusion length.

Estrogen stimulates leptin receptor expression in ATDC5 cells via the estrogen receptor and extracellular signal-regulated kinase pathways.

Regulation of the physiological processes of endochondral bone formation during long bone growth is controlled by various factors including the hormones estrogen and leptin. The effects of estrogen are mediated not only through the direct activity of estrogen receptors (ERs) but also through cross talk with other signaling systems implicated in chondrogenesis. The receptors of both estrogen and leptin (OBR (LEPR)) are detectable in growth plate chondrocytes of all zones. In this study, the expression of mRNA and protein of OBR in chondrogenic ATDC5 cells and the effect of 17ß-estradiol (E(2)) stimulation were assessed using quantitative PCR and western blotting. We have found that the mRNA of Obr was dynamically expressed during the differentiation of ATDC5 cells over 21 days. Application of E(2) (10(-7) M) at day 14 for 48 h significantly upregulated OBR mRNA and protein levels (P<0.05). The upregulation of Obr mRNA by E(2) was shown to take place in a concentration-dependent manner, with a concentration of 10(-7) M E(2) having the greatest effect. Furthermore, we have confirmed that E(2) affected the phosphorylation of ERK1/2 (MAPK1/MAPK3) in a time-dependent manner where a maximal fourfold change was observed at 10 min following application of E(2). Finally, pretreatment of the cells with either U0126 (ERK1/2 inhibitor) or ICI 182 780 (ER antagonist) blocked the upregulation of OBR by E(2) and prevented the E(2)-induced phosphorylation of ERK. These data demonstrate, for the first time, the existence of cross talk between estrogen and OBR in the regulation of bone growth whereby estrogen regulates the expression of Obr in growth plate chondrocytes via ERs and the activation of ERK1/2 signaling pathways.

Reliability of the measurement of thoracolumbar burst fracture kyphosis with Cobb angle, Gardner angle, and sagittal index.

INTRODUCTION: Kyphotic deformity plays a key role in our evaluation of patients with thoracolumbar burst fracture, and there are several variables available for kyphotic deformity assessment, including Cobb angle, Gardner angle, and sagittal index. However, it remains unknown about intra- and inter-observer variability of sagittal index. AIM: The purpose of this study is to determine the reliability of the measurement for thoracolumbar burst fracture kyphosis using Cobb angle, Gardner angle, and sagittal index. Thirty-five patients with thorocolumbar burst fractures treated in our institute were identified. The lateral spine radiographs and midsagittal CT images of the 35 patients were measured on two separate occasions, in random order, by five attending spine surgeons using Cobb angle, Gardner angle, and sagittal index. METHOD: We statistically assessed the agreement, the intra-observer and the inter-observer reliability of the three methods. The intra-observer reliability is always better than the inter-observer reliability, regardless of the parameter being measured or the imaging modality. RESULTS: Intra-class correlation coefficients (ICC) were the most consistent for Cobb angle, followed by Gardner angle and sagittal index. Midsagittal CT images had better intra- and inter-observer reliabilities than lateral plain radiography. Also, agreement was better using midsagittal CT images as compared with lateral plain radiography. CONCLUSION: The results of our study suggest that Cobb angle is the most consistent in terms of intra- and inter-observer reliabilities in the assessment of thoracolumbar burst fracture kyphosis.

Gender- and region-specific variations of estrogen receptor α and ß expression in the growth plate of spine and limb during development and adulthood.

Although estrogen action is indispensable for normal bone growth in both genders, the roles of estrogen receptors (ERs) in mediating bone growth are not fully understood. The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and ß in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and ß expression were shown in the growth plates. Mainly nuclear staining of ERα and ß immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. Moreover, our study indicated significant effect of gender on temporal ERα and ß expression and of region on temporal ERα/ERß expression ratio. However, spatial differences of region-related ERα and ß expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and ß immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males. Gender- and region-specific expression patterns of ERα and ß gene might be one possible reason for differences in sex- and region-related body growth phenotypes. Gender, age and region differences should be taken into consideration when the roles of ERs in the growth plate are investigated.

