TNFR1-mediated senescence and lack of TNFR2-signaling limit human intervertebral disc cell repair in back pain conditions.

Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of TNFα-receptors (TNFRs) in contributing to poor healing in painful IVDD. We first isolated IVDD tissue of back pain subjects and determined the complex pro-inflammatory mixture contained many chemokines for recruiting inflammatory cells. Single-cell RNA-sequencing of human IVDD tissues revealed these pro-inflammatory cytokines were dominantly expressed by a small macrophage-population. Human annulus fibrosus (hAF) cells treated with IVDD-conditioned media (CM) underwent senescence with greatly reduced metabolic rates and limited inflammatory responses. TNFR1 inhibition partially restored hAF cell metabolism sufficiently to enable a robust chemokine and cytokine response to CM. We showed that the pro-reparative TNFR2 was very limited on hIVD cell membranes so that TNFR2 inhibition with blocking antibodies or activation using Atsttrin had no effect on hAF cells with CM challenge. However, TNFR2 was expressed in high levels on macrophages identified in scRNA-seq analyses, suggesting their role in repair responses. Results therefore point to therapeutic strategies for painful IVDD involving immunomodulation of TNFR1 signaling in IVD cells to enhance metabolism and enable a more robust inflammatory response including recruitment or delivery of TNFR2 expressing immune cells to enhance IVD repair.

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.

WNT7A suppresses adipogenesis of skeletal muscle mesenchymal stem cells and fatty infiltration through the alternative Wnt-Rho-YAP/TAZ signaling axis.

Intramuscular fatty infiltration in muscle injuries and diseases, caused by aberrant adipogenesis of fibro-adipogenic progenitors, negatively impacts function. Intramuscular delivery of wingless-type MMTV integration site family 7a (WNT7A) offers a promising strategy to stimulate muscle regeneration, but its effects on adipogenic conversion of fibro-adipogenic progenitors remain unknown. Here, we show that WNT7A decreases adipogenesis of fibro-adipogenic progenitors (FAPs) by inducing nuclear localization of Yes-associated protein (YAP) through Rho in a ß-CATENIN-independent manner and by promoting nuclear retention of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) in differentiating FAPs. Furthermore, intramuscular injection of WNT7A in vivo effectively suppresses fatty infiltration in mice following glycerol-induced injury. Our results collectively suggest WNT7A as a potential protein-based therapeutic for diminishing adipogenesis of FAPs and intramuscular fatty infiltration in pathological muscle injuries or diseases.

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.

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.

Tendon progenitor cells as biological augmentation improve functional gait and reduce scar formation after rotator cuff repair.

BACKGROUND: High rates of structural failure are reported after rotator cuff repairs due to inability to recreate the native enthesis during healing. The development of biological augmentation methods that mitigate scar formation and regenerate the enthesis is still an unmet need. Since neonatal enthesis is capable of regeneration after injury, this study tested whether delivery of neonatal tendon progenitor cells (TPCs) into the adult injured environment can enhance functional and structural supraspinatus enthesis and tendon healing. METHODS: TPCs were isolated from Ai14 Rosa26-TdTomato mouse Achilles tendons and labeled using adenovirus-Cre. Fifty-two CB57BL/6J mice underwent detachment and acute repair of the supraspinatus tendon and received either a fibrin-only or TPC-fibrin gel. Immunofluorescence analysis was carried out to determine cellularity (DAPI), fibrocartilage (SOX9), macrophages (F4/80), myofibroblasts (α-smooth muscle actin), and scar (laminin). Assays for function (gait and biomechanical testing) and structure (micro-computed tomography imaging, picrosirius red/Alcian Blue staining, type I and III collagen staining) were carried out. RESULTS: Analysis of TdTomato cells after injury showed minimal retention of TPCs by day 7 and day 14, with detected cells localized near the bursa and deltoid rather than the enthesis/tendon. However, TPC delivery led to significantly increased %Sox9+ cells in the enthesis at day 7 after injury and decreased laminin intensity across almost all time points compared to fibrin-only treatment. Similarly, TPC-treated mice showed gait recovery by day 14 (paw area and stride length) and day 28 (stance time), while fibrin-treated mice failed to recover gait parameters. Despite improved gait, biomechanical testing showed no differences between groups. Structural analysis by micro-computed tomography suggests that TPC application improves cortical thickness after surgery compared to fibrin. Superior collagen alignment at the neo-enthesis was also observed in the TPC-augmented group at day 28, but no difference was detected in type I and III collagen intensity. CONCLUSION: We found that neonatal TPCs improved and restored functional gait by reducing overall scar formation, improving enthesis collagen alignment, and altering bony composition response after supraspinatus tendon repair. TPCs did not appear to integrate into the healing tissue, suggesting improved healing may be due to paracrine effects at early stages. Future work will determine the factors secreted by TPCs to develop translational targets.

