Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

Painful musculoskeletal disorders such as intervertebral disc (IVD) degeneration associated with chronic low back pain (termed "Discogenic back pain", DBP), are a significant socio-economic burden worldwide and contribute to the growing opioid crisis. Yet there are very few if any successful interventions that can restore the tissue's structure and function while also addressing the symptomatic pain. Here we have developed a novel non-viral gene therapy, using engineered extracellular vesicles (eEVs) to deliver the developmental transcription factor FOXF1 to the degenerated IVD in an in vivo model. Injured IVDs treated with eEVs loaded with FOXF1 demonstrated robust sex-specific reductions in pain behaviors compared to control groups. Furthermore, significant restoration of IVD structure and function in animals treated with FOXF1 eEVs were observed, with significant increases in disc height, tissue hydration, proteoglycan content, and mechanical properties. This is the first study to successfully restore tissue function while modulating pain behaviors in an animal model of DBP using eEV-based non-viral delivery of transcription factor genes. Such a strategy can be readily translated to other painful musculoskeletal disorders.

Annulus Fibrosus Injury Induces Acute Neuroinflammation and Chronic Glial Response in Dorsal Root Ganglion and Spinal Cord-An In Vivo Rat Discogenic Pain Model.

Chronic painful intervertebral disc (IVD) degeneration (i.e., discogenic pain) is a major source of global disability needing improved knowledge on multiple-tissue interactions and how they progress in order improve treatment strategies. This study used an in vivo rat annulus fibrosus (AF) injury-driven discogenic pain model to investigate the acute and chronic changes in IVD degeneration and spinal inflammation, as well as sensitization, inflammation, and remodeling in dorsal root ganglion (DRG) and spinal cord (SC) dorsal horn. AF injury induced moderate IVD degeneration with acute and broad spinal inflammation that progressed to DRG to SC changes within days and weeks, respectively. Specifically, AF injury elevated macrophages in the spine (CD68) and DRGs (Iba1) that peaked at 3 days post-injury, and increased microglia (Iba1) in SC that peaked at 2 weeks post-injury. AF injury also triggered glial responses with elevated GFAP in DRGs and SC at least 8 weeks post-injury. Spinal CD68 and SC neuropeptide Substance P both remained elevated at 8 weeks, suggesting that slow and incomplete IVD healing provides a chronic source of inflammation with continued SC sensitization. We conclude that AF injury-driven IVD degeneration induces acute spinal, DRG, and SC inflammatory crosstalk with sustained glial responses in both DRGs and SC, leading to chronic SC sensitization and neural plasticity. The known association of these markers with neuropathic pain suggests that therapeutic strategies for discogenic pain need to target both spinal and nervous systems, with early strategies managing acute inflammatory processes, and late strategies targeting chronic IVD inflammation, SC sensitization, and remodeling.

Galectin-3 and RAGE differentially control advanced glycation endproduct-induced collagen damage in murine intervertebral disc organ culture.

Background: Back and neck pain are leading causes of global disability that are associated with intervertebral disc (IVD) degeneration. Causes of IVD degeneration are multifactorial, and diet, age, and diabetes have all been linked to IVD degeneration. Advanced glycation endproducts (AGEs) accumulate in the IVD as a result of aging, diet, and diabetes, and AGE accumulation in the IVD has been shown to induce oxidative stress and catabolic activity that result in collagen damage. An association between AGE accumulation and IVD degeneration is emerging, yet mechanism behind this association remains unclear. The Receptor for AGEs (RAGE) is thought to induce catabolic responses in the IVD, and the AGE receptor Galectin 3 (Gal3) had a protective effect in other tissue systems but has not been evaluated in the IVD. Methods: This study used an IVD organ culture model with genetically modified mice to analyze the roles of RAGE and Gal3 in an AGE challenge. Results: Gal3 was protective against an AGE challenge in the murine IVD ex vivo, limiting collagen damage and biomechanical property changes. Gal3 receptor levels in the AF significantly decreased upon an AGE challenge. RAGE was necessary for AGE-induced collagen damage in the IVD, and RAGE receptor levels in the AF significantly increased upon AGE challenge. Discussion: These findings suggest both RAGE and Gal3 are important in the IVD response to AGEs and highlight Gal3 as an important receptor with protective effects on collagen damage. This research improves understanding the mechanisms of AGE-induced IVD degeneration and suggests Gal3 receptor modulation as a potential target for preventative and therapeutic treatment for IVD degeneration.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - an in vivo rat model.