Leptin regulates estrogen receptor gene expression in ATDC5 cells through the extracellular signal regulated kinase signaling pathway.

Both estrogen and leptin play an important role in the regulation of physiological processes of endochondral bone formation in linear growth. Estrogen receptors (ERα and ERß) are known as members of the superfamily of nuclear steroid hormone receptors and are detected in all zones of growth plate chondrocytes. They can be regulated in a ligand-independent manner. Whether leptin regulates ERs in the growth plate is still not clear. To explore this issue, chondrogenic ATDC5 cells were used in the present study. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting. We found that both ERα and ERß were dynamically expressed during the ATDC5 cell differentiation for 21 days. Leptin (50 ng/ml) significantly upregulated ERα and ERß mRNA and protein levels 48 h after leptin stimulation (P<0.05) at day 14. The up-regulation of ERα and ERß mRNA by leptin was shown in a dose-dependent manner, but the most effective dose of leptin was different (100 and 1,000 ng/ml, respectively). Furthermore, we confirmed that leptin augmented the phosphorylation of ERK1/2 in a time-dependent manner. A maximum eightfold change was observed at 15 min. Finally, a specific ERK1/2 inhibitor, UO126, blocked leptin-induced ERs regulation in ATDC5 cells, indicating that ERK1/2 mediates, partly, the effects of leptin on ERs. These data demonstrate, for the first time, that leptin regulates the expression of ERs in growth plate chondrocytes via ERK signaling pathway, thereby suggesting a crosstalk between leptin and estrogen receptors in the regulation of bone formation.

Cage subsidence does not, but cervical lordosis improvement does affect the long-term results of anterior cervical fusion with stand-alone cage for degenerative cervical disc disease: a retrospective study.

OBJECTIVE: Clinical outcomes of the stand-alone cage have been encouraging when used in anterior cervical discectomy and fusion (ACDF), but concerns remain regarding its complications, especially cage subsidence. This retrospective study was undertaken to investigate the long-term radiological and clinical outcomes of the stand-alone titanium cage and to evaluate the incidence of cage subsidence in relation to the clinical outcome in the surgical treatment of degenerative cervical disc disease. METHODS: A total of 57 consecutive patients (68 levels) who underwent ACDF using a titanium box cage for the treatment of cervical radiculopathy and/or myelopathy were reviewed for the radiological and clinical outcomes. They were followed for at least 5 years. Radiographs were obtained before and after surgery, 3 months postoperatively, and at the final follow-up to determine the presence of fusion and cage subsidence. The Cobb angle of C2-C7 and the vertebral bodies adjacent to the treated disc were measured to evaluate the cervical sagittal alignment and local lordosis. The disc height was measured as well. The clinical outcomes were evaluated using the Japanese Orthopaedic Association (JOA) score for cervical myelopathy, before and after surgery, and at the final follow-up. The recovery rate of JOA score was also calculated. The Visual Analogue Scale (VAS) score of neck and radicular pain were evaluated as well. The fusion rate was 95.6% (65/68) 3 months after surgery. RESULTS: Successful bone fusion was achieved in all patients at the final follow-up. Cage subsidence occurred in 13 cages (19.1%) at 3-month follow-up; however, there was no relation between fusion and cage subsidence. Cervical and local lordosis improved after surgery, with the improvement preserved at the final follow-up. The preoperative disc height of both subsidence and non-subsidence patients was similar; however, postoperative posterior disc height (PDH) of subsidence group was significantly greater than of non-subsidence group. Significant improvement of the JOA score was noted immediately after surgery and at the final follow-up. There was no significant difference of the recovery rate of JOA score between subsidence and non-subsidence groups. The recovery rate of JOA score was significantly related to the improvement of the C2-C7 Cobb angle. The VAS score regarding neck and radicular pain was significantly improved after surgery and at the final follow-up. There was no significant difference of the neck and radicular pain between both subsidence and non-subsidence groups. CONCLUSIONS: The results suggest that the clinical and radiological outcomes of the stand-alone titanium box cage for the surgical treatment of one- or two-level degenerative cervical disc disease are satisfactory. Cage subsidence does not exert significant impact upon the long-term clinical outcome although it is common for the stand-alone cages. The cervical lordosis may be more important for the long-term clinical outcome than cage subsidence.

