Serum and nutrient deprivation increase autophagic flux in intervertebral disc annulus fibrosus cells: an in vitro experimental study.

PURPOSE: The loss of nutrient supply is a suspected contributor of intervertebral disc degeneration. However, the extent to which low nutrition affects disc annulus fibrosus (AF) cells is unknown as nutrient deprivation has mainly been investigated in disc nucleus pulposus cells. Hence, an experimental study was designed to clarify the effects of limited nutrients on disc AF cell fate, including autophagy, the process by which cells recycle their own damaged components. METHODS: Rabbit disc AF cells were cultured in different media with varying serum concentrations under 5% oxygen. Cellular responses to changes in serum and nutrient concentrations were determined by measuring proliferation and metabolic activity. Autophagic flux in AF cells was longitudinally monitored using imaging cytometry and Western blotting for LC3, HMGB1, and p62/SQSTM1. Apoptosis (TUNEL staining and cleaved caspase-3 immunodetection) and cellular senescence (senescence-associated ß-galactosidase assay and p16/INK4A immunodetection) were measured. RESULTS: Markers of apoptosis and senescence increased, while cell proliferation and metabolic activity decreased under the withdrawal of serum and of nutrients other than oxygen, confirming cellular stress. Time-dependent increases in autophagy markers, including LC3 puncta number per cell, LC3-II expression, and cytoplasmic HMGB1, were observed under conditions of reduced nutrition, while an autophagy substrate, p62/SQSTM1, decreased over time. Collectively, these findings suggest increased autophagic flux in disc AF cells under serum and nutrient deprivation. CONCLUSION: Disc AF cells exhibit distinct responses to serum and nutrient deprivation. Cellular responses include cell death and quiescence in addition to reduced proliferation and metabolic activity, as well as activation of autophagy under conditions of nutritional stress. These slides can be retrieved under Electronic Supplementary Material.

Verification of a Portable Motion Tracking System for Remote Management of Physical Rehabilitation of the Knee.

Rehabilitation following knee injury or surgery is critical for recovery of function and independence. However, patient non-adherence remains a significant barrier to success. Remote rehabilitation using mobile health (mHealth) technologies have potential for improving adherence to and execution of home exercise. We developed a remote rehabilitation management system combining two wireless inertial measurement units (IMUs) with an interactive mobile application and a web-based clinician portal (interACTION). However, in order to translate interACTION into the clinical setting, it was first necessary to verify the efficacy of measuring knee motion during rehabilitation exercises for physical therapy and determine if visual feedback significantly improves the participant's ability to perform the exercises correctly. Therefore, the aim of this study was to verify the accuracy of the IMU-based knee angle measurement system during three common physical therapy exercises, quantify the effect of visual feedback on exercise performance, and understand the qualitative experience of the user interface through survey data. A convenience sample of ten healthy control participants were recruited for an IRB-approved protocol. Using the interACTION application in a controlled laboratory environment, participants performed ten repetitions of three knee rehabilitation exercises: heel slides, short arc quadriceps contractions, and sit-to-stand. The heel slide exercise was completed without feedback from the mobile application, then all exercises were performed with visual feedback. Exercises were recorded simultaneously by the IMU motion tracking sensors and a video-based motion tracking system. Validation showed moderate to good agreement between the two systems for all exercises and accuracy was within three degrees. Based on custom usability survey results, interACTION was well received. Overall, this study demonstrated the potential of interACTION to measure range of motion during rehabilitation exercises for physical therapy and visual feedback significantly improved the participant's ability to perform the exercises correctly.

Influence of follower load application on moment-rotation parameters and intradiscal pressure in the cervical spine.

