Osteoblast-lineage calcium/calmodulin-dependent kinase 2 delta and gamma regulates bone mass and quality.

Bone regulates its mass and quality in response to diverse mechanical, hormonal, and local signals. The bone anabolic or catabolic responses to these signals are often received by osteocytes, which then coordinate the activity of osteoblasts and osteoclasts on bone surfaces. We previously established that calcium/calmodulin-dependent kinase 2 (CaMKII) is required for osteocytes to respond to some bone anabolic cues in vitro. However, a role for CaMKII in bone physiology in vivo is largely undescribed. Here, we show that conditional codeletion of the most abundant isoforms of CaMKII (delta and gamma) in mature osteoblasts and osteocytes [Ocn-cre:Camk2d/Camk2g double-knockout (dCKO)] caused severe osteopenia in both cortical and trabecular compartments by 8 wk of age. In addition to having less bone mass, dCKO bones are of worse quality, with significant deficits in mechanical properties, and a propensity to fracture. This striking skeletal phenotype is multifactorial, including diminished osteoblast activity, increased osteoclast activity, and altered phosphate homeostasis both systemically and locally. These dCKO mice exhibited decreased circulating phosphate (hypophosphatemia) and increased expression of the phosphate-regulating hormone fibroblast growth factor 23. Additionally, dCKO mice expressed less bone-derived tissue nonspecific alkaline phosphatase protein than control mice. Consistent with altered phosphate homeostasis, we observed that dCKO bones were hypo-mineralized with prominent osteoid seams, analogous to the phenotypes of mice with hypophosphatemia. Altogether, these data reveal a fundamental role for osteocyte CaMKIIδ and CaMKIIγ in the maintenance of bone mass and bone quality and link osteoblast/osteocyte CaMKII to phosphate homeostasis.

Emergency department and transport predictors of neurological deterioration in patients with spontaneous intracranial hemorrhage.

BACKGROUND: Patients with spontaneous intracranial hemorrhage (sICH) and intracranial hypertension are associated with poor outcomes. Blood pressure variability (BPV) and neurological deterioration (ND) are known factors associated with sICH outcomes, but the relationship between BPV and ND in the hyperacute phase remains poorly described. We hypothesized that BPV is associated with ND during patients' initial emergency department (ED) stay and during interhospital transport (IHT) to a tertiary care center. METHODS: A retrospective study of adult patients with sICH was performed. Patients who were transferred from an ED to a tertiary care center between 01/01/2011 and 09/30/2015 and underwent external ventricular drainage were eligible. The outcome was ND at any time before arrival at a tertiary care center. Classification and Regression Tree (CART) analysis, a machine learning algorithm, was used to assign "relative variable importance" (RVI) for important predictive clinical factors. RESULTS: 153 eligible patients were analyzed. Sixty-five (42%) patients developed ND. Maximum ED systolic blood pressure (ED SBPMax) was most predictive of sICH patients developing ND (RVI = 100%). Other important factors for ND included standard deviation in SBP (SBPSD) during ED stay and IHT, with RVI of 43% and 20%, respectively. CONCLUSION: ED SBPMax was the strongest predictive factor of ND, while other BPV components were also significant. Our study found evidence that BPV should be prioritized as it may also increase the risk of ND among patients with sICH who required external ventricular drain placement. Future studies should examine whether fluctuations in BP in an ED or IHT setting are associated with increased risk of worsening outcomes.

Tenomodulin and Chondromodulin-1 Are Both Required to Maintain Biomechanical Function and Prevent Intervertebral Disc Degeneration.

