The Contribution of Biomechanical-Biological Interactions of the Spine to Low Back Pain.

OBJECTIVE: The objective of this mini-review is to examine a subset of literature that demonstrates multiple interactions between mechanics and biology within the spine and propose how incorporation of these mechano-biologic interactions can be applied to improve the conceptual understanding of tissue tolerances. BACKGROUND: Low back pain represents a major musculoskeletal problem in the workplace. Traditional biomechanical assessments have employed tissue tolerances as an approach for reducing workplace injuries; however, development of more universal biologically sensitive tolerances requires incorporation of mechano-biologic interactions. METHODS: A focused literature review addressing the interactions between mechanical loading and biology in the spine. RESULTS: Mechanical loads applied to the body are distributed across all spatial scales from the body to the tissues to the cells. These mechanical loads regulate cellular metabolism and over time can lead to tissue strengthening or weakening. Mechanical loading also interacts with the biologic environment (e.g., tissue inflammation, nerve sensitization) to influence the perception of pain, thereby changing the risk of experiencing pain. Biologic tissues also exhibit time-dependent changes in mechanical behaviors that occur throughout the day and with disease, suggesting tissue tolerances are time dependent. CONCLUSION: Incorporating mechano-biologic interactions into the traditional tissue tolerance paradigm through describing tissue tolerances as a function of multiple factors (e.g., preexisting risk factors, underlying pathology, and time) may lead to the development of tissue tolerances that are more representative of the in vivo situation. APPLICATION: Efforts must work toward incorporating biological concepts into tissue tolerances in order to improve risk assessment tools.

Modic changes in the lumbar vertebral column of chondrodystrophic and non-chondrodystrophic dogs with intervertebral disc disease.

Introduction: Modic changes (MC) are signs of vertebral pathology visible on magnetic resonance (MR) images that have been associated with low back pain (LBP) and disc degeneration in people. Multiple breeds of dogs also develop MCs and coincident back pain. However, the association between breed, MC, and spinal pathologies has yet to be fully elucidated. This study aimed to identify the prevalence of MC that occur spontaneously in the lumbar vertebral column of dogs diagnosed with intervertebral disc disease (IVDD) and examine their association with demographic criteria and the disc width index (DWI). Methods: Medical records and lumbar vertebral column MR images were examined from 104 dogs (831 intervertebral disc spaces and adjacent vertebrae), which were divided into three groups: chondrodystrophic dogs (CD; n =54) and non-chondrodystrophic dogs (NCD; n =30) with IVDD as the primary diagnosis, and control dogs (n =20) with other spinal diseases as their primary diagnosis. Results: Increasing age and a diagnosis of IVDD were significantly associated with MC in dogs (p < 0.001 and p = 0.0062, respectively). In CD dogs with IVDD, Type 2 MC were most prevalent, whereas, in NCD dogs, Type 3 MC were the most prevalent type. Type 2 MC were distributed nearly equally across the lumbar vertebral column, while Type 3 MC were primarily detected at the level of L7-S1. Discussion: This study demonstrated that MC developed spontaneously in dogs, are common in dogs diagnosed with IVDD, and the type observed varies by breed. Further research is needed to understand the pathogenesis of MC; however, the increased presence of Type 2 MC in CD dogs, similar to what is found in people with disc degeneration, suggests that CD dogs could serve as models for MC in people.

Transcriptional profiling of human cartilage endplate cells identifies novel genes and cell clusters underlying degenerated and non-degenerated phenotypes.