BACKGROUND CONTEXT: Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSE: Establish in vivo rat lumbar EP microfracture model and assess crosstalk between IVD, vertebra and spinal cord. STUDY DESIGN/SETTING: In vivo rat EP microfracture injury model with characterization of IVD degeneration, vertebral remodeling, spinal cord substance P (SubP), and pain-related behaviors. METHODS: EP-injury was induced in 5 month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs by puncturing through the cephalad vertebral body and EP into the NP of the IVDs followed by intradiscal injections of TNFα (n=7) or PBS (n=6), compared with Sham (surgery without EP-injury, n=6). The EP-injury model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and µCT, and spinal cord SubP. RESULTS: Surgically-induced EP microfracture with PBS and TNFα injection induced IVD degeneration with decreased IVD height and MRI T2 signal, vertebral remodeling, and secondary damage to cartilage EP adjacent to the injury. Both EP injury groups showed MC-like changes around defects with hypointensity on T1-weighted and hyperintensity on T2-weighted MRI, suggestive of MC type 1. EP injuries caused significantly decreased paw withdrawal threshold, reduced axial grip, and increased spinal cord SubP, suggesting axial spinal discomfort and mechanical hypersensitivity and with spinal cord sensitization. CONCLUSIONS: Surgically-induced EP microfracture can cause crosstalk between IVD, vertebra, and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCE: This rat EP microfracture model was validated to induce broad spinal degenerative changes that may be useful to improve understanding of MC-like changes and for therapeutic screening.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - An In Vivo Rat Model.

BACKGROUND CONTEXT : Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSE : Establish in vivo rat lumbar EP microfracture model with painful phenotype. STUDY DESIGN/SETTING : In vivo rat study to characterize EP-injury model with characterization of IVD degeneration, vertebral bone marrow remodeling, spinal cord sensitization, and pain-related behaviors. METHODS : EP-driven degeneration was induced in 5-month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs through the proximal vertebral body injury with intradiscal injections of TNFα (n=7) or PBS (n=6), compared to Sham (surgery without EP-injury, n=6). The EP-driven model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and µCT analyses, and spinal cord substance P (SubP). RESULTS : EP injuries induced IVD degeneration with decreased IVD height and MRI T2 values. EP injury with PBS and TNFα both showed MC type1-like changes on T1 and T2-weighted MRI, trabecular bone remodeling on µCT, and damage in cartilage EP adjacent to the injury. EP injuries caused significantly decreased paw withdrawal threshold and reduced grip forces, suggesting increased pain sensitivity and axial spinal discomfort. Spinal cord dorsal horn SubP was significantly increased, indicating spinal cord sensitization. CONCLUSIONS : EP microfracture can induce crosstalk between vertebral bone marrow, IVD and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCE : This rat EP microfracture model of IVD degeneration was validated to induce MC-like changes and pain-like behaviors that we hope will be useful to screen therapies and improve treatment for EP-drive pain.

Spinal Cord Sensitization and Spinal Inflammation from an In Vivo Rat Endplate Injury Associated with Painful Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration with Modic-like changes is strongly associated with pain. Lack of effective disease-modifying treatments for IVDs with endplate (EP) defects means there is a need for an animal model to improve understanding of how EP-driven IVD degeneration can lead to spinal cord sensitization. This rat in vivo study determined whether EP injury results in spinal dorsal horn sensitization (substance P, SubP), microglia (Iba1) and astrocytes (GFAP), and evaluated their relationship with pain-related behaviors, IVD degeneration, and spinal macrophages (CD68). Fifteen male Sprague Dawley rats were assigned into sham or EP injury groups. At chronic time points, 8 weeks after injury, lumbar spines and spinal cords were isolated for immunohistochemical analyses of SubP, Iba1, GFAP, and CD68. EP injury most significantly increased SubP, demonstrating spinal cord sensitization. Spinal cord SubP-, Iba1- and GFAP-immunoreactivity were positively correlated with pain-related behaviors, indicating spinal cord sensitization and neuroinflammation play roles in pain responses. EP injury increased CD68 macrophages in the EP and vertebrae, and spinal cord SubP-, Iba1- and GFAP-ir were positively correlated with IVD degeneration and CD68-ir EP and vertebrae. We conclude that EP injuries result in broad spinal inflammation with crosstalk between spinal cord, vertebrae and IVD, suggesting that therapies must address neural pathologies, IVD degeneration, and chronic spinal inflammation.