The objective of this study was to implement a follower load (FL) device within a robotic (universal force-moment sensor) testing system and utilize the system to explore the effect of FL on multi-segment cervical spine moment-rotation parameters and intradiscal pressure (IDP) at C45 and C56. Twelve fresh-frozen human cervical specimens (C3-C7) were biomechanically tested in a robotic testing system to a pure moment target of 2.0 Nm for flexion and extension (FE) with no compression and with 100 N of FL. Application of FL was accomplished by loading the specimens with bilateral cables passing through cable guides inserted into the vertebral bodies and attached to load controlled linear actuators. FL significantly increased neutral zone (NZ) stiffness and NZ width but resulted in no change in the range of motion (ROM) or elastic zone stiffness. C45 and C56 IDP measured in the neutral position were significantly increased with application of FL. The change in IDP with increasing flexion rotation was not significantly affected by the application of FL, whereas the change in IDP with increasing extension rotation was significantly reduced by the application of FL. Application of FL did not appear to affect the specimen's quantity of motion (ROM) but did affect the quality (the shape of the curve). Regarding IDP, the effects of adding FL compression approximates the effect of the patient going from supine to a seated position (FL compression increased the IDP in the neutral position). The change in IDP with increasing flexion rotation was not affected by the application of FL, but the change in IDP with increasing extension rotation was, however, significantly reduced by the application of FL.

Biomechanical Evaluation of Transpedicular Nucleotomy With Intact Annulus Fibrosus.

STUDY DESIGN: Biomechanical testing of partially nucleotomized ovine cadaveric spines. OBJECTIVE: To explore how the nucleus pulposus (NP) affects the biomechanical behavior of the intervertebral disc (IVD) by performing a partial nucleotomy via the transpedicular approach. SUMMARY OF BACKGROUND DATA: Mechanical loading represents a crucial part of IVD homeostasis. However, traditional regenerative strategies require violation of the annulus fibrosus (AF) resulting in significant alteration of joint mechanics. The transpedicular nucleotomy represents a suitable method to create a cavity into the NP, as a model to study IVD regeneration with intact AF. METHODS: A total of 30 ovine-lumbar- functional spinal units (FSUs) (L1-L6) randomly assigned to 5 groups: control; transpedicular tunnel (TT); TT + polymethylmethacrylate (PMMA) to repair the bone tunnel; nucleotomy; nucleotomy + PMMA. Flexion/extension, lateral-bending, and axial-rotation were evaluated under adaptive displacement control. Axial compression was applied for 15 cycles of preconditioning followed by 1 hour of constant compression. Viscoelastic behavior was modeled and parameterized. RESULTS: TT has minimal effects on rotational biomechanics. The nucleotomy increases ROM and neutral zone (NZ) displacement width whereas decreasing NZ stiffness. TT + PMMA has small effects in terms of ROM. Nucleotomy + PMMA brings ROM back to the control, increases NZ stiffness, and decreases NZ displacement width. The nucleotomy tends to increase the rate of early creep. TT reduces early and late damping. The use of PMMA increased late elastic stiffness (S2) and reduced viscous damping (η2) culminating in faster resolution of creep. CONCLUSION: Biomechanical properties of NP are crucial for IVD repair. This study demonstrated that TT does not affect rotational stability whereas partial nucleotomy with intact AF induce rotational instability, highlighting the central role of NP in early stages of IDD. Therefore, this model represents a successful platform to validate and optimize disc regeneration strategies. LEVEL OF EVIDENCE: N/A.

Mechanical role of the posterior column components in the cervical spine.

PURPOSE: To quantify the mechanical role of posterior column components in human cervical spine segments. METHODS: Twelve C6-7 segments were subjected to resection of (1) suprasinous/interspinous ligaments (SSL/ISL), (2) ligamenta flavum (LF), (3) facet capsules, and (4) facets. A robot-based testing system performed repeated flexibility testing of flexion-extension (FE), axial rotation (AR), and lateral bending (LB) to 2.5Nm and replayed kinematics from intact flexibility tests for each state. Range-of-motion, stiffness, moment resistance and resultant forces were calculated. RESULTS: The LF contributes largely to moment resistance, particularly in flexion. Facet joints were primary contributors to AR and LB mechanics. Moment/force responses were more sensitive and precise than kinematic outcomes. CONCLUSIONS: The LF is mechanically important in the cervical spine; its injury could negatively impact load distribution. Damage to facets in a flexion injury could lead to AR or LB hypermobility. Quantifying the contribution of spinal structures to moment resistance is a sensitive, precise process for characterizing structural mechanics.