BACKGROUND: Low back pain is a leading cause of disability worldwide and is frequently attributed to intervertebral disc (IVD) degeneration. Though the contributions of the adjacent cartilage endplates (CEP) to IVD degeneration are well documented, the phenotype and functions of the resident CEP cells are critically understudied. To better characterize CEP cell phenotype and possible mechanisms of CEP degeneration, bulk and single-cell RNA sequencing of non-degenerated and degenerated CEP cells were performed. METHODS: Human lumbar CEP cells from degenerated (Thompson grade ≥ 4) and non-degenerated (Thompson grade ≤ 2) discs were expanded for bulk (N=4 non-degenerated, N=4 degenerated) and single-cell (N=1 non-degenerated, N=1 degenerated) RNA sequencing. Genes identified from bulk RNA sequencing were categorized by function and their expression in non-degenerated and degenerated CEP cells were compared. A PubMed literature review was also performed to determine which genes were previously identified and studied in the CEP, IVD, and other cartilaginous tissues. For single-cell RNA sequencing, different cell clusters were resolved using unsupervised clustering and functional annotation. Differential gene expression analysis and Gene Ontology, respectively, were used to compare gene expression and functional enrichment between cell clusters, as well as between non-degenerated and degenerated CEP samples. RESULTS: Bulk RNA sequencing revealed 38 genes were significantly upregulated and 15 genes were significantly downregulated in degenerated CEP cells relative to non-degenerated cells (|fold change| ≥ 1.5). Of these, only 2 genes were previously studied in CEP cells, and 31 were previously studied in the IVD and other cartilaginous tissues. Single-cell RNA sequencing revealed 11 unique cell clusters, including multiple chondrocyte and progenitor subpopulations with distinct gene expression and functional profiles. Analysis of genes in the bulk RNA sequencing dataset showed that progenitor cell clusters from both samples were enriched in "non-degenerated" genes but not "degenerated" genes. For both bulk- and single-cell analyses, gene expression and pathway enrichment analyses highlighted several pathways that may regulate CEP degeneration, including transcriptional regulation, translational regulation, intracellular transport, and mitochondrial dysfunction. CONCLUSIONS: This thorough analysis using RNA sequencing methods highlighted numerous differences between non-degenerated and degenerated CEP cells, the phenotypic heterogeneity of CEP cells, and several pathways of interest that may be relevant in CEP degeneration.

Characterization and modulation of the pro-inflammatory effects of immune cells in the canine intervertebral disk.

Background: Intervertebral disk (IVD) degeneration affects both humans and canines and is a major cause of low back pain (LBP). Mast cell (MC) and macrophage (MØ) infiltration has been identified in the pathogenesis of IVD degeneration (IVDD) in the human and rodent model but remains understudied in the canine. MC degranulation in the IVD leads to a pro-inflammatory cascade and activates protease activated receptor 2 (PAR2) on IVD cells. The objectives of the present study are to: (1) highlight the pathophysiological changes observed in the degenerate canine IVD, (2) further characterize the inflammatory effect of MCs co-cultured with canine nucleus pulposus (NP) cells, (3) evaluate the effect of construct stiffness on NP and MCs, and (4) identify potential therapeutics to mitigate pathologic changes in the IVD microenvironment. Methods: Canine IVD tissue was isolated from healthy autopsy research dogs (beagle) and pet dogs undergoing laminectomy for IVD herniation. Morphology, protein content, and inflammatory markers were assessed. NP cells isolated from healthy autopsy (Mongrel hounds) tissue were co-cultured with canine MCs within agarose constructs and treated with cromolyn sodium (CS) and PAR2 antagonist (PAR2A). Gene expression, sulfated glycosaminoglycan content, and stiffness of constructs were assessed. Results: CD 31+ blood vessels, mast cell tryptase, and macrophage CD 163+ were increased in the degenerate surgical canine tissue compared to healthy autopsy. Pro-inflammatory genes were upregulated when canine NP cells were co-cultured with MCs and the stiffer microenvironment enhanced these effects. Treatment with CS and PAR2 inhibitors mediated key pro-inflammatory markers in canine NP cells. Conclusion: There is increased MC, MØs, and vascular ingrowth in the degenerate canine IVD tissue, similar to observations in the clinical population with IVDD and LBP. MCs co-cultured with canine NP cells drive inflammation, and CS and PAR2A are potential therapeutics that may mitigate the pathophysiology of IVDD in vitro.

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.

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.

Equine peripheral blood CD14+ monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation.

Macrophages are a heterogeneous population of immune cells that exhibit dynamic plasticity, polarize into inflammatory or regulatory/pro-resolving macrophages, and influence the healing tissue microenvironment. This study evaluated the in-vitro morphological, proliferative, cell surface marker expression and cytokine/soluble factor secretion characteristics of control, GM-CSF pretreated and inflammatory (LPS+IFN-γ) and regulatory (IL-4 + IL-10) differentiated equine CD14+ monocyte-derived macrophages. Phase contrast microscopy demonstrated that LPS+IFN-γ-primed macrophages exhibited a rounded, granular morphology, whereas IL-4 +IL-10-primed macrophages were elongated with a spindle-shaped morphology. GM-CSF enhanced the proliferation