OBJECTIVE: The underlying mechanisms and molecular factors influencing intervertebral disc (IVD) homeostasis and degeneration remain clinically relevant. Tenomodulin (Tnmd) and chondromodulin (Chm1) are antiangiogenic transmembrane glycoproteins, with cleavable C-terminus, expressed by IVD cells that are implicated in the onset of degenerative processes. We evaluate the organ-level biomechanical impact of knocking out Tnmd alone, and Tnmd and Chm1, simultaneously. DESIGN: Caudal (c5-8) and lumbar vertebrae (L1-4) of skeletally mature male and female 9-month-old wildtype (WT), Tnmd knockout (Tnmd-/-), and Tnmd/Chm1 double knockout (Tnmd-/-/Chm-/-) mice were used (n = 9-13 per group). Disc height index (DHI), histomorphological changes, and axial, torsional, creep, and failure biomechanical properties were evaluated. Differences were assessed by one-way ANOVA with post hoc Bonferroni-corrected comparisons (P < 0.05). RESULTS: Tnmd-/-/Chm1-/- IVDs displayed increased DHI and histomorphological scores that indicated increased IVD degeneration compared to the WT and Tnmd-/- groups. Double knockout IVDs required significantly less torque and energy to initiate torsional failure. Creep parameters were comparable between all groups, except for the slow time constant, which indicated faster outward fluid flow. Tnmd-/- IVDs lost fluid faster than the WT group, and this effect was amplified in the double knockout IVDs. CONCLUSION: Knocking out Tnmd and Chm1 affects IVD fluid flow and organ-level biomechanical function and therefore may play a role in contributing to IVD degeneration. Larger effects of the Tnmd and Chm1 double knockout mice compared to the Tnmd single mutant suggest that Chm1 may play a compensatory role in the Tnmd single mutant IVDs.

The cytoskeleton and connected elements in bone cell mechano-transduction.

Bone is a mechano-responsive tissue that adapts to changes in its mechanical environment. Increases in strain lead to increased bone mass acquisition, whereas decreases in strain lead to a loss of bone mass. Given that mechanical stress is a regulator of bone mass and quality, it is important to understand how bone cells sense and transduce these mechanical cues into biological changes to identify druggable targets that can be exploited to restore bone cell mechano-sensitivity or to mimic mechanical load. Many studies have identified individual cytoskeletal components - microtubules, actin, and intermediate filaments - as mechano-sensors in bone. However, given the high interconnectedness and interaction between individual cytoskeletal components, and that they can assemble into multiple discreet cellular structures, it is likely that the cytoskeleton as a whole, rather than one specific component, is necessary for proper bone cell mechano-transduction. This review will examine the role of each cytoskeletal element in bone cell mechano-transduction and will present a unified view of how these elements interact and work together to create a mechano-sensor that is necessary to control bone formation following mechanical stress.

Disparate bone anabolic cues activate bone formation by regulating the rapid lysosomal degradation of sclerostin protein.

The downregulation of sclerostin in osteocytes mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation of the Sost gene hours after stimulation. Using mouse models and rodent cell lines, we describe the rapid, minute-scale post-translational degradation of sclerostin protein by the lysosome following mechanical load and PTH. We present a model, integrating both new and established mechanically and hormonally activated effectors into the regulated degradation of sclerostin by lysosomes. Using a mouse forelimb mechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation by preventing sclerostin degradation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using human Gaucher disease iPSCs. These results reveal how bone anabolic cues post-translationally regulate sclerostin abundance in osteocytes to regulate bone formation.

In vitro Fluid Shear Stress Induced Sclerostin Degradation and CaMKII Activation in Osteocytes.

Bone is a dynamic tissue that adapts to changes in its mechanical environment. Mechanical stimuli pressurize interstitial fluid in the lacunar-canalicular system within the bone matrix, causing fluid shear stress (FSS) across bone embedded, mechano-sensitive osteocytes. Therefore, modeling this mechanical stimulus in vitro is vital for identifying mechano-transduction cascades that contribute to the regulation of mechano-responsive proteins, such as the Wnt/ß-catenin antagonist, sclerostin, which is reduced in response to FSS. Recently, we reported the rapid post-translational degradation of sclerostin protein in bone cells following FSS. Given the fundamental nature of sclerostin to bone physiology and the nuances of studying its rapid post-translational control, here, we detail our FSS protocol, and adaptations that can be made, to stimulate Ocy454 osteocyte-like cells to study sclerostin protein in vitro. While this protocol is optimized for detecting sclerostin degradation by western blot, this protocol can be adapted to examine transcriptional changes with RT-qPCR, cellular dynamics with live cell imaging, or secreted factors in the FSS buffer. This protocol utilizes 3D-printed FSS tips that are compatible with commercially available 96-well plates, allowing for high experimental accessibility, versatility, and throughput. However, this protocol can be adapted for any FSS chamber. It can also be combined with pharmacological inhibitors or genetic manipulations to interrogate the role of specific cellular components. In all, this experimental set-up and protocol is highly adaptable to allow for many experimental outcomes to examine many aspects of cell mechano-transduction.