Molecular mechanisms of biological aging in intervertebral discs.

Advanced age is the greatest risk factor for the majority of human ailments, including spine-related chronic disability and back pain, which stem from age-associated intervertebral disc degeneration (IDD). Given the rapid global rise in the aging population, understanding the biology of intervertebral disc aging in order to develop effective therapeutic interventions to combat the adverse effects of aging on disc health is now imperative. Fortunately, recent advances in aging research have begun to shed light on the basic biological process of aging. Here we review some of these insights and organize the complex process of disc aging into three different phases to guide research efforts to understand the biology of disc aging. The objective of this review is to provide an overview of the current knowledge and the recent progress made to elucidate specific molecular mechanisms underlying disc aging. In particular, studies over the last few years have uncovered cellular senescence and genomic instability as important drivers of disc aging. Supporting evidence comes from DNA repair-deficient animal models that show increased disc cellular senescence and accelerated disc aging. Additionally, stress-induced senescent cells have now been well documented to secrete catabolic factors, which can negatively impact the physiology of neighboring cells and ECM. These along with other molecular drivers of aging are reviewed in depth to shed crucial insights into the underlying mechanisms of age-related disc degeneration. We also highlight molecular targets for novel therapies and emerging candidate therapeutics that may mitigate age-associated IDD. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1289-1306, 2016.

Biological responses to flexion/extension in spinal segments ex-vivo.

Mechanical loading is a salient factor in the progression of spinal disorders that contribute to back pain. Biological responses to loading modes like flexion/extension (F/E) in relevant spinal tissues remain unstudied. A novel, multi-axial experimental system was developed to subject viable functional spinal units (FSUs) to complex, in-situ loading. The objective was to determine biological effects of F/E in multiple spinal tissues-annulus fibrosus, nucleus pulposus, facet cartilage, and ligamentum flavum. Rabbit lumbar FSUs were mounted in a bioreactor within a robotic testing system. FSUs underwent small (0.17/0.05 Nm) and large (0.5/0.15 Nm) range-of-motion F/E for 1 or 2 h of cycling. Outcomes in each tissue, compared to unloaded FSUs, included (i) relative mRNA expression of catabolic (MMP-1, 3 and ADAMTS-5), pro-inflammatory (COX-2), and anabolic (ACAN) genes and (ii) immunoblotting of aggrecan degradation. Total energy applied to FSUs increased in groups subject to large range-of-motion and 2-h cycling, and moment relaxation was higher with large range-of-motion. F/E significantly modulated MMP1,-3 and COX-2 in facet cartilage and MMP-3 and ACAN in annulus fibrosus. Large range-of-motion loading increased MMP-mediated aggrecan fragmentation in annulus fibrosus. Biological responses to complex loading ex vivo showed variation among spinal tissues that reflect tissue structure and mechanical loading in F/E.

Needle puncture in rabbit functional spinal units alters rotational biomechanics.