Genipin-crosslinked fibrin seeded with oxidized alginate microbeads as a novel composite biomaterial strategy for intervertebral disc cell therapy.

Discectomy procedures alleviate disability caused by intervertebral disc (IVD) herniation, but do not repair herniation-induced annulus fibrosus (AF) defects. Cell therapy shows promise for IVD repair, yet cell delivery biomaterials capable of sealing AF defects and restoring biomechanical function have poor biological performance. To balance the biomechanical and biological demands of IVD cell delivery biomaterials, we engineered an injectable composite biomaterial using cell-laden, degradable oxidized alginate (OxAlg) microbeads (MBs) to deliver AF cells within high-modulus genipin-crosslinked fibrin (FibGen) hydrogels (FibGen + MB composites). Conceptually, the high-modulus FibGen would immediately stabilize injured IVDs, while OxAlg MBs would protect and release cells required for long-term healing. We first showed that AF cells microencapsulated in OxAlg MBs maintained high viability and, upon release, displayed phenotypic AF cell morphology and gene expression. Next, we created cell-laden FibGen + MB composites and demonstrated that OxAlg MBs functionalized with RGD peptides (MB-RGD) minimized AF cell apoptosis and retained phenotypic gene expression. Further, we showed that cell-laden FibGen + MB composites are biomechanically stable and promote extracellular matrix (ECM) synthesis in long-term in vitro culture. Lastly, we evaluated cell-laden FibGen + MB-RGD composites in a long-term bovine caudal IVD organ culture bioreactor and found that composites had low herniation risk, provided superior biomechanical and biological repair to discectomy controls, and retained anabolic cells within the IVD injury space. This novel injectable composite hydrogel strategy shows promise as an IVD cell delivery sealant with potentially broad applications for its capacity to balance biomechanical and biological performance.

Ex vivo biomechanical evaluation of Acute lumbar endplate injury and comparison to annulus fibrosus injury in a rat model.

Back pain is often associated with intervertebral disc (IVD) degeneration, and IVD degeneration phenotypes are commonly characterized by annulus fibrosus (AF)-driven and endplate (EP)-driven phenotypes. Few studies of EP injury exist in animal models, even though clinical studies show EP lesions are strongly associated with IVD pathology and pain. This project established an ex-vivo rat lumbar EP injury model and characterized effects of EP injury on motion segment biomechanical properties, as compared to AF injury, a common way of inducing IVD degeneration. Lumbar motion segments (39 total vertebra-IVD-vertebra sections) assigned to Intact (L1/L2), AF injury and EP injury (L3/L4 and L5/L6 randomly selected), and biomechanically tested in axial tension-compression, stress-relaxation and torsional testing in pre-injury and post-injury conditions using a repeated-measures design. EP injury involved superior vertebra endplate puncture transcorporeally and obliquely. AF injury involved mid-line punctures anterior and bilaterally. Axial ROM, tensile stiffness, hysteresis, and neutral zone stiffness were significantly affected by EP injury but not AF injury. Torque range, torsional stiffness and torsional neutral zone stiffness were significantly affected by AF injury but not EP injury. Stress-relaxation fast time constant was decreased for EP injury. EP and AF injuries induced distinct biomechanical changes in lumbar motion segments with EP injury having the largest impact on axial biomechanical properties and AF injury most prominently affecting torsional properties. This study deepens the understanding of biomechanical mechanism of EP-driven low back pain and provides methods and biomechanical characterization for future in vivo studies.

High fat diet causes inferior vertebral structure and function without disc degeneration in RAGE-KO mice.

Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences. This study determined if high fat (HF) diet causes structural and functional changes to vertebrae and intervertebral discs (IVDs); if these changes are modulated in mice with systematic ablation for the receptor for advanced glycation endproducts (RAGE-KO); and if these changes are sex-dependent. Wild-type (WT) and RAGE-KO mice were fed a low fat (LF) or HF diet for 12 weeks starting at 6 weeks, representing the juvenile population. HF diet led to weight/fat gain, glucose intolerance, and increased cytokine levels (IL-5, MIG, and RANTES); with less fat gain in RAGE-KO females. Most importantly, HF diet reduced vertebral trabecular bone volume fraction and compressive and shear moduli, without a modifying effect of RAGE-KO, but with a more pronounced effect in females. HF diet caused reduced cortical area fraction only in WT males. Neither HF diet nor RAGE-KO affected IVD degeneration grade. Biomechanical properties of coccygeal motion segments were affected by RAGE-KO but not diet, with some interactions identified. In conclusion, HF diet resulted in inferior vertebral structure and function with some sex differences, no IVD degeneration, and few modifying effects of RAGE-KO. These structural and functional deficiencies with HF diet provide further evidence that diet can affect spinal structures and may increase the risk for spinal injury and degeneration with aging and additional stressors. Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences.

Development of a two-part biomaterial adhesive strategy for annulus fibrosus repair and ex vivo evaluation of implant herniation risk.

Intervertebral disc (IVD) herniation causes pain and disability, but current discectomy procedures alleviate pain without repairing annulus fibrosus (AF) defects. Tissue engineering strategies seal AF defects by utilizing hydrogel systems to prevent recurrent herniation, however current biomaterials are limited by poor adhesion to wetted tissue surfaces or low failure strength resulting in considerable risk of implant herniation upon spinal loading. Here, we developed a two-part repair strategy comprising a dual-modified (oxidized and methacrylated) glycosaminoglycan that can chemically adsorb an injectable interpenetrating network hydrogel composed of fibronectin-conjugated fibrin and poly (ethylene glycol) diacrylate (PEGDA) to covalently bond the hydrogel to AF tissue. We show that dual-modified hyaluronic acid imparts greater adhesion to AF tissue than dual-modified chondroitin sulfate, where the degree of oxidation is more strongly correlated with adhesion strength than methacrylation. We apply this strategy to an ex vivo bovine model of discectomy and demonstrate that PEGDA molecular weight tunes hydrogel mechanical properties and affects herniation risk, where IVDs repaired with low-modulus hydrogels composed of 20kDa PEGDA failed at levels at or exceeding discectomy, the clinical standard of care. This strategy bonds injectable hydrogels to IVD extracellular matrix proteins, is optimized to seal AF defects, and shows promise for IVD repair.

Advanced glycation end products cause RAGE-dependent annulus fibrosus collagen disruption and loss identified using in situ second harmonic generation imaging in mice intervertebral disk in vivo and in organ culture models.

Aging and diabetes are associated with increased low-back pain and intervertebral disk (IVD) degeneration yet causal mechanisms remain uncertain. Advanced glycation end products (AGEs), which accumulate in IVDs from aging and are implicated in diabetes-related disorders, alter collagen and induce proinflammatory conditions. A need exists for methods that assess IVD collagen quality and degradation in order to better characterize specific structural changes in IVDs due to AGE accumulation and to identify roles for the receptor for AGEs (RAGE). We used multiphoton microscopy with second harmonic generation (SHG), collagen-hybridizing peptide (CHP), and image analysis methods to characterize effects of AGEs and RAGE on collagen quality and quantity in IVD annulus fibrosus (AF). First, we used SHG imaging on thin sections with an in vivo dietary mouse model and determined that high-AGE (H-AGE) diets increased AF fibril disruption and collagen degradation resulting in decreased total collagen content, suggesting an early degenerative cascade. Next, we used in situ SHG imaging with an ex vivo IVD organ culture model of AGE challenge on wild type and RAGE-knockout (RAGE-KO) mice and determined that early degenerative changes to collagen quality and degradation were RAGE dependent. We conclude that AGE accumulation leads to RAGE-dependent collagen disruption in the AF and can initiate molecular and tissue level collagen disruption. Furthermore, SHG and CHP analyzes were sensitive to collagenous alterations at multiple hierarchical levels due to AGE and may be useful in identifying additional contributors to collagen damage in IVD degeneration processes.

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.

Composite biomaterial repair strategy to restore biomechanical function and reduce herniation risk in an ex vivo large animal model of intervertebral disc herniation with varying injury severity.