Measuring the neutral zone of spinal motion segments: Comparison of multiple analysis methods to quantify spinal instability.

PURPOSE: Neutral zone (NZ) parameters in spinal biomechanics studies are sensitive to spinal instability, disc degeneration, and repair. Multiple methods in the literature quantify NZ, yet no consensus exists on applicability and comparability of methods. This study compares five different NZ quantification methods using two different load-deflection profiles. METHODS: Rat caudal and lumbar motion segments were tested in axial rotation to generate load-deflection curves with profiles exhibiting prominent distinction between elastic and NZ regions (ie, triphasic) and profiles that did not (ie, viscoelastic). NZ was quantified using five methods: trilinear, double sigmoid (DS), zero load, stiffness threshold (ST), and extrapolated elastic zone. Absolute agreement and consistency of NZ parameters were assessed using intraclass correlation (ICC), Bland-Altman analyses, and analysis of variance. RESULTS: For triphasic profiles, NZ magnitude exhibited high consistency (methods correlate but differ in absolute values), and only some methods exhibited agreement. For viscoelastic profiles, NZ magnitude showed limited consistency and no absolute agreement. NZ stiffness had high agreement and consistency across most methods and profiles. For triphasic profiles, the linear NZ regions for all methods were not well-described by a linear fit yet for viscoelastic profiles all methods characterized a linear NZ region. CONCLUSION: This NZ comparison study showed surprisingly limited agreement and consistency among NZ parameters with approximately 5% to 100% difference depending on the method and load-deflection profile. Nevertheless, the DS and ST methods appeared to be most comparable. We conclude that most NZ quantification methods cannot be applied interchangeably, highlighting a need to clearly state NZ calculation methods. Future studies are required to identify which methods are most sensitive to disc degeneration and repair in order to identify a "best" method.

Neonatal annulus fibrosus regeneration occurs via recruitment and proliferation of Scleraxis-lineage cells.

Intervertebral disc (IVD) injuries are a cause of degenerative changes in adults which can lead to back pain, a leading cause of disability. We developed a model of neonatal IVD regeneration with full functional restoration and investigate the cellular dynamics underlying this unique healing response. We employed genetic lineage tracing in mice using Scleraxis (Scx) and Sonic hedgehog (Shh) to fate-map annulus fibrosus (AF) and nucleus pulposus (NP) cells, respectively. Results indicate functional AF regeneration after severe herniation injury occurs in neonates and not adults. AF regeneration is mediated by Scx-lineage cells that lose ScxGFP expression and adopt a stem/progenitor phenotype (Sca-1, days 3-14), proliferate, and then redifferentiate towards type I collagen producing, ScxGFP+ annulocytes at day 56. Non Scx-lineage cells were also transiently observed during neonatal repair, including Shh-lineage cells, macrophages, and myofibroblasts; however, these populations were no longer detected by day 56 when annulocytes redifferentiate. Overall, repair did not occur in adults. These results identify an exciting cellular mechanism of neonatal AF regeneration that is predominantly driven by Scx-lineage annulocytes.

Phlpp1 is associated with human intervertebral disc degeneration and its deficiency promotes healing after needle puncture injury in mice.

Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD.

Biomechanical test protocols to detect minor injury effects in intervertebral discs.