Anterior cervical discectomy and fusion versus anterior cervical corpectomy and fusion for multilevel cervical spondylosis: a systematic review.

INTRODUCTION: There is considerable controversy as to which technique is best option for reconstruction after multilevel anterior decompression for cervical spondylosis. The aim of this study was to compare the clinical and radiographic results and complications of anterior cervical discectomy fusion (ACDF) and anterior cervical corpectomy fusion (ACCF) in the treatment of multi-level cervical spondylosis. METHOD: We reviewed and analyzed papers published from Jan 1969 to Dec 2010 regarding the comparison of ACDF and ACCF for multilevel cervical spondylosis. Statistical comparisons were made when appropriate. RESULTS: Twelve studies were included in this systematic review. Blood loss was greater for ACCF compared with ACDF. Similarly, the rate of graft dislodgement in ACCF was higher than that in ACDF. Nonunion rates were 18.4% for 2-level ACDF and 37.3% for 3-level ACDF, whereas nonfusion rates were 5.1% for single-level ACCF and 15.2% for 2-level ACCF. In addition, nonunion rates for three disc levels fused were much higher than that for two disc levels fused, regardless of discectomy or corpectomy. Clinical outcome was compared between ACDF and ACCF in nine studies. Of these, similar outcome was found between ACDF and ACCF in six studies, whereas three studies reported better outcome in ACCF compared with ACDF. CONCLUSION: Nonunion rates of ACDF are higher than those of ACCF for multilevel cervical spondylosis. Sometimes, clinical outcome of ACCF was better than ACDF for multilevel cervical spondylosis.

A systematic review of complications in cervical spine surgery for ossification of the posterior longitudinal ligament.

BACKGROUND CONTEXT: Surgical management of ossification of the posterior longitudinal ligament (OPLL) is associated with complications. However, surgical complications for OPLL have not been clearly documented. PURPOSE: To review and summarize the incidence of surgical complications of OPLL in the cervical spine and evaluate the impact of surgical approach (anterior or posterior), year of study publication, follow-up duration, and the surgical outcome on complication incidence. STUDY DESIGN: Systematic review. METHODS: An English literature review was conducted especially on surgical complications of cervical OPLL. The incidence of complications was statistically summarized, with its correlation to surgical approaches, year of study publications, follow-up duration, and the surgical outcome was statistically evaluated. RESULTS: Twenty-seven retrospective studies, including a total of 1,558 patients, were reviewed. The overall incidence of surgical complications of cervical OPLL was 21.8%. Neurologic deficit (8.3%, overall rate), cerebrospinal fluid leakage (CFL) (5.1%), axial pain (3.5%), and implant complications (3.5%) were relatively common. The incidence of complications for posterior approach was not statistically different from those for anterior procedures. However, with regard to individual complication, C5 palsy and axial pain occurred more frequently in patients approached posteriorly, whereas CFL, implant complications, hoarseness, dysphagia, and dyspnea appeared more common in anterior cases. CONCLUSIONS: There is a relatively high incidence of surgical complications for cervical OPLL compared with other cervical degeneration diseases. It is, therefore, necessary for surgeons to take into consideration the risk of surgical complications when communicating with patients for decision making and to alert complications during or after surgical procedures.

Autophagy in rat annulus fibrosus cells: evidence and possible implications.

