Robert A. Hartman

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.

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.

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.

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.

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.

Alterations in gene expression in response to compression of nucleus pulposus cells

BACKGROUND CONTEXT It is clear that mechanical forces are involved in initiating disc degeneration but also have the potential to exert beneficial effects. However, the signaling pathways initiated by mechanical stress and thresholds for these responses have not been elucidated. We have developed a metabolically active compression system with the advantages of having the ability to test cells in vitro as well as within their native matrix and control exposure to environmental factors. We hypothesized that nucleus pulposus cells would respond to compressive stress with different thresholds for alterations in catabolic and anabolic gene expression. PURPOSE The purpose of the study was to establish the utility of a novel compression chamber and examine the effects of various magnitudes and durations of compression on nucleus pulposus inflammatory, catabolic, and anabolic gene expression. STUDY DESIGN In vitro controlled examination of intervertebral disc cell responses to compression. METHODS A chamber capable of imparting 0 to 20 MPa of hydrostatic compression onto nucleus pulposus cells was fabricated. Healthy rabbit nucleus pulposus cells were cultured in alginate beads and exposed to static compression at 0.7, 2, and 4 MPa for 4 or 24 hours. Gene expression analysis (real-time polymerase chain reaction) was performed to compare markers of inflammation (inducible nitric oxide synthase, cyclooxygenase-2), matrix catabolism (matrix metalloproteinase-3), and anticatabolic/anabolic metabolism (tissue inhibitor of metalloproteinase-1, aggrecan) in control and compressed cells. RESULTS Compression resulted in magnitude- and duration-dependent changes in gene expression. Increasing magnitudes showed more anticatabolic gene expression changes, whereas increasing duration resulted in increases in procatabolic gene expression. CONCLUSION These data demonstrate favorable effects of compression in relation to genes involved in matrix homeostasis and procatabolic gene expression in response to sustained loading levels, consistent with traumatic effects. These data provide an improved understanding of how compression affects cell signaling, which has the potential to be exploited to initiate repair and prevent matrix breakdown.

Effects of secreted factors in culture medium of annulus fibrosus cells on microvascular endothelial cells: elucidating the possible pathomechanisms of matrix degradation and nerve in-growth in disc degeneration

OBJECTIVE To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators. METHODS Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media. RESULTS Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture. DISCUSSION AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration.

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.

Glucosamine supplementation demonstrates a negative effect on intervertebral disc matrix in an animal model of disc degeneration.

Study Design. Laboratory based controlled in vivo study. Objective. To determine the in vivo effects of oral glucosamine sulfate on intervertebral disc degeneration. Summary of Background Data. Although glucosamine has demonstrated beneficial effect in articular cartilage, clinical benefit is uncertain. A Centers for Disease Control report from 2009 reported that many patients are using glucosamine supplementation for low back pain, without significant evidence to support its use. Because disc degeneration is a major contributor of low back pain, we explored the effects of glucosamine on disc matrix homeostasis in an animal model of disc degeneration. Methods. Eighteen skeletally mature New Zealand White rabbits were divided into 4 groups: control, annular puncture, glucosamine, and annular puncture + glucosamine. Glucosamine treated rabbits received daily oral supplementation with 107 mg/d (weight based equivalent to human 1500 mg/d). Annular puncture surgery involved puncturing the annulus fibrosus of 3 lumbar discs with a 16-gauge needle to induce degeneration. Serial magnetic resonance images were obtained at 0, 4, 8, 12, and 20 weeks. Discs were harvested at 20 weeks for determination of glycosaminoglycan content, relative gene expression measured by real time polymerase chain reaction, and histological analyses. Results. The magnetic resonance imaging index and nucleus pulposus area of injured discs of glucosamine treated animals with annular puncture was found to be lower than that of degenerated discs from rabbits not supplemented with glucosamine. Consistent with this, decreased glycosaminoglycan was demonstrated in glucosamine fed animals, as determined by both histological and glycosaminoglycan content. Gene expression was consistent with a detrimental effect on matrix. Conclusion. These data demonstrate that the net effect on matrix in an animal model in vivo, as measured by gene expression, magnetic resonance imaging, histology, and total proteoglycan is antianabolic. This raises concern about this commonly used supplement, and future research is needed to establish the clinical relevance of these findings.

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. Subjects 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 subjects 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.