Intervertebral discs (IVDs) maintain flexibility of the spine and bear mechanical load. Annulus fibrosus (AF) defects are associated with IVD degeneration and herniation which disrupt biomechanical function and can cause pain. AF puncture injuries can induce IVD degeneration but are needed to inject therapies. Identifying small AF defects with biomechanical testing can be difficult because IVDs have a complex, composite structure and nonlinear biomechanical properties that are dependent on AF fiber tension. It remains unclear how choice of biomechanical testing protocols affect the sensitivity of biomechanical properties to AF injuries. This study determined whether axial preload or magnitude of cyclic axial or torsional testing affected the ability to detect minor AF defects in rat caudal motion segments using ex vivo biomechanical testing. Intact and injured motion segments were subjected to a repeated measures study design with multiple biomechanical testing protocols that varied axial tension-compression force amplitude (±1.6 N, ±8.0 N, ±16.0 N), axial preload (-1.6 N, -8.0 N, -16.0 N, corresponding to -0.1 MPa, -0.5 MPa, and -1.0 MPa, respectively), and torsional rotation angle (±10°, ±15°, and ±20°). Biomechanical properties obtained from the lowest force testing conditions for axial tension-compression (±1.6 N), axial preload (-1.6 N), and angular rotation (±10°) exhibited the largest differences in biomechanical properties between intact and injured conditions. Biomechanical properties determined under low axial force or torsion amplitudes involve less AF fiber tension and were most sensitive to injury. Low force testing protocols are recommended for detecting minor structural AF defects and may enable more precise assessments of IVD injuries, healing or repair.

Annulus fibrosus cell phenotypes in homeostasis and injury: implications for regenerative strategies.

Despite considerable efforts to develop cellular, molecular, and structural repair strategies and restore intervertebral disk function after injury, the basic biology underlying intervertebral disk healing remains poorly understood. Remarkably, little is known about the origins of cell populations residing within the annulus fibrosus, or their phenotypes, heterogeneity, and roles during healing. This review focuses on recent literature highlighting the intrinsic and extrinsic cell types of the annulus fibrosus in the context of the injury and healing environment. Spatial, morphological, functional, and transcriptional signatures of annulus fibrosus cells are reviewed, including inner and outer annulus fibrosus cells, which we propose to be referred to as annulocytes. The annulus also contains peripheral cells, interlamellar cells, and potential resident stem/progenitor cells, as well as macrophages, T lymphocytes, and mast cells following injury. Phases of annulus fibrosus healing include inflammation and recruitment of immune cells, cell proliferation, granulation tissue formation, and matrix remodeling. However, annulus fibrosus healing commonly involves limited remodeling, with granulation tissues remaining, and the development of chronic inflammatory states. Identifying annulus fibrosus cell phenotypes during health, injury, and degeneration will inform reparative regeneration strategies aimed at improving annulus fibrosus healing.

Dietary advanced glycation end-product consumption leads to mechanical stiffening of murine intervertebral discs.

Back pain is a leading cause of disability and is strongly associated with intervertebral disc (IVD) degeneration. Reducing structural disruption and catabolism in IVD degeneration remains an important clinical challenge. Pro-oxidant and structure-modifying advanced glycation end-products (AGEs) contribute to obesity and diabetes, which are associated with increased back pain, and accumulate in tissues due to hyperglycemia or ingestion of foods processed at high heat. Collagen-rich IVDs are particularly susceptible to AGE accumulation due to their slow metabolic rates, yet it is unclear whether dietary AGEs can cross the endplates to accumulate in IVDs. A dietary mouse model was used to test the hypothesis that chronic consumption of high AGE diets results in sex-specific IVD structural disruption and functional changes. High AGE diet resulted in AGE accumulation in IVDs and increased IVD compressive stiffness, torque range and failure torque, particularly for females. These biomechanical changes were likely caused by significantly increased AGE crosslinking in the annulus fibrosus, measured by multiphoton imaging. Increased collagen damage measured with collagen hybridizing peptide did not appear to influence biomechanical properties and may be a risk factor as these animals age. The greater influence of high AGE diet on females is an important area of future investigation that may involve AGE receptors known to interact with estrogen. We conclude that high AGE diets can be a source for IVD crosslinking and collagen damage known to be important in IVD degeneration. Dietary modifications and interventions that reduce AGEs warrant further investigation and may be particularly important for diabetics, in whom AGEs accumulate more rapidly.

