Thermoresponsive, redox-polymerized cellulosic hydrogels undergo in situ gelation and restore intervertebral disc biomechanics post discectomy.

Back and neck pain are commonly associated with intervertebral disc (IVD) degeneration. Structural augmentation of diseased nucleus pulposus (NP) tissue with biomaterials could restore degeneration-related IVD height loss and degraded biomechanical behaviors; however, effective NP replacement biomaterials are not commercially available. This study developed a novel, crosslinked, dual-polymer network (DPN) hydrogel comprised of methacrylated carboxymethylcellulose (CMC) and methylcellulose (MC), and used in vitro, in situ and in vivo testing to assess its efficacy as an injectable, in situ gelling, biocompatible material that matches native NP properties and restores IVD biomechanical behaviors. Thermogelling MC was required to enable consistent and timely gelation of CMC in situ within whole IVDs. The CMC-MC hydrogel was tuned to match compressive and swelling NP tissue properties. When injected into whole IVDs after discectomy injury, CMC-MC restored IVD height and compressive biomechanical behaviors, including range of motion and neutral zone stiffness, to intact levels. Subcutaneous implantation of the hydrogels in rats further demonstrated good biocompatibility of CMC-MC with a relatively thin fibrous capsule, similar to comparable biomaterials. In conclusion, CMC-MC is an injectable, tunable and biocompatible hydrogel with strong potential to be used as an NP replacement biomaterial since it can gel in situ, match NP properties, and restore IVD height and biomechanical function. Future investigations will evaluate herniation risk under severe loading conditions and assess long-term in vivo performance.

Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells.

The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1ß (IL-1ß) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease.

Form and function of the intervertebral disc in health and disease: a morphological and stain comparison study.

Multiple histologic measurements are commonly used to assess degenerative changes in intervertebral disc (IVD) structure; however, there is no consensus on which stains offer the clearest visualization of specific areas within the IVD. The objective of this study was to compare multiple tinctorial stains, evaluate their ability to highlight structural features within the IVD, and investigate how they influence the capacity to implement a degeneration scoring system. Lumbar IVDs from seven human autopsy specimens were stained using six commonly used stains (Hematoxylin/Eosin, Toluidine Blue, Safranin-O/Fast Green, Extended FAST, modified Gomori's Trichrome, and Picrosirius Red Alcian Blue). All IVDs were evaluated by three separate graders to independently determine which stains (i) were most effective at discerning different structural features within different regions of the IVDs and (ii) allowed for the most reproducible assessment of degeneration grade, as assessed via the Rutges histological scoring system (Rutges et al. A validated new histological classification for intervertebral disc degeneration. Osteoarthritis Cartilage, 21, 2039-47). Although Trichrome, XFAST and PR/AB stains were all effective at highlighting different regions of whole IVDs, we recommend the use of PR/AB because it had the highest degree of rater agreement on assigned degeneration grade, allowed greater resolution of degeneration grade, has an inferential relationship between color and composition, and allowed clear differentiation of the different regions and structural disruptions within the IVD. The use of a standard set of stains together with a histological grading scheme can aid in the characterization of structural changes in different regions of the IVD and may simplify comparisons across the field. This collection of human IVD histological images highlights how IVD degeneration is not a single disease but a composite of multiple processes such as aging, injury, repair, and disease, each of which are unique to the individual.

Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests.

Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation.

A role for TNFα in intervertebral disc degeneration: a non-recoverable catabolic shift.

This study examines the effect of TNFα on whole bovine intervertebral discs in organ culture and its association with changes characteristic of intervertebral disc degeneration (IDD) in order to inform future treatments to mitigate the chronic inflammatory state commonly found with painful IDD. Pro-inflammatory cytokines such as TNFα contribute to disc pathology and are implicated in the catabolic phenotype associated with painful IDD. Whole bovine discs were cultured to examine cellular (anabolic/catabolic gene expression, cell viability and senescence using ß-galactosidase) and structural (histology and aggrecan degradation) changes in response to TNFα treatment. Control or TNFα cultures were assessed at 7 and 21 days; the 21 day group also included a recovery group with 7 days TNFα followed by 14 days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7 days and this persisted until 21 days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased ß-galactosidase staining at 21 days without any recovery. TNFα treatment of whole bovine discs for 7 days induced changes similar to the degeneration processes that occur in human IDD: aggrecan degradation, increased catabolism, pro-inflammatory cytokines and nerve growth factor expression. TNFα significantly reduced anabolism in cultured IVDs and a possible mechanism may be associated with cell senescence. Results therefore suggest that successful treatments must promote anabolism and cell proliferation in addition to limiting inflammation.

Does the neutral zone quantification method matter? Efficacy of evaluating neutral zone during destabilization and restabilization in human spine implant testing.