Back pain commonly arises from intervertebral disc (IVD) damage including annulus fibrosus (AF) defects and nucleus pulposus (NP) loss. Poor IVD healing motivates developing tissue engineering repair strategies. This study evaluated a composite injectable IVD biomaterial repair strategy using carboxymethylcellulose-methylcellulose (CMC-MC) and genipin-crosslinked fibrin (FibGen) that mimic NP and AF properties, respectively. Bovine ex vivo caudal IVDs were evaluated in cyclic compression-tension, torsion, and compression-to-failure tests to determine IVD biomechanical properties, height loss, and herniation risk following experimentally-induced severe herniation injury and discectomy (4 mm biopsy defect with 20% NP removed). FibGen with and without CMC-MC had failure strength similar to discectomy injury suggesting no increased risk compared to surgical procedures, yet no biomaterials improved axial or torsional biomechanical properties suggesting they were incapable of adequately restoring AF tension. FibGen had the largest failure strength and was further evaluated in additional discectomy injury models with varying AF defect types (2 mm biopsy, 4 mm cruciate, 4 mm biopsy) and NP removal volume (0%, 20%). All simulated discectomy defects significantly compromised failure strength and biomechanical properties. The 0% NP removal group had mean values of axial biomechanical properties closer to intact levels than defects with 20% NP removed but they were not statistically different and 0% NP removal also decreased failure strength. FibGen with and without CMC-MC failed at super-physiological stress levels above simulated discectomy suggesting repair with these tissue engineered biomaterials may perform better than discectomy alone, although restored biomechanical function may require additional healing with the potential application of these biomaterials as sealants and cell/drug delivery carriers.

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.

Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N-cadherin expression.

The nucleus pulposus (NP) of intervertebral discs (IVD) undergoes dramatic changes with aging including loss of its gelatinous structure and large, vacuolated notochordal cells (NCs) in favor of a matrix-rich structure populated by small NP cells (sNPCs). NP maturation also involves a loading-pattern shift from pressurization to matrix deformations, and these events are thought to predispose to degeneration. Little is known of the triggering events and cellular alterations involved with NP maturation, which remains a fundamental open spinal mechanobiology question. A mouse IVD organ culture model was used to test the hypotheses that hyperosmotic overloading will induce NP maturation with transition of NCs to sNPCs while also increasing matrix accumulation and altering osmoregulatory and mechanotransductive proteins. Results indicated that static hyperosmolarity, as might occur during growth, caused maturation of NCs to sNPCs and involved a cellular differentiation process since known NC markers (cytokeratin-8, -19, and sonic hedgehog) persisted without increased cell apoptosis. Osmosensitive channels Aquaporin 3 (Aqp3) and transient receptor potential vanilloid-4 (TRPV4) expression were both modified with altered osmolarity, but increased Aqp3 with hyperosmolarity was associated with NC to sNPC differentiation. NC to sNPC differentiation was accompanied by a shift in cellular mechanotransduction proteins with decreased N-cadherin adhesions and increased Connexin 43 connexons. We conclude that hyperosmotic overloading can promote NC differentiation into sNPCs. This study identified osmolarity as a triggering mechanism for notochordal cell differentiation with associated shifts in osmoregulatory and mechanotransductive proteins that are likely to play important roles in intervertebral disc aging. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:788-798, 2018.

Accumulation and localization of macrophage phenotypes with human intervertebral disc degeneration.