Neonatal mouse intervertebral discs heal with restored function following herniation injury.

Adult intervertebral discs (IVDs) have poor endogenous healing capacity, because of their challenging microenvironment and complex mechanical demands, which can result in painful IVD herniation. There are no regenerative strategies available to improve IVD healing and restore its function. Neonatal mice are excellent models of mammalian regeneration, but there are no studies of the regenerative capacity of neonatal IVDs. In this study, we developed a neonatal model of improved IVD healing to inform repair strategies after herniation. In vivo puncture injuries were performed to simulate herniation with complete annulus fibrosus (AF) tears in caudal IVDs of neonatal (postnatal d 5) and adult (4-6 mo) Scleraxis green fluorescent protein ( ScxGFP) mice. Acute and long-term healing responses were assessed with histologic, radiologic, and biomechanical measurements. Neonates underwent accelerated IVD healing compared to adults with functional restoration and enhanced structural repair after herniation. A population of ScxGFP- cells identified in the neonatal repair site may be associated with this improved healing and warrants future investigation. In summary, function of neonatal IVDs was restored after herniation injury, whereas that of adult discs was not. This improved healing response is likely driven by multiple mechanisms that may include differences in mechanical loading and available repair cells during growth.-Torre, O. M., Das, R., Berenblum, R. E., Huang, A. H., Iatridis, J. C. Neonatal mouse intervertebral discs heal with restored function following herniation injury.

Cell-Seeded Adhesive Biomaterial for Repair of Annulus Fibrosus Defects in Intervertebral Discs.

Defects in the annulus fibrosus (AF) of intervertebral discs allow nucleus pulposus tissue to herniate causing painful disability. Microdiscectomy procedures remove herniated tissue fragments, but unrepaired defects remain allowing reherniation or progressive degeneration. Cell therapies show promise to enhance repair, but methods are undeveloped and carriers are required to prevent cell leakage. To address this challenge, this study developed and evaluated genipin-crosslinked fibrin (FibGen) as an adhesive cell carrier optimized for AF repair that can deliver cells, match AF material properties, and have low risk of extrusion during loading. Part 1 determined that feasibility of bovine AF cells encapsulated in high concentration FibGen (F140G6: 140 mg/mL fibrinogen; 6 mg/mL genipin) for 7 weeks could maintain high viability, but had little proliferation or matrix deposition. Part 2 screened tissue mechanics and in situ failure testing of nine FibGen formulations (fibrin: 35-140 mg/mL; genipin: 1-6 mg/mL). F140G6 formulation matched AF shear and compressive properties and significantly improved failure strength in situ. Formulations with reduced genipin also exhibited satisfactory material properties and failure behaviors warranting further biological screening. Part 3 screened AF cells encapsulated in four FibGen formulations for 1 week and found that reduced genipin concentrations increased cell viability and glycosaminoglycan production. F70G1 (70 mg/mL fibrinogen; 1 mg/mL genipin) demonstrated balanced biological and biomechanical performance warranting further testing. We conclude that FibGen has potential to serve as an adhesive cell carrier to repair AF defects with formulations that can be tuned to enhance biomechanical and biological performance; future studies are required to develop strategies to enhance matrix production.

Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?