STUDY DESIGN: An in vitro biomechanical study for rabbit lumbar functional spinal units (FSUs) using a robot-based spine testing system. OBJECTIVE: To elucidate the effect of annular puncture with a 16 G needle on mechanical properties in flexion/extension, axial rotation, and lateral bending. SUMMARY OF BACKGROUND DATA: Needle puncture of the intervertebral disk has been shown to alter mechanical properties of the disk in compression, torsion, and bending. The effect of needle puncture in FSUs, where intact spinal ligaments and facet joints may mitigate or amplify these changes in the disk, on spinal motion segment stability subject to physiological rotations remains unknown. METHODS: Rabbit FSUs were tested using a robot testing system whose force/moment and position precision were assessed to demonstrate system capability. Flexibility testing methods were developed by load-to-failure testing in flexion/extension, axial rotation, and lateral bending. Subsequent testing methods were used to examine a 16 G needle disk puncture and No. 11 blade disk stab (positive control for mechanical disruption). Flexibility testing was used to assess segmental range-of-motion (degrees), neutral zone stiffness (N m/degrees) and width (degrees and N m), and elastic zone stiffness before and after annular injury. RESULTS: The robot-based system was capable of performing flexibility testing on FSUs-mean precision of force/moment measurements and robot system movements were <3% and 1%, respectively, of moment-rotation target values. Flexibility moment targets were 0.3 N m for flexion and axial rotation and 0.15 N m for extension and lateral bending. Needle puncture caused significant (P<0.05) changes only in flexion/extension range-of-motion and neutral zone stiffness and width (N m) compared with preintervention. No. 11 blade-stab significantly increased range-of-motion in all motions, decreased neutral zone stiffness and width (N m) in flexion/extension, and increased elastic zone stiffness in flexion and lateral bending. CONCLUSIONS: These findings suggest that disk puncture and stab can destabilize FSUs in primary rotations.

In vitro spine testing using a robot-based testing system: comparison of displacement control and "hybrid control".

The two leading control algorithms for in-vitro spine biomechanical testing-"load control" and "displacement control"-are limited in their lack of adaptation to changes in the load-displacement response of a spine specimen-pointing to the need for sufficiently sophisticated control algorithms that are able to govern the application of loads/motions to a spine specimen in a more realistic, adaptive manner. A robotics-based spine testing system was programmed with a novel hybrid control algorithm combining "load control" and "displacement control" into a single, robust algorithm. Prior to in-vitro cadaveric testing, preliminary testing of the new algorithm was performed using a rigid-body-spring model with known structural properties. The present study also offers a direct comparison between "hybrid control" and "displacement control". The hybrid control algorithm enabled the robotics-based spine testing system to apply pure moments to an FSU (in flexion/extension, lateral bending, or axial rotation) in an unconstrained manner through active control of secondary translational/rotational degrees-of-freedom-successfully minimizing coupled forces/moments. The characteristic nonlinear S-shaped curves of the primary moment-rotation responses were consistent with previous reports of the FSU having a region of low stiffness (neutral zone) bounded by regions of increasing stiffness (elastic zone). Direct comparison of "displacement control" and "hybrid control" showed that hybrid control was able to actively minimize off-axis forces and resulted in larger neutral zone and range of motion.

Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration.

BACKGROUND CONTEXT: Destruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD). PURPOSE: To summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD. METHODS: A comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems. RESULTS: Upregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9. CONCLUSIONS: Upregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.

Injection of human umbilical tissue-derived cells into the nucleus pulposus alters the course of intervertebral disc degeneration in vivo.

