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.

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.

Single-cell RNA-sequencing atlas of bovine caudal intervertebral discs: Discovery of heterogeneous cell populations with distinct roles in homeostasis.

Back and neck pain are significant healthcare burdens that are commonly associated with pathologies of the intervertebral disc (IVD). The poor understanding of the cellular heterogeneity within the IVD makes it difficult to develop regenerative IVD therapies. To address this gap, we developed an atlas of bovine (Bos taurus) caudal IVDs using single-cell RNA-sequencing (scRNA-seq). Unsupervised clustering resolved 15 unique clusters, which we grouped into the following annotated partitions: nucleus pulposus (NP), outer annulus fibrosus (oAF), inner AF (iAF), notochord, muscle, endothelial, and immune cells. Analyzing the pooled gene expression profiles of the NP, oAF, and iAF partitions allowed us to identify novel markers for NP (CP, S100B, H2AC18, SNORC, CRELD2, PDIA4, DNAJC3, CHCHD7, and RCN2), oAF (IGFBP6, CTSK, LGALS1, and CCN3), and iAF (MGP, COMP, SPP1, GSN, SOD2, DCN, FN1, TIMP3, WDR73, and GAL) cells. Network analysis on subpopulations of NP and oAF cells determined that clusters NP1, NP2, NP4, and oAF1 displayed gene expression profiles consistent with cell survival, suggesting these clusters may uniquely support viability under the physiological stresses of the IVD. Clusters NP3, NP5, oAF2, and oAF3 expressed various extracellular matrix (ECM)-associated genes, suggesting their role in maintaining IVD structure. Lastly, transcriptional entropy and pseudotime analyses found that clusters NP3 and NP1 had the most stem-like gene expression signatures of the NP partition, implying these clusters may contain IVD progenitor cells. Overall, results highlight cell type diversity within the IVD, and these novel cell phenotypes may enhance our understanding of IVD development, homeostasis, degeneration, and regeneration.

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

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

Structural and Chemical Modification to Improve Adhesive and Material Properties of Fibrin-Genipin for Repair of Annulus Fibrosus Defects in Intervertebral Disks.

Annulus fibrosus (AF) defects from intervertebral disk (IVD) herniation and degeneration are commonly associated with back pain. Genipin-crosslinked fibrin hydrogel (FibGen) is an injectable, space-filling AF sealant that was optimized to match AF shear properties and partially restored IVD biomechanics. This study aimed to enhance mechanical behaviors of FibGen to more closely match AF compressive, tensile, and shear properties by adjusting genipin crosslink density and by creating a composite formulation by adding Poly(D,L-lactide-co-glycolide) (PDLGA). This study also evaluated effects of thrombin concentration and injection technique on gelation kinetics and adhesive strength. Increasing FibGen genipin concentration from 1 to 36 mg/mL significantly increased adhesive strength (∼5 to 35 kPa), shear moduli (∼10 to 110 kPa), and compressive moduli (∼25 to 150 kPa) with concentration-dependent effects, and spanning native AF properties. Adding PDLGA to FibGen altered the material microstructure on electron microscopy and nearly tripled adhesive strength, but did not increase tensile moduli, which remained nearly 5× below native AF, and had a small increase in shear moduli and significantly decreased compressive moduli. Increased thrombin concentration decreased gelation rate to < 5 min and injection methods providing a structural FibGen cap increased pushout strength by ∼40%. We conclude that FibGen is highly modifiable with tunable mechanical properties that can be formulated to be compatible with human AF compressive and shear properties and gelation kinetics and injection techniques compatible with clinical discectomy procedures. However, further innovations, perhaps with more efficient fiber reinforcement, will be required to enable FibGen to match AF tensile properties.

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

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

Does type 2 diabetes mellitus promote intervertebral disc degeneration?

PURPOSE: LDD is an important cause of low back pain. Many people believe there is an adverse influence of type 2 diabetes (T2D) on lumbar intervertebral disc degeneration (LDD). We examined a population sample for epidemiological evidence of association. METHODS: Twin volunteers from the TwinsUK cohort having spine magnetic resonance (MR) scans coded for LDD and information about T2D were investigated in two ways. First, as a population sample and second as a cotwin case control study in twin pairs discordant for T2D. Other risk factors for LDD considered were age, body-mass index (BMI), smoking, and alcohol. RESULTS: In 956 twin volunteers T2D had a prevalence of 6.6 %. LDD score was higher in T2D twins (14.9 vs 13.1 p = 0.04) but was not an independent risk factor if the influence of age and BMI were included in the model. Discordant twin analysis (n = 33 pairs) showed no significant difference in LDD between twins having T2D and their unaffected cotwins. CONCLUSIONS: Twins having T2D did manifest higher LDD scores but the effect was abrogated once BMI was included in multivariable analysis, showing it is not an independent risk factor for LDD. The population study had 80 % power at 0.1 significance level to detect a difference of 1.8 in LDD score (range of 0-60), so if there is an effect of T2D on LDD, it is likely to be small.

The role of the complement system in disc degeneration and Modic changes.