During intervertebral disc (IVD) injury and degeneration, annulus fibrosus (AF) cells experience large mechanical strains in a pro-inflammatory milieu. We hypothesized that TNF-α, an initiator of IVD inflammation, modifies AF cell mechanobiology via cytoskeletal changes, and interacts with mechanical strain to enhance pro-inflammatory cytokine production. Human AF cells (N=5, Thompson grades 2-4) were stretched uniaxially on collagen-I coated chambers to 0%, 5% (physiological) or 15% (pathologic) strains at 0.5Hz for 24h under hypoxic conditions with or without TNF-α (10ng/mL). AF cells were treated with anti-TNF-α and anti-IL-6. ELISA assessed IL-1ß, IL-6, and IL-8 production and immunocytochemistry measured F-actin, vinculin and α-tubulin in AF cells. TNF-α significantly increased AF cell pro-inflammatory cytokine production compared to basal conditions (IL-1ß:2.0±1.4-84.0±77.3, IL-6:10.6±9.9-280.9±214.1, IL-8:23.9±26.0-5125.1±4170.8pg/ml for basal and TNF-α treatment, respectively) as expected, but mechanical strain did not. Pathologic strain in combination with TNF-α increased IL-1ß, and IL-8 but not IL-6 production of AF cells. TNF-α treatment altered F-actin and α-tubulin in AF cells, suggestive of altered cytoskeletal stiffness. Anti-TNF-α (infliximab) significantly inhibited pro-inflammatory cytokine production while anti-IL-6 (atlizumab) did not. In conclusion, TNF-α altered AF cell mechanobiology with cytoskeletal remodeling that potentially sensitized AF cells to mechanical strain and increased TNF-α-induced pro-inflammatory cytokine production. Results suggest an interaction between TNF-α and mechanical strain and future mechanistic studies are required to validate these observations.

Design Requirements for Annulus Fibrosus Repair: Review of Forces, Displacements, and Material Properties of the Intervertebral Disk and a Summary of Candidate Hydrogels for Repair.

There is currently a lack of clinically available solutions to restore functionality to the intervertebral disk (IVD) following herniation injury to the annulus fibrosus (AF). Microdiscectomy is a commonly performed surgical procedure to alleviate pain caused by herniation; however, AF defects remain and can lead to accelerated degeneration and painful conditions. Currently available AF closure techniques do not restore mechanical functionality or promote tissue regeneration, and have risk of reherniation. This review determined quantitative design requirements for AF repair materials and summarized currently available hydrogels capable of meeting these design requirements by using a series of systematic PubMed database searches to yield 1500+ papers that were screened and analyzed for relevance to human lumbar in vivo measurements, motion segment behaviors, and tissue level properties. We propose a testing paradigm involving screening tests as well as more involved in situ and in vivo validation tests to efficiently identify promising biomaterials for AF repair. We suggest that successful materials must have high adhesion strength (∼0.2 MPa), match as many AF material properties as possible (e.g., approximately 1 MPa, 0. 3 MPa, and 30 MPa for compressive, shear, and tensile moduli, respectively), and have high tensile failure strain (∼65%) to advance to in situ and in vivo validation tests. While many biomaterials exist for AF repair, few undergo extensive mechanical characterization. A few hydrogels show promise for AF repair since they can match at least one material property of the AF while also adhering to AF tissue and are capable of easy implantation during surgical procedures to warrant additional optimization and validation.

Fibrin-genipin annulus fibrosus sealant as a delivery system for anti-TNFα drug.