BACKGROUND CONTEXT: Patients often present to spine clinic with evidence of intervertebral disc degeneration (IDD). If conservative management fails, a safe and effective injection directly into the disc might be preferable to the risks and morbidity of surgery. PURPOSE: To determine whether injecting human umbilical tissue-derived cells (hUTC) into the nucleus pulposus (NP) might improve the course of IDD. DESIGN: Prospective, randomized, blinded placebo-controlled in vivo study. PATIENT SAMPLE: Skeletally mature New Zealand white rabbits. OUTCOME MEASURES: Degree of IDD based on magnetic resonance imaging (MRI), biomechanics, and histology. METHODS: Thirty skeletally mature New Zealand white rabbits were used in a previously validated rabbit annulotomy model for IDD. Discs L2-L3, L3-L4, and L4-L5 were surgically exposed and punctured to induce degeneration and then 3 weeks later the same discs were injected with hUTC with or without a hydrogel carrier. Serial MRIs obtained at 0, 3, 6, and 12 weeks were analyzed for evidence of degeneration qualitatively and quantitatively via NP area and MRI Index. The rabbits were sacrificed at 12 weeks and discs L4-L5 were analyzed histologically. The L3-L4 discs were fixed to a robotic arm and subjected to uniaxial compression, and viscoelastic displacement curves were generated. RESULTS: Qualitatively, the MRIs demonstrated no evidence of degeneration in the control group over the course of 12 weeks. The punctured group yielded MRIs with the evidence of disc height loss and darkening, suggestive of degeneration. The three treatment groups (cells alone, carrier alone, or cells+carrier) generated MRIs with less qualitative evidence of degeneration than the punctured group. MRI Index and area for the cell and the cell+carrier groups were significantly distinct from the punctured group at 12 weeks. The carrier group generated MRI data that fell between control and punctured values but failed to reach a statistically significant difference from the punctured values. There were no statistically significant MRI differences among the three treatment groups. The treated groups also demonstrated viscoelastic properties that were distinct from the control and punctured values, with the cell curve more similar to the punctured curve and the carrier curve and carrier+cells curve more similar to the control curve (although no creep differences achieved statistical significance). There was some histological evidence of improved cellularity and disc architecture in the treated discs compared with the punctured discs. CONCLUSIONS: Treatment of degenerating rabbit intervertebral discs with hUTC in a hydrogel carrier solution might help restore the MRI, histological, and biomechanical properties toward those of nondegenerated controls. Treatment with cells in saline or a hydrogel carrier devoid of cells also might help restore some imaging, architectural, and physical properties to the degenerating disc. These data support the potential use of therapeutic cells in the treatment of disc degeneration.

In vivo analysis of cervical range of motion after 4- and 5-level subaxial cervical spine fusion.

STUDY DESIGN: A cohort study analyzing the cervical range of motion (ROM) of subjects with 4- or 5-level posterior laminectomy and fusion or anterior and posterior decompression and fusion operation. OBJECTIVE: The purpose of this study was to evaluate the effect of extending a C3-C7 fusion to C3-T1 on subject's ROM and level of disability. SUMMARY OF BACKGROUND DATA: Cadaveric studies show a reduction in the ROM of C3-C7 cervical fusion spines. In vivo, surgeons treat symptomatic cervical subaxial spine with either a C3-C7 fusion or C3-T1 fusion. While in some cases extending the fusion level to T1 is merited due to pathology, most cases are due to surgeon's preference to avoid future degeneration and reoperation of the C7-T1 junction. METHODS: This study involved 44 4-level fusion and 20 5-level fusion subjects along with 18 nonoperative controls. Operative subjects were divided according to early or late postoperative clinical visit. Subjects were asked to complete the neck disability index survey and their maximum ROM during flexion/extension, axial rotation, and lateral bending was measured using a virtual reality assisted electromagnetic tracking system. In addition, the helical axis of motion was calculated for flexion and extension motions. An analysis of variance statistical test was used to determine significant differences between study groups. RESULTS: Five- level subjects had significantly less ROM than 4-level subjects and both groups were significantly less than control group during all motions. There was no effect of postoperative time on subject's ROM. In addition, there was no difference in the center of helical axis of rotation across the 3 groups. Finally, both operative groups exhibited similar levels of mild disability as measured by the neck disability index. CONCLUSIONS: Extending the subaxial fusion from C3-C7 to include C7-T1 resulted in a significant loss of ROM, while postoperative time healing, center of rotation, and level of disability were similar across groups. This finding merits further investigation of the intersegmental motions of the cervical spine.

Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model.