INTRODUCTION: Programmed cell death of intervertebral disc (IVD) cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented. The current study was designed to investigate the effect of interleukin (IL)-1ß on the occurrence of autophagy of rat annulus fibrosus (AF) cells and the interrelationship between autophagy and apoptosis. METHODS: Rat AF cells were isolated and exposed, in tissue cultures with or without serum, to IL-1ß in different concentrations for 24 hours. Ultrastructural analysis, flow cytometry and lysosomal activity assessment were performed after the in vitro treatment to determine the presence and levels of autophagy. The mRNA expression of autophagy-related proteins (Beclin-1, Bcl-2 and microtubule associated protein 1 light chain 3 (LC3)) were evaluated using real-time PCR. 3-methyladenine (3-MA), a PI3K inhibitor, was used to determine the interaction between autophagy and apoptosis via the suppression of autophagy. RESULTS: Autophagy was detected in rat AF cells under serum starvation condition by transmission electron microscopy. PCR and flow cytometry results showed that IL-1ß enhanced the autophagy-induction effect of serum deprivation in a dose-dependent manner. However, IL-1ß alone failed to induce autophagy in AF cells cultured without serum starvation. When autophagy was suppressed by 3-MA, the apoptosis incidence was increased. Serum supplement also partly reversed the autophagy incidence without affecting the apoptosis incidence in the same cells. CONCLUSIONS: IL-1ß up-regulates serum deprivation-induced autophagy of AF cells in a dose-dependent manner. Autophagy may represent a protective mechanism against apoptosis in AF cells and IVD degeneration.

Lentiviral shRNA silencing of CHOP inhibits apoptosis induced by cyclic stretch in rat annular cells and attenuates disc degeneration in the rats.

The expression of CHOP (C/EBP homologous protein), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress induced by cyclic stretch and leads to rat AF cells apoptosis. However, whether the suppression of CHOP can inhibit apoptosis and attenuates disc degeneration by cyclic stretch remains unclear. The aim of this study was to evaluate the suppressive effects of lentiviral CHOP shRNA on apoptosis induced by cyclic stretch in rat annulus fibrosus (AF) cells in vitro and disc degeneration of rat lumber spine in vivo. Lentiviral CHOP shRNA was constructed and introduced into AF cells. After stretched by the Flexcell Tension Plus system with 20% elongation for 36 h, silencing of the CHOP gene was identified by RT-PCR and Western blot. Inhibition of apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining. The effect of CHOP shRNA on disc degeneration was determined in vivo by using a rat model. At 7 weeks after intradiscal injection of the control or CHOP shRNA in the L4/L5 and L5/L6 discs, disc degeneration was assessed by X-ray examination, magnetic resonance imaging (MRI) assessment, and HE and TUNEL staining. A significant decrease in CHOP mRNA and protein expression was detected in AF cells with CHOP shRNA transfection after 36 h stretch. There was a significant decrease in apoptotic incidence in cells treated with CHOP shRNA, which was parallel to the expression of CHOP. Injection of CHOP shRNA in vivo resulted in the improvement in MRI and histologic score, and decrease in the apoptosis in the disc. No significant change in disc height was observed. In conclusion, a novel lentiviral vector expressing CHOP shRNA efficiently inhibits apoptosis in rat AF cells by silencing CHOP expression. In a rat model, intradiscal injection of CHOP shRNA induces the suppression of disc degeneration. The therapeutic effects of lentiviral CHOP shRNA should be further explored.

Age-related variations of leptin receptor expression in the growth plate of spine and limb: gender- and region-specific changes.