BACKGROUND CONTEXT: Intervertebral discs (IVDs) are attractive targets for local drug delivery because they are avascular structures with limited transport. Painful IVDs are in a chronic inflammatory state. Although anti-inflammatories show poor performance in clinical trials, their efficacy treating IVD cells suggests that sustained, local drug delivery directly to painful IVDs may be beneficial. PURPOSE: The purpose of this study was to determine if genipin cross-linked fibrin (FibGen) with collagen Type I hollow spheres (CHS) can serve as a drug-delivery carrier for infliximab, the anti-tumor necrosis factor α (TNFα) drug. Infliximab was chosen as a model drug because of the known role of TNFα in increasing downstream production of several pro-inflammatory cytokines and pain mediators. Genipin cross-linked fibrin was used as drug carrier because it is adhesive, injectable, and slowly degrading hydrogel with the potential to seal annulus fibrosus (AF) defects. CHS allow simple and nondamaging drug loading and could act as a drug reservoir to improve sustained delivery. STUDY DESIGN/SETTING: This is a study of biomaterials and human AF cell culture to determine drug release kinetics and efficacy. METHODS: Infliximab was delivered at low and high concentrations using FibGen with and without CHS. Gels were analyzed for structure, drug release kinetics, and efficacy treating human AF cells after release. RESULTS: Fibrin showed rapid infliximab drug release but degraded quickly. CHS alone showed a sustained release profile, but the small spheres may not remain in a degenerated IVD with fissures. Genipin cross-linked fibrin showed steady and low levels of infliximab release that was increased when loaded with higher drug concentrations. Infliximab was bound in CHS when delivered within FibGen and was only released after enzymatic degradation. The infliximab released over 20 days retained its bioactivity as confirmed by the sustained reduction of interleukin (IL)-1ß, IL-6, IL-8, and TNFα concentrations produced by AF cells. CONCLUSIONS: Direct mixing of infliximab into FibGen was the simplest drug-loading protocol capable of sustained release. Results show feasibility of using drug-loaded FibGen for delivery of infliximab and, in the context with the literature, show potential to seal AF defects and partially restore IVD biomechanics. Future investigations are required to determine if drug-loaded FibGen can effectively deliver drugs, seal AF defects, and promote IVD repair or prevent further IVD degeneration in vivo.

Comparative mechanisms of cancer cell migration through 3D matrix and physiological microtracks.

Tumor cell invasion through the stromal extracellular matrix (ECM) is a key feature of cancer metastasis, and understanding the cellular mechanisms of invasive migration is critical to the development of effective diagnostic and therapeutic strategies. Since cancer cell migration is highly adaptable to physiochemical properties of the ECM, it is critical to define these migration mechanisms in a context-specific manner. Although extensive work has characterized cancer cell migration in two- and three-dimensional (3D) matrix environments, the migration program employed by cells to move through native and cell-derived microtracks within the stromal ECM remains unclear. We previously reported the development of an in vitro model of patterned type I collagen microtracks that enable matrix metalloproteinase-independent microtrack migration. Here we show that collagen microtracks closely resemble channel-like gaps in native mammary stroma ECM and examine the extracellular and intracellular mechanisms underlying microtrack migration. Cell-matrix mechanocoupling, while critical for migration through 3D matrix, is not necessary for microtrack migration. Instead, cytoskeletal dynamics, including actin polymerization, cortical tension, and microtubule turnover, enable persistent, polarized migration through physiological microtracks. These results indicate that tumor cells employ context-specific mechanisms to migrate and suggest that selective targeting of cytoskeletal dynamics, but not adhesion, proteolysis, or cell traction forces, may effectively inhibit cancer cell migration through preformed matrix microtracks within the tumor stroma.

Characterization of mechanics and cytocompatibility of fibrin-genipin annulus fibrosus sealant with the addition of cell adhesion molecules.

There is an unmet clinical need for a biomaterial sealant capable of repairing small annulus fibrosus (AF) defects. Causes of these defects include painful intervertebral disc herniations, microdiscectomy procedures, morbidity associated with needle puncture injury from discography, and future nucleus replacement procedures. This study describes the enhancements of a fibrin gel through genipin crosslinking (FibGen) and the addition of the cell adhesion molecules (CAMs), fibronectin and collagen. The gel's performance as a potential AF sealant is assessed using a series of in vitro tests. FibGen gels with CAMs had equivalent adhesive strength, gene expression, cytomorphology, and cell proliferation as fibrin alone. However, FibGen gels had enhanced material behaviors that were tunable to higher shear stiffness values and approximated human annulus tissue as compared with fibrin alone, were more dimensionally stable, and had a slower in vitro degradation rate. Cytomorphology of human AF cells cultured on FibGen gels exhibited increased elongation compared with fibrin alone, and the addition of CAMs to FibGen did not significantly affect elongation. This FibGen gel offers the promise of being used as a sealant material to repair small AF defects or to be used in combination with other biomaterials as an adhesive for larger defects.