BACKGROUND CONTEXT: Intervertebral disc degeneration (IDD) is a common cause of back pain. Patients who fail conservative management may face the morbidity of surgery. Alternative treatment modalities could have a significant impact on disease progression and patients' quality of life. PURPOSE: To determine if the injection of a virus vector carrying a therapeutic gene directly into the nucleus pulposus improves the course of IDD. STUDY DESIGN: Prospective randomized controlled animal study. METHODS: Thirty-four skeletally mature New Zealand white rabbits were used. In the treatment group, L2-L3, L3-L4, and L4-L5 discs were punctured in accordance with a previously validated rabbit annulotomy model for IDD and then subsequently treated with adeno-associated virus serotype 2 (AAV2) vector carrying genes for either bone morphogenetic protein 2 (BMP2) or tissue inhibitor of metalloproteinase 1 (TIMP1). A nonoperative control group, nonpunctured sham surgical group, and punctured control group were also evaluated. Serial magnetic resonance imaging (MRI) studies at 0, 6, and 12 weeks were obtained, and a validated MRI analysis program was used to quantify degeneration. The rabbits were sacrificed at 12 weeks, and L4-L5 discs were analyzed histologically. Viscoelastic properties of the L3-L4 discs were analyzed using uniaxial load-normalized displacement testing. Creep curves were mathematically modeled according to a previously validated two-phase exponential model. Serum samples obtained at 0, 6, and 12 weeks were assayed for biochemical evidence of degeneration. RESULTS: The punctured group demonstrated MRI and histologic evidence of degeneration as expected. The treatment groups demonstrated less MRI and histologic evidence of degeneration than the punctured group. The serum biochemical marker C-telopeptide of collagen type II increased rapidly in the punctured group, but the treated groups returned to control values by 12 weeks. The treatment groups demonstrated several viscoelastic properties that were distinct from control and punctured values. CONCLUSIONS: Treatment of punctured rabbit intervertebral discs with AAV2-BMP2 or AAV2-TIMP1 helps delay degenerative changes, as seen on MRI, histologic sampling, serum biochemical analysis, and biomechanical testing. Although data from animal models should be extrapolated to the human condition with caution, this study supports the potential use of gene therapy for the treatment of IDD.

Novel ex-vivo mechanobiological intervertebral disc culture system.

Intervertebral disc degeneration, a leading cause of low back pain, poses a significant socioeconomic burden with a broad array of costly treatment options. Mechanical loading is important in disease progression and treatment. Connecting mechanics and biology is critical for determining how loading parameters affect cellular response and matrix homeostasis. A novel ex-vivo experimental platform was developed to facilitate in-situ loading of rabbit functional spinal units (FSUs) with relevant biological outcome measures. The system was designed for motion outside of an incubator and validated for rigid fixation and physiologic environmental conditions. Specimen motion relative to novel fixtures was assessed using a digitizer; fixture stiffness exceeded specimen stiffness by an order of magnitude. Intradiscal pressure (IDP), measured using a fiber-optic pressure transducer, confirmed rigidity and compressive force selection. Surrounding media was controlled at 37 °C, 5% O(2)/CO(2) using a closed flow loop with an hypoxic incubator and was validated with probes in the specimen chamber. FSUs were subjected to cyclic compression (20 cycles) and four-hour creep at 1.0 MPa. Disc tissue was analyzed for cell viability using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which showed high viability (>90%) regardless of loading. Conditioned media was assayed for type-II collagen degradation fragments (CTX-II) and an aggrecan epitope (CS-846) associated with new aggrecan synthesis. CTX-II concentrations were not associated with loading, but CS-846 concentrations appeared to be increased with loading. Preservation of the full FSU allows physiologic load transmission and future multi-axis motion and identification of load-responsive proteins, thereby forming a new niche in intervertebral disc organ culture.

Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system.