BACKGROUND CONTEXT: Chronic inflammation is an important component of intervertebral disc (IVD) degeneration, but there is limited knowledge about the identity and source of inflammatory cells involved with the degenerative processes. Macrophages can exhibit multiple phenotypes and are known inflammatory regulators in many tissues, but their phenotypes have not been characterized in IVD degeneration. PURPOSE: We aimed to characterize accumulation and localization of macrophages in IVD degeneration. STUDY DESIGN/SETTING: This is an exploratory study to characterize macrophage phenotypes in human cadaver IVDs and the effects of injury and degeneration using multiple immunohistochemistry methods. OUTCOME MEASURES: Percent positivity of immunohistochemical markers specific for CCR7, CD163, and CD206, and qualitative assessments of dual immunofluorescence and immunostaining localization were the outcome measures. METHODS: Macrophages were identified in human cadaveric IVDs with immunohistochemistry using cell surface markers CCR7, CD163, and CD206, which are associated with proinflammatory M1, remodeling M2c, and anti-inflammatory M2a phenotypes, respectively. Variations in the accumulation and localization of macrophage markers with degenerative grade across subjects and within donors are described. RESULTS: Cells expressing all three macrophage markers were found in all degenerative IVDs, but not in the healthiest IVDs. Cells expressing CCR7 and CD163, but not CD206, significantly increased with degenerative grade. Many cells also co-expressed multiple macrophage markers. Across all degenerative grades, CCR7+ and CD163+ were significantly more present in unhealthy nucleus pulposus (NP), annulus fibrosus (AF), and end plate (EP) regions exhibiting structural irregularities and defects. Positively stained cells in the NP and AF closely resembled resident IVD cells, suggesting that IVD cells can express macrophage cell surface markers. In the EP, there were increasing trends of positively stained cells with atypical morphology and distribution, suggesting a source for exogenous macrophage infiltration into the IVD. CONCLUSIONS: Chronic inflammatory conditions of IVD degeneration appear to involve macrophages or macrophage-like cells, as expression of multiple macrophage markers increased with degeneration, especially around unhealthy regions with defects and the EP. Knowledge of macrophage phenotypes and their localization better elucidates the complex injury and repair processes in IVDs and may eventually lead to novel treatments.

Annular puncture with tumor necrosis factor-alpha injection enhances painful behavior with disc degeneration in vivo.

BACKGROUND CONTEXT: Painfulintervertebral disc degeneration is extremely common and costly. Effective treatments are lacking because the nature of discogenic pain is complex with limited capacity to distinguish painful conditions from age-related changes in the spine. Hypothesized sources of discogenic pain include chronic inflammation, neurovascular ingrowth, and structural disruption. PURPOSE: This study aimed to investigate inflammation, pro-neurovascular growth factors, and structural disruption as sources of painful disc degeneration STUDY DESIGN/SETTING: This study used an in vivo study to address these hypothesized mechanisms with anterior intradiscal injections of tumor necrosis factor-alpha (TNFα), pro-neurovascular growth factors: nerve growth factor and vascular endothelial growth factor (NGF and VEGF), and saline with additional sham surgery and naïve controls. Depth of annular puncture was also evaluated for its effects on structural and painful degeneration. METHODS: Rat lumbar discs were punctured (shallow or deeper puncture) and intradiscally injected with saline, TNFα, or NGF and VEGF. Structural disc degeneration was assessed using X-ray, magnetic resonance imaging (MRI), and histology. The rat painful condition was evaluated using Von Frey hyperalgesia measurements, and substance P immunostaining in dorsal root ganglion (DRG) was performed to determine the source of pain. RESULTS: Saline injection increased painful responses with degenerative changes in disc height, MRI intensity, and morphologies of disc structure and cell. TNFα and NGF/VEGF accelerated painful behavior, and TNFα-injected animals had increased substance P in DRGs. Deeper punctures led to more severe disc degeneration. Multiple regression analysis showed that the painful behavior was correlated with disc height loss. CONCLUSIONS: We concluded that rate and severity of structural disc degeneration was associated with the amount of annular disruption and puncture depth. The painful behavior was associated with disc height loss and discal inflammatory state, whereas pro-inflammatory cytokines might play a more important role in the level of pain, which might have resulted from enhanced DRG sensitization. These in vivo painful disc degeneration models with different severities of structural changes may be useful for investigating discogenic pain mechanisms and for screening therapies, although interpretations must note the differences between all surgically induced animal models and the human condition.

Development of a bovine decellularized extracellular matrix-biomaterial for nucleus pulposus regeneration.