Leptin is a potent growth-stimulating factor of bone. The effects of leptin on bone growth differ significantly between axial and appendicular regions. Gender differences of leptin function have also been suggested in normal pubertal development. To explore the mechanisms underlying these effects, we investigated the spatial and temporal expressions of the active form of the leptin receptor (Ob-Rb) in the tibial and spinal growth plates of the female and male rats during postnatal development. The 1-, 4-, 7-, 12- and 16-week age stages are representative for early life, puberty and early adulthood after puberty, respectively. Quantitative real-time PCR was used for Ob-Rb mRNA examination and comparison. The spatial location of Ob-Rb was determined by immunohistochemical analysis. There were gender- and region-specific differences in Ob-Rb mRNA expression in the growth plate. Mainly cytoplasm staining of Ob-Rb immunoreactivity was observed in the spinal and tibial growth plate chondrocytes of both genders. Spatial differences of region- and gender-related Ob-Rb expression were not observed. Ob-Rb immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in early life stage and during puberty. After puberty, staining was mainly located in the late proliferative and hypertrophic chondrocytes. The results of Ob-Rb HSCORE analysis were similar to those obtained from quantitative real-time PCR. Our study indicated direct effects on the chondrocytes of the growth plate in different development stages. The region-specific expression patterns of Ob-Rb gene might be one possible reason for contrasting phenotypes in limb and spine. Different Ob-Rb expression patterns might partly contribute to age- and gender- related differences in trabecular bone mass.

Small-animal PET/CT assessment of bone microdamage in ovariectomized rats.

UNLABELLED: Microdamage in bone contributes to bone fragility in postmenopausal women. Therefore, it is important to find a noninvasive method to detect microdamage in living bone. PET with (18)F-fluoride has been used for skeletal imaging in clinical studies. However, few studies are undertaken to investigate bone microdamage associated with osteoporosis in vivo using noninvasive means. The aim of our study was to analyze the impact of osteoporosis due to estrogen deficiency on the occurrence of microdamage by observing the change in the uptake of (18)F-fluoride in the tibiae of ovariectomized rats after fatigue loading with small-animal PET/CT. We also explored the feasibility of noninvasive detection of bone microdamage in vivo using a small-animal PET/CT scanner specially designed for rodent study. METHODS: Rats were randomized into 2 groups: ovariectomy and sham surgery. These rats were imaged using a dedicated small-animal PET scanner with (18)F-fluoride after the left tibiae were loaded cyclically under the axial compression. The fluoride uptake values were quantified in the tibial mid shafts, and the tibia was obtained for histomorphometric measurements of bone microdamage and osteocyte density. Bone mineral density at the fourth lumbar vertebra and right femur were measured using dual-energy x-ray absorptiometry. RESULTS: PET image intensity was significantly increased (P < 0.05) in the loaded tibia of the ovariectomy group, compared with that of the sham group. Histomorphometry showed that both crack density and crack length in the loaded tibia were significantly higher (P < 0.05) in ovariectomized rats than in sham rats. The PET image intensity in the loaded tibia was significantly positively correlated with crack length and crack density (which show in histomorphometric measurement) (P < 0.05). CONCLUSION: Both small-animal PET/CT and histomorphometric measurement provided evidence that bone microdamage is significantly increased after estrogen depletion. The strong correlation between these 2 measurements suggests that small-animal PET/CT is a useful noninvasive means to detect bone microdamage in vivo.

ADAMTS-5 and intervertebral disc degeneration: the results of tissue immunohistochemistry and in vitro cell culture.

Matrix metalloproteinases (MMPs) are known to be involved in IVD degeneration by hydrolyzing the extracellular matrix (ECM), especially the collagens. The degradation of proteoglycans, which is another main ECM component in the IVD, however, has not been extensively investigated. This study aimed to determine the expression of ADAMTS-5 in human herniated intervertebral disc (IVD) tissues and to investigate whether interleukin-1ß (IL-1ß)-induced expression of ADAMTS-5 is mediated by nitric oxide (NO). Forty-five herniated IVDs were harvested and immunostained to determine the distribution and type of ADAMTS-5 expressing cells. Rat NP cells maintained in alginate beads were treated with IL-1ß, accumulation of NO was detected by Griess reaction, the expression of ADAMTS-5 and inducible nitric oxide synthase (iNOS) was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), the content of proteoglycans in alginate beads was visualized by alcian blue staining, and the effect of aminoguanidine on the changes in alginate beads induced by IL-1ß treatment were also examined. Immunohistochemical results from 45 herniated discs showed that ADAMTS-5-positive cells are commonly seen in cell clusters, that the percentage of ADAMTS-5-positive cells was higher in uncontained herniated discs than in contained ones, and that the percentage of ADAMTS-5-positive cells correlated with the age of the patients. IL-1ß treatment resulted in increased accumulation of NO, increased expression of ADAMTS-5 and iNOS, whereas the accumulation of proteoglycan in alginate beads decreased. Aminoguanidine significantly reversed the changes in alginate beads induced by IL-1ß treatment. We thus suggested that ADAMTS-5 is probably involved in the process of IVD degeneration, and that IL-1ß-induced expression of ADAMTS-5 is mediated by NO.

Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production.

Various mechanical stresses in vivo induce disc cell apoptosis and intervertebral disc (IVD) degeneration, but the underlying molecular mechanism is not fully known. The aim of this study was to investigate the role of endoplasmic reticulum stress in cyclic stretch-induced apoptosis of rat annulus fibrosus (AF) cells. Flexercell Tension Plus system was used to apply cyclic stretch to rat annulus fibrosus cells at a frequency of 0.5 Hz with 20% elongation for 12, 24, 36, or 48 h. Apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining and caspase-8, 9 activity assays. The expression of the markers of endoplasmic reticulum stress including CHOP, GRP78, and caspase-12 were determined by RT-PCR and Western blot. Mitochondrial membrane potential change was observed by JC-1 staining in situ. In addition, the levels of the nitric oxide (NO) were determined with the Griess reaction and fluorescence staining. The results indicated that cyclic stretch at a frequency of 0.5 Hz with 20% elongation-induced apoptosis in rat AF cells. Prolonged exposure of the unphysiologically cyclic stretch to AF cells caused NO overproduction, up-regulation of endoplasmic reticulum stress markers including CHOP, GRP78, and caspase-12, depolarization of mitochondria and activation of caspase-9. However, cyclic stretch at this level had no effect on caspase-8 activity. In addition, specific inhibitor of caspase-12 (Z-ATAD-FMK) and caspase-9 (Z-LEHD-FMK) partly suppressed cyclic stretch-induced AF cell apoptosis and the anti-apoptotic effects of the caspase inhibitors were additive. Our data suggest that endoplasmic reticulum stress, likely mediated by NO, contributes to the AF cell apoptosis induced by cyclic stretch in addition to the mitochondrial pathway. These findings could be helpful to understand the mechanism of disc cell apoptosis, the root cause of IVD degeneration.

The roles of the sympathetic nervous system in osteoporotic diseases: A review of experimental and clinical studies.

With the rapid aging of the world population, the issue of skeletal health is becoming more prominent and urgent. The bone remodeling mechanism has sparked great interest among bone research societies. At the same time, increasing clinical and experimental evidence has driven attention towards the pivotal role of the sympathetic nervous system (SNS) in bone remodeling. Bone remodeling is thought to be partially controlled by the hypothalamus, a process which is mediated by the adrenergic nerves and neurotransmitters. Currently, new knowledge about the role of the SNS in the development and pathophysiology of osteoporosis is being generated. The aim of this review is to summarize the evidence that proves the involvement of the SNS in bone metabolism and to outline some common osteoporotic diseases that occur under different circumstances. The adrenergic signaling pathway and its neurotransmitters are involved to various degrees of importance in the development of osteoporosis in postmenopause, as well as in spinal cord injury, depression, unloading and the complex regional pain syndrome. In addition, clinical and pharmacological studies have helped to increase the comprehension of the adrenergic signaling pathway. We try to individually examine the contributions of the SNS in osteoporotic diseases from a different perspective. It is our hope that a further understanding of the adrenergic signaling by the SNS will pave the way for conceptualizing optimal treatment regimens for osteoporosis in the near future.