BACKGROUND CONTEXT: Bupivacaine is a local anesthetic commonly used for back pain management in interventional procedures. Cytotoxic effects of bupivacaine have been reported in articular cartilage and, recently, in intervertebral disc cell culture. However, the relevance of these effects to discs in vivo remains unclear. This study examines the effect of bupivacaine on disc cell metabolism using an organotypic culture model system that mimics the in vivo environment. PURPOSE: To assess the effect of bupivacaine on disc cell viability and matrix protein synthesis using an organotypic model system and to determine whether this anesthetic has toxic effects. STUDY DESIGN: Mouse intervertebral discs were isolated and maintained ex vivo in an organotypic culture then exposed to clinically relevant concentrations of bupivacaine, and the impact on disc cell viability and matrix proteoglycan (PG) and collagen syntheses were measured in the presence and absence of the drug. SUBJECTS: Mouse functional spine units (FSUs) were isolated from the lumbar spines of 10-week-old mice. OUTCOME MEASURES: Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Total PG and collagen syntheses were determined by measuring the incorporation of radioactive (35)S-sulfate and (3)H-l-proline into PG and collagen, respectively. METHODS: Organotypic cultures of mouse FSUs were exposed to different concentrations (0%-0.5%) of bupivacaine for variable amounts of time (0-2 hours). Cell viability within disc tissue was quantified by MTT staining and histologic assay. Matrix protein synthesis was measured by incorporation of radioactive (35)S-sulfate (for PG synthesis) and (3)H-l-proline (for collagen synthesis). RESULTS: Untreated mouse disc organs were maintained in culture for up to 1 month with minimal changes in tissue histology, cell viability, and matrix protein synthesis. Exposure to bupivacaine decreased cell viability in a dose- and time-dependent manner. Exposure to bupivacaine at concentrations less than or equal to 0.25% did not significantly affect matrix protein synthesis. However, at 0.5% bupivacaine, collagen synthesis was reduced by fourfold and PG synthesis by threefold. CONCLUSIONS: Mouse discs can be successfully maintained ex vivo for upward of 4 weeks with little cell death, change in histologic structure, or matrix protein synthesis. This organotypic model system closely mimics the in vivo environment of the disc. Exposure of these cultures to bupivacaine dramatically decreased cell viability and matrix protein synthesis in a dose- and time-dependent manner. These findings corroborate those previously reported by Lee et al. using disc cell culture and demonstrate that this anesthetic at clinically relevant doses is toxic to intervertebral discs in both cell culture and disc organ models representative of the native architectural context.

Influence of number of operated levels and postoperative time on active range of motion following anterior cervical decompression and fusion procedures.

STUDY DESIGN: A cohort study analyzing the cervical range of motion of subjects with anterior cervical decompression and fusion operation (ACDF). OBJECTIVE: The purpose of this study was to compare the cervical range of motion of subjects who underwent an ACDF operation to age-matched healthy nonoperative subjects. Subjects were divided according to the number of operated levels, postoperative time point, and level of disability. SUMMARY OF BACKGROUND DATA: ACDF is an operative treatment aimed at expansion of the spinal canal and relief of cord compression. In addition to alleviating pain, 2 common tools are used to measure postoperative success; cervical range of motion kinematic analysis and subjective evaluation questionnaires (Neck Disability Index [NDI]). METHODS: This study involved 25 preoperative and 110 postoperative ACDF subjects as well as 18 control volunteers with no prior history of neck complaints. ACDF subjects were divided according to the number of operated levels; 1-, 2-, 3-, and 4-levels as well as time of their clinical visit; preoperative, early, and late postoperative. Before kinematic testing, the subjects were asked to complete the NDI survey. A virtual reality assisted electromagnetic tracking was used to measure an active voluntary motion of the head relative to the torso. The subjects' maximum range of motion was calculated and compared as they executed 3 to 5 consecutive cycles of the primary motions, flexion/extension, axial rotation, and lateral bending. An analysis of variance statistical test (P < 0.01) was used to determine significant differences between study groups. RESULTS.: Subject's range of motion decreased relative to control as the number of operated levels increased. Moreover, 1- and 2-level subjects increased their range motion relative to preoperative. Finally, there was a decrease in range of motion as the subject's level of disability increased as measured by an NDI score but all subjects reported a lower score relative to preoperative time point. CONCLUSION: The active range of motion of subjects who underwent an ACDF surgery increased postoperative and was dependent on the number of operated levels. In addition, there was an improvement in the disability level after the surgery as measured by the NDI score.

