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.

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.

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.

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.

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.

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.

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.

Structural, compositional, and biomechanical alterations of the lumbar spine in rats with mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome).

Mucopolysaccharidosis (MPS) VI is an inherited lysosomal storage disorder resulting from deficiency of N-acetylgalactosamine-4-sulfatase activity and subsequent accumulation of incompletely degraded dermatan sulfate (DS) containing glycosaminoglycans (GAGs). Painful spinal deformities are commonly found in MPS VI patients. We characterized lumbar spine structure, composition, and biomechanics in a naturally occurring rat MPS VI model and evaluated the role of MMP-13, ADAMTS-5 and TNF-α in modulating the observed changes. MPS VI rats had discs with large vacuolated cells and sizable nuclear defects. MPS spine segments also had structural and functional changes suggestive of spinal instability, including decreased nuclear pressurization, increased joint laxity and increased disc height index. These functional changes were at least partly associated with elevated ADAMTS-5, MMP-13, and TNF-α. Vertebral and endplate biomechanics were also affected by MPS VI with decreased failure load and stiffness. The discal and vertebral dysfunctions observed in MPS VI rats are likely to be associated with pathological spinal conditions, similar to those that afflict MPS patients. Our findings also suggest more broadly that abnormal accumulation of GAGs and the associated chronic pro-inflammatory and catabolic cascade may also be a source of spinal dysfunction.

Activation of resorption in fatigue-loaded bone involves both apoptosis and active pro-osteoclastogenic signaling by distinct osteocyte populations.

Osteocyte apoptosis is required to initiate osteoclastic bone resorption following fatigue-induced microdamage in vivo; however, it is unclear whether apoptotic osteocytes also produce the signals that induce osteoclast differentiation. We determined the spatial and temporal patterns of osteocyte apoptosis and expression of pro-osteoclastogenic signaling molecules in vivo. Ulnae from female Sprague-Dawley rats (16-18weeks old) were cyclically loaded to a single fatigue level, and tissues were analyzed 3 and 7days later (prior to the first appearance of osteoclasts). Expression of genes associated with osteoclastogenesis (RANKL, OPG, VEGF) and apoptosis (caspase-3) were assessed by qPCR using RNA isolated from 6mm segments of ulnar mid-diaphysis, with confirmation and spatial localization of gene expression performed by immunohistochemistry. A novel double staining immunohistochemistry method permitted simultaneous localization of apoptotic osteocytes and osteocytes expressing pro-osteoclastogenic signals relative to microdamage sites. Osteocyte staining for caspase-3 and osteoclast regulatory signals exhibited different spatial distributions, with apoptotic (caspase 3-positive) cells highest in the damage region and declining to control levels within several hundred microns of the microdamage focus. Cells expressing RANKL or VEGF peaked between 100 and 300µm from the damage site, then returned to control levels beyond this distance. Conversely, osteocytes in non-fatigued control bones expressed OPG. However, OPG staining was reduced markedly in osteocytes immediately surrounding microdamage. These results demonstrate that while osteocyte apoptosis triggers the bone remodeling response to microdamage, the neighboring non-apoptotic osteocytes are the major source of pro-osteoclastogenic signals. Moreover, both the apoptotic and osteoclast-signaling osteocyte populations are localized in a spatially and temporally restricted pattern consistent with the targeted nature of this remodeling response.

BMP-12 treatment of adult mesenchymal stem cells in vitro augments tendon-like tissue formation and defect repair in vivo.

We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ~80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering.

Physiological loading of joints prevents cartilage degradation through CITED2.

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.