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.

Cell lineage tracing and functional assessment of supraspinatus tendon healing in an acute repair murine model.

Rotator cuff supraspinatus tendon injuries are common with high rates of anatomic failure after surgical repair. The purpose of the study was to define clinically relevant features of a mouse model of supraspinatus tendon injury to determine painful, functional, and structural outcomes; we further investigated two cell populations mediating healing using genetic lineage tracing after full detachment and repair of the supraspinatus tendon in mice. The pain was assessed using the mouse grimace scale and function by gait analysis and tensile testing. Histological and microCT analyses were used to determine enthesis/tendon and bone structure, respectively. Lineage tracing was carried out using inducible Cre lines for ScxCreERT2 (tendon cells) and αSMACreERT2 (myofibroblasts and mesenchymal progenitors). Mice only expressed pain transiently after surgery despite long-term impairment of functional and structural properties. Gait, tensile mechanical properties, and bone properties were significantly reduced after injury and repair. Lineage tracing showed relatively few Scx lin tendon cells while αSMA lin cells contributed strongly to scar formation. Despite surgical reattachment of healthy tendon, lineage tracing revealed poor preservation of supraspinatus tendon after acute injury and loss of tendon structure, suggesting that tendon degeneration is also a key impediment of successful rotator cuff repair. Scar formation after surgery is mediated largely by αSMA lin cells and results in permanently reduced functional and structural properties.

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.

Spatial mapping of collagen content and structure in human intervertebral disk degeneration.

Collagen plays a key structural role in both the annulus fibrosus (AF) and nucleus pulposus (NP) of intervertebral disks (IVDs). Changes in collagen content with degeneration suggest a shift from collagen type II to type I within the NP, and the activation of pro-inflammatory factors is indicative of fibrosis throughout. While IVD degeneration is considered a fibrotic process, an increase in collagen content with degeneration, reflective of fibrosis, has not been demonstrated. Additionally, changes in collagen content and structure in human IVDs with degeneration have not been characterized with high spatial resolution. The collagen content of 23 human lumbar L2/3 or L3/4 IVDs was quantified using second harmonic generation imaging (SHG) and multiple image processing algorithms, and these parameters were correlated with the Rutges histological degeneration grade. In the NP, SHG intensity increased with degeneration grade, suggesting fibrotic collagen deposition. In the AF, the entropy of SHG intensity was reduced with degeneration indicating increased collagen uniformity and suggesting less-organized lamellar structure. Collagen orientation entropy decreased throughout most IVD regions with increasing degeneration grade, further supporting a loss in collagen structural complexity. Overall, SHG imaging enabled visualization and quantification of IVD collagen content and organization with degeneration. There was an observed shift from an initially complex structure to more uniform structure with loss of microstructural elements and increased NP collagen polarity, suggesting fibrotic remodeling.

Correction: Dietary polyphenols as a safe and novel intervention for modulating pain associated with intervertebral disc degeneration in an in-vivo rat model.

[This corrects the article DOI: 10.1371/journal.pone.0223435.].

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.

Dietary polyphenols as a safe and novel intervention for modulating pain associated with intervertebral disc degeneration in an in-vivo rat model.

Developing effective therapies for back pain associated with intervertebral disc (IVD) degeneration is a research priority since it is a major socioeconomic burden and current conservative and surgical treatments have limited success. Polyphenols are naturally occurring compounds in plant-derived foods and beverages, and evidence suggests dietary supplementation with select polyphenol preparations can modulate diverse neurological and painful disorders. This study tested whether supplementation with a select standardized Bioactive-Dietary-Polyphenol-Preparation (BDPP) may alleviate pain symptoms associated with IVD degeneration. Painful IVD degeneration was surgically induced in skeletally-mature rats by intradiscal saline injection into three consecutive lumbar IVDs. Injured rats were given normal or BDPP-supplemented drinking water. In-vivo hindpaw mechanical allodynia and IVD height were assessed weekly for 6 weeks following injury. Spinal column, dorsal-root-ganglion (DRG) and serum were collected at 1 and 6 weeks post-operative (post-op) for analyses of IVD-related mechanical and biological pathogenic processes. Dietary BDPP significantly alleviated the typical behavioral sensitivity associated with surgical procedures and IVD degeneration, but did not modulate IVD degeneration nor changes of pro-inflammatory cytokine levels in IVD. Gene expression analyses suggested BDPP might have an immunomodulatory effect in attenuating the expression of pro-inflammatory cytokines in DRGs. This study supports the idea that dietary supplementation with BDPP has potential to alleviate IVD degeneration-related pain, and further investigations are warranted to identify the mechanisms of action of dietary BDPP.