Assessing range of motion to evaluate the adverse effects of ill-fitting cervical orthoses.

BACKGROUND CONTEXT: Although previous studies have primarily focused on testing the effectiveness of cervical orthoses under properly fit conditions, this study focuses on analyzing the effects of an ill-fitted cervical orthosis (Miami J). This may have significance to health-care providers in understanding the effects of an improperly fitted neck brace. PURPOSE: The aims of this study were threefold: first, to apply virtual reality (VR) feedback control to repeatedly measure orthoses effectiveness in the primary motions; second, to use this control methodology to test the orthoses ability to restrict flexion/extension (FE) as a function of axial rotation (AR); third, to test the effects of an ill-fitting Miami J on cervical motion. STUDY DESIGN/SETTING: This study combines six degrees of freedom electromagnetic trackers and VR feedback to analyze the effectiveness of common cervical orthoses under less than optimal conditions. PATIENT SAMPLE: Twelve healthy male subjects aged 21 to 35 (mean 29.44 years, SD 6.598) years with no previous spinal cord injuries or current neck pain participated in the study. OUTCOME MEASURES: Cervical range of motion (CRoM) measurements were used to determine the amount of motion restriction for each of the fitted (too small, correct size, and too big) Miami J orthoses. METHODS: One Nest of Birds (NOB) electromagnetic sensor (Ascension Technology) was placed on the head and another on the upper back to measure motion of the head relative to the torso. The VR goggles (i-O Display Systems) were worn so that real-time feedback was available to the subject for motion control. The subject executed the primary motions of FE, AR, and lateral bending (LB) in separate sets of five trials each. Next, in combined motion, the subject axially rotated to a set point and then FE to his maximums. This entire set of motions was repeated for each (soft collar, Miami J, Miami J with chest extension, Sternal Occipital Mandibular Immobilizer (AliMed, Inc.), (SOMI and Halo) as well as the Miami J (one size too small and one size too big); the fitting of each brace was done by a board certified orthotist. A repeated measures analysis of variance was used to determine differences between the tested states (*p=.05). RESULTS: For the validation test, the primary motions recorded for subjects wearing each cervical brace, which demonstrated that the various orthoses all restricted CRoM. The soft collar restricted less motion than the other devices, whereas the Halo restricted the most motion throughout. For the ill-fitting cervical collar comparison, motion in the correct size collar was normalized to 1.0, and the correct size allowed less motion than either the too big or too small braces. In FE, the too big brace tended to allow more motion than the too small, but only the too big brace in extension was significantly different from the correct size. In AR, the too small brace seemed to allow more motion than the too big. Both the too big and too small braces were significantly different than the correct size in both left and right AR. In LB, the too big brace and too small brace were very similar in the amount of motion they were able to restrict. Both braces were significantly different than the correct size in right LB, whereas only the too small brace was significantly different from the correct size in left LB. In the combined motion data, both the too big and too small braces allowed more motion than the correct size. The too small brace seemed to allow more FE at all degrees of AR except for extreme right AR. CONCLUSIONS: To our knowledge, the effects of improperly fitted cervical orthoses on CRoM are still unknown. Using the NOB electromagnetic tracking system combined with VR feedback, we were able to consider the motion restriction of ill-fitting Miami J orthoses for both primary and combined motions. For both motion types, increased motion was possible when the subject was improperly fitted with the Miami J. If not considered, these excessive motions could potentially have detrimental effects on patient satisfaction, clinical outcomes, or even lead to increased secondary injury.