Neutral zone (NZ) is an important biomechanical parameter when evaluating spinal instability following destabilizing and restabilizing events, with particular relevance for implant efficacy testing. It remains unclear what NZ calculation methods are most sensitive at capturing NZ changes across treatment conditions and a direct comparison is needed. The purpose of this study was to determine the most sensitive method at quantifying instability in human spines. Six cadaveric lumbar motion segments were subjected to a repeated measures implant testing schema of four sequential conditions: (1) Intact, (2) injury by herniation, (3) device implantation, (4) long-term cyclic fatigue loading. NZ was expected to increase after destabilization (steps 2 & 4) and decrease after restabilization (step 3). NZ methods compared in this study were: trilinear (TL), double sigmoid (DS), zero load (ZL), stiffness threshold (ST), and extrapolated elastic zone (EEZ). TL, ZL, and EEZ identified statistically significant NZ differences after each condition in flexion/extension and lateral bending. The ZL method also captured differences in axial rotation. All methods identified expected NZ changes after destabilization and restabilization, except DS in axial rotation. The TL, ZL, and EEZ methods were the most sensitive methods with this human cadaveric dataset. Future investigations comparing methods with additional datasets will clarify outcome generalizability and determine what curve profiles are most suitable for DS and ST methods. Understanding the applicability of NZ methods can enhance rigor and reliability of spinal instability measurements when quantifying the efficacy of novel implants and permits insight into clinically relevant biomechanical changes.

Development and validation of a bioreactor system for dynamic loading and mechanical characterization of whole human intervertebral discs in organ culture.

Intervertebral disc (IVD) degeneration is a common cause of back pain, and attempts to develop therapies are frustrated by lack of model systems that mimic the human condition. Human IVD organ culture models can address this gap, yet current models are limited since vertebral endplates are removed to maintain cell viability, physiological loading is not applied, and mechanical behaviors are not measured. This study aimed to (i) establish a method for isolating human IVDs from autopsy with intact vertebral endplates, and (ii) develop and validate an organ culture loading system for human or bovine IVDs. Human IVDs with intact endplates were isolated from cadavers within 48h of death and cultured for up to 21 days. IVDs remained viable with ~80% cell viability in nucleus and annulus regions. A dynamic loading system was designed and built with the capacity to culture 9 bovine or 6 human IVDs simultaneously while applying simulated physiologic loads (maximum force: 4kN) and measuring IVD mechanical behaviors. The loading system accurately applied dynamic loading regimes (RMS error <2.5N and total harmonic distortion <2.45%), and precisely evaluated mechanical behavior of rubber and bovine IVDs. Bovine IVDs maintained their mechanical behavior and retained >85% viable cells throughout the 3 week culture period. This organ culture loading system can closely mimic physiological conditions and be used to investigate response of living human and bovine IVDs to mechanical and chemical challenges and to screen therapeutic repair techniques.

C5 nerve root palsy following decompression of the cervical spine: a systematic evaluation of the literature.

C5 nerve root palsy is a rare and potentially debilitating complication of cervical spine surgery. Currently, however, there are no guidelines to help surgeons to prevent or treat this complication. We carried out a systematic review of the literature to identify the causes of this complication and options for its prevention and treatment. Searches of PubMed, Embase and Medline yielded 60 articles for inclusion, most of which addressed C5 palsy as a complication of surgery. Although many possible causes were given, most authors supported posterior migration of the spinal cord with tethering of the nerve root as being the most likely. Early detection and prevention of a C5 nerve root palsy using neurophysiological monitoring and variations in surgical technique show promise by allowing surgeons to minimise or prevent the incidence of C5 palsy. Conservative treatment is the current treatment of choice; most patients make a full recovery within two years.

Notochordal cell-derived therapeutic strategies for discogenic back pain.

An understanding of the processes that occur during development of the intervertebral disk can help inform therapeutic strategies for discogenic pain. This article reviews the literature to identify candidates that are found in or derived from the notochord or notochordal cells and evaluates the theory that such factors could be isolated and used as biologics to target the structural disruption, inflammation, and neurovascular ingrowth often associated with discogenic back pain. A systematic review using PubMed was performed with a primary search using keywords "(notochordal OR notochord) And (nerves OR blood vessels OR SHH OR chondroitin sulfate OR notch OR CTGF) NOT chordoma." Secondary searches involved keywords associated with the intervertebral disk and pain. Several potential therapeutic candidates from the notochord and their possible targets were identified. Studies are needed to further identify candidates, explore mechanisms for effect, and to validate the theory that these candidates can promote structural restoration and limit or inhibit neurovascular ingrowth using in vivo studies.

Diagnosis and treatment of unicameral and aneurysmal bone cysts in children.

Bone cysts are commonly encountered clinical problems in the pediatric age group. The most common types of cysts are the unicameral and aneurysmal bone cysts. These benign lesions vary in their aggressiveness, clinical behavior and treatment. Both of these lesions are poorly understood in terms of their etiology, but effective treatment exists. These lesions represent a source of great consternation to both clinicians and families, for they weaken the bone and may be confused with malignant lesions. This review will focus on the two most commonly encountered cystic lesions in the pediatric population.

A matrix metalloproteinase proenzyme activator produced by articular cartilage.

Matrix metalloproteinases (MMPs) are involved in connective tissue turnover under physiological and pathological conditions. MMP activity is regulated by the requirement for zymogen activation. This report describes a proMMP-3 activator produced by articular cartilage. The activator initiates a step-wise processing of proMMP-3 to generate an array of active species. Sequencing of activation intermediates demonstrated cleavage on the NH2-terminal side of certain basic residues in the MMP-3 propeptide. Metal ion chelators inhibited activator-dependent proteolysis, and activity was restored by low levels of ZnCl2. These catalytic properties suggest similarity to members of the insulinase superfamily of metalloendopeptidases with in vitro specificity for single arginine or paired basic processing sites in a variety of prohormones. Dibasic sites also exist in the propeptides of several MMPs including proMMP-3. This is the first report that cartilage produces a potent MMP proenzyme activator, opening the possibility of a novel intrinsic activation pathway for catabolic processes in this avascular tissue.