Painful intervertebral disc (IVD) degeneration is a common cause for spinal surgery. There is a clinical need to develop injectable biomaterials capable of promoting IVD regeneration, yet many available biomaterials do not mimic the native extracellular matrix (ECM) or promote matrix production. This study aimed to develop a decellularized injectable bovine ECM material that maintains structural and compositional features of native tissue and promotes nucleus pulposus (NP) cell (NPC) and mesenchymal stem cell (MSC) adaption. Injectable decellularized ECM constructs were created using 3 NP tissue decellularization methods (con.A: sodium deoxycholate, con.B: sodium deoxycholate & sodium dodecyl sulfate, con.C: sodium deoxycholate, sodium dodecyl sulfate & TritonX-100) and evaluated for protein, microstructure, and for cell adaptation in 21 day human NPC and MSC culture experiments. Con.A was most efficient at DNA depletion, preserved best collagen microstructure and content, and maintained the highest glycosaminoglycan (GAG) content. NPCs in decellularized constructs of con.A&B demonstrated newly synthesized GAG production, which was apparent from "halos" of GAG staining surrounding seeded NPCs. Con.A also promoted MSC adaption with high cell viability and ECM production. The injectable decellularized NP biomaterial that used sodium deoxycholate without additional decellularization steps maintained native NP tissue structure and composition closest to natural ECM and promoted cellular adaptation of NP cells and MSCs. This natural decellularized biomaterial warrants further investigation for its potential as an injectable cell seeded supplement to augment NP replacement biomaterials and deliver NPCs or MSCs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:876-888, 2016.

Osteocyte apoptosis is required for production of osteoclastogenic signals following bone fatigue in vivo.

Osteocyte apoptosis is spatially, temporally and functionally linked to the removal and replacement of microdamage in the bone. Recently we showed that microdamage elicits distinct responses in two populations of osteocytes near the injury site. Osteocytes directly adjacent to microdamage undergo apoptosis, whereas there is a second group of osteocytes located adjacent to the apoptotic population that upregulate expression of osteoclastogenic signaling molecules. In this study we used the pan-caspase inhibitor QVD to test the hypothesis that osteocyte apoptosis is an obligatory step in the production of key osteoclastogenic signals by in situ osteocytes in fatigue-damaged bone. We found, based on real-time PCR and immunohistochemistry assays, that expression of the apoptosis marker caspase-3 as well osteoclastogenic proteins RANKL and VEGF were increased following fatigue, while expression of the RANKL antagonist OPG decreased. However, when apoptosis was inhibited using QVD, these changes in gene expression were completely blocked. This dependence on apoptosis for neighboring non-apoptotic cells to produce signals that promote tissue remodeling also occurs in response to focal ischemic injury in the brain and heart, indicating that osteoclastic bone remodeling follows a common paradigm for localized tissue repair.

Combined anti-inflammatory and anti-AGE drug treatments have a protective effect on intervertebral discs in mice with diabetes.

OBJECTIVE: Diabetes and low back pain are debilitating diseases and modern epidemics. Diabetes and obesity are also highly correlated with intervertebral disc (IVD) degeneration and back pain. Advanced-glycation-end-products (AGEs) increase reactive-oxygen-species (ROS) and inflammation, and are one cause for early development of diabetes mellitus. We hypothesize that diabetes results in accumulation of AGEs in spines and associated spinal pathology via increased catabolism. We present a mouse model showing that: 1) diabetes induces pathological changes to structure and composition of IVDs and vertebrae; 2) diabetes is associated with accumulation of AGEs, TNFα, and increased catabolism spinal structures; and 3) oral-treatments with a combination of anti-inflammatory and anti-AGE drugs mitigate these diabetes-induced degenerative changes to the spine. METHODS: Three age-matched groups of ROP-Os mice were compared: non-diabetic, diabetic (streptozotocin (STZ)-induced), or diabetic mice treated with pentosan-polysulfate (anti-inflammatory) and pyridoxamine (AGE-inhibitor). Mice were euthanized and vertebra-IVD segments were analyzed by µCT, histology and Immunohistochemistry. RESULTS: Diabetic mice exhibited several pathological changes including loss in IVD height, decreased vertebral bone mass, decreased glycosaminoglycan content and morphologically altered IVDs with focal deposition of tissues highly expressing TNFα, MMP-13 and ADAMTS-5. Accumulation of larger amounts of methylglyoxal suggested that AGE accumulation was associated with these diabetic degenerative changes. However, treatment prevented or reduced these pathological effects on vertebrae and IVD. CONCLUSION: This is the first study to demonstrate specific degenerative changes to nucleus pulposus (NP) morphology and their association with AGE accumulation in a diabetic mouse model. Furthermore, this is the first study to demonstrate that oral-treatments can inhibit AGE-induced ROS and inflammation in spinal structures and provide a potential treatment to slow progression of degenerative spine changes in diabetes. Since diabetes, IVD degeneration, and accumulation of AGEs are frequent consequences of aging, early treatments to reduce AGE-induced ROS and Inflammation may have broad public-health implications.