Disc degeneration and vertebral endplate bone marrow lesions called Modic changes are prevalent spinal pathologies found in chronic low back pain patients. Their pathomechanisms are complex and not fully understood. Recent studies have revealed that complement system proteins and interactors are dysregulated in disc degeneration and Modic changes. The complement system is part of the innate immune system and plays a critical role in tissue homeostasis. However, its dysregulation has also been associated with various pathological conditions such as rheumatoid arthritis and osteoarthritis. Here, we review the evidence for the involvement of the complement system in intervertebral disc degeneration and Modic changes. We found that only a handful of studies reported on complement factors in Modic changes and disc degeneration. Therefore, the level of evidence for the involvement of the complement system is currently low. Nevertheless, the complement system is tightly intertwined with processes known to occur during disc degeneration and Modic changes, such as increased cell death, autoantibody production, bacterial defense processes, neutrophil activation, and osteoclast formation, indicating a contribution of the complement system to these spinal pathologies. Based on these mechanisms, we propose a model how the complement system could contribute to the vicious cycle of tissue damage and chronic inflammation in disc degeneration and Modic changes. With this review, we aim to highlight a currently understudied but potentially important inflammatory pathomechanism of disc degeneration and Modic changes that may be a novel therapeutic target.

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.

Stress and matrix-responsive cytoskeletal remodeling in fibroblasts.

In areolar "loose" connective tissue, fibroblasts remodel their cytoskeleton within minutes in response to static stretch resulting in increased cell body cross-sectional area that relaxes the tissue to a lower state of resting tension. It remains unknown whether the loosely arranged collagen matrix, characteristic of areolar connective tissue, is required for this cytoskeletal response to occur. The purpose of this study was to evaluate cytoskeletal remodeling of fibroblasts in, and dissociated from, areolar and dense connective tissue in response to 2 h of static stretch in both native tissue and collagen gels of varying crosslinking. Rheometric testing indicated that the areolar connective tissue had a lower dynamic modulus and was more viscous than the dense connective tissue. In response to stretch, cells within the more compliant areolar connective tissue adopted a large "sheet-like" morphology that was in contrast to the smaller dendritic morphology in the dense connective tissue. By adjusting the in vitro collagen crosslinking, and the resulting dynamic modulus, it was demonstrated that cells dissociated from dense connective tissue are capable of responding when seeded into a compliant matrix, while cells dissociated from areolar connective tissue can lose their ability to respond when their matrix becomes stiffer. This set of experiments indicated stretch-induced fibroblast expansion was dependent on the distinct matrix material properties of areolar connective tissues as opposed to the cells' tissue of origin. These results also suggest that disease and pathological processes with increased crosslinks, such as diabetes and fibrosis, could impair fibroblast responsiveness in connective tissues.

Analysis of quantitative magnetic resonance imaging and biomechanical parameters on human discs with different grades of degeneration.

PURPOSE: To establish relationships between quantitative MRI (qMRI) and biomechanical parameters in order to help inform and interpret alterations of human intervertebral discs (IVD) with different grades of degeneration. MATERIALS AND METHODS: The properties of the nucleus pulposus (NP) and annulus fibrosus (AF) of each IVD of 10 lumbar spines (range, 32-77 years) were analyzed by qMRI (relaxation times T1 and T2, magnetization transfer ratio [MTR], and apparent diffusion coefficient [ADC]), and tested in confined compression and dynamic shear. RESULTS: T1 and T2 significantly decreased in both the NP and AF with increasing degeneration grades while the MTR increased significantly with grade 4. In contrast to the other qMRI parameters, the ADC had a tendency to decrease with increasing grade. Disc degeneration caused a decrease in the aggregate modulus, hydraulic permeability and shear modulus magnitude along with an increase in phase angle in the AF. In contrast, disc degeneration of NPs demonstrated decreases in shear modulus and phase angle. CONCLUSION: Our studies indicate that qMRI can be used as a noninvasive diagnostic tool in the detection of IVD properties with the potential to help interpret and detect early, middle, and late stages of degeneration. QMRI of human IVD can therefore become a very important diagnostic assessment tool in determining the functional state of the disc.

Degenerative grade affects the responses of human nucleus pulposus cells to link-N, CTGF, and TGFß3.

STUDY DESIGN: Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. OBJECTIVE: To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. SUMMARY OF BACKGROUND DATA: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. METHODS: Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor ß3 (TGFß3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. RESULTS: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1ß, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGFßR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFß3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments upregulated IL-1ß expression and had a more catabolic response than TGFß3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. CONCLUSIONS: Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFß receptor 1 expression and better responded to anabolic stimulation.

Orthogonal plating of distal femur fractures: A biomechanical comparison with plate-nail and parallel plating constructs.

Purpose: This study compared the biomechanical properties of orthogonal plating with plate-nail and parallel plating constructs for supracondylar distal femur fractures. Methods: A supracondylar distal femur fracture was simulated using 15 synthetic osteoporotic femurs. Constructs included: (1) plate-nail (lateral locked distal femoral plate + retrograde intramedullary nail); (2) parallel plating (lateral locked distal femoral plate + medial 4.0 mm compression plate); and (3) orthogonal plating (lateral locked distal femoral plate + posterior one-third tubular plate). Specimens underwent nondestructive loading, fatigue loading, and loading to failure. Gapping at the fracture was measured using a three-dimensional motion capture system. Baseline torsional and axial stiffness, stiffness and strain after fatigue loading, and load to failure were determined. A case example of orthogonal plating is also presented. Results: There was no difference in baseline torsional (p = 0.51) and axial stiffness (p = 0.53). Stiffness after fatigue loading was highest with parallel plating, with no difference between the plate-nail and orthogonal plating constructs (p = 0.84). Strain after fatigue loading was lowest in the parallel plating group (0.54 ± 0.19%), followed by the plate-nail (2.89 ± 0.83%) and orthogonal plating groups (3.04 ± 0.51%). Conclusion: Orthogonal plating demonstrated comparable baseline stiffness to plate-nail and parallel plating constructs, and similar biomechanical performance in fatigue loading to plate-nail constructs. All specimens had ≤3% strain after fatigue loading, suggesting sufficient stability for fracture healing. The benefits of enhanced stability from dual-implant fixation may be achieved through orthogonal plating while avoiding an additional medial surgical approach, and therefore warrants further investigation as a novel alternative for distal femur fracture fixation.

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.

Teaching Tissue Repair Through an Inquiry-Based Learning Bioadhesives Module.

Bioadhesives are an important class of biomaterials for wound healing, hemostasis, and tissue repair. To develop the next generation of bioadhesives, there is a societal need to teach trainees about their design, engineering, and testing. This study designed, implemented, and evaluated a hands-on, inquiry-based learning (IBL) module to teach bioadhesives to undergraduate, master's, and PhD/postdoctoral trainees. Approximately 30 trainees across three international institutions participated in this IBL bioadhesives module, which was designed to last approximately 3 h. This IBL module was designed to teach trainees about how bioadhesives are used for tissue repair, how to engineer bioadhesives for different biomedical applications, and how to assess the efficacy of bioadhesives. The IBL bioadhesives module resulted in significant learning gains for all cohorts; whereby, trainees scored an average of 45.5% on the pre-test assessment and 69.0% on the post-test assessment. The undergraduate cohort experienced the greatest learning gains of 34.2 points, which was expected since they had the least theoretical and applied knowledge about bioadhesives. Validated pre/post-survey assessments showed that trainees also experienced significant improvements in scientific literacy from completing this module. Similar to the pre/post-test, improvements in scientific literacy were most significant for the undergraduate cohort since they had the least amount of experience with scientific inquiry. Instructors can use this module, as described, to introduce undergraduate, master's, and PhD/postdoctoral trainees to principles of bioadhesives.

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.

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.

Combining adhesive and nonadhesive injectable hydrogels for intervertebral disc repair in an ovine discectomy model.

Background: Intervertebral disc (IVD) disorders (e.g., herniation) directly contribute to back pain, which is a leading cause of global disability. Next-generation treatments for IVD herniation need advanced preclinical testing to evaluate their ability to repair large defects, prevent reherniation, and limit progressive degeneration. This study tested whether experimental, injectable, and nonbioactive biomaterials could slow IVD degeneration in an ovine discectomy model. Methods: Ten skeletally mature sheep (4-5.5 years) experienced partial discectomy injury with cruciate-style annulus fibrosus (AF) defects and 0.1 g nucleus pulposus (NP) removal in the L1-L2, L2-L3, and L3-L4 lumbar IVDs. L4-L5 IVDs were Intact controls. IVD injury levels received: (1) no treatment (Injury), (2) poly (ethylene glycol) diacrylate (PEGDA), (3) genipin-crosslinked fibrin (FibGen), (4) carboxymethylcellulose-methylcellulose (C-MC), or (5) C-MC and FibGen (FibGen + C-MC). Animals healed for 12 weeks, then IVDs were assessed using computed tomography (CT), magnetic resonance (MR) imaging, and histopathology. Results: All repaired IVDs retained ~90% of their preoperative disc height and showed minor degenerative changes by Pfirrmann grading. All repairs had similar disc height loss and Pfirrmann grade as Injury IVDs. Adhesive AF sealants (i.e., PEGDA and FibGen) did not herniate, although repair caused local endplate (EP) changes and inflammation. NP repair biomaterials (i.e., C-MC) and combination repair (i.e., FibGen + C-MC) exhibited lower levels of degeneration, less EP damage, and less severe inflammation; however, C-MC showed signs of herniation via biomaterial expulsion. Conclusions: All repair IVDs were noninferior to Injury IVDs by IVD height loss and Pfirrmann grade. C-MC and FibGen + C-MC IVDs had the best outcomes, and may be appropriate for enhancement with bioactive factors (e.g., cells, growth factors, and miRNAs). Such bioactive factors appear to be necessary to prevent injury-induced IVD degeneration. Application of AF sealants alone (i.e., PEGDA and FibGen) resulted in EP damage and inflammation, particularly for PEGDA IVDs, suggesting further material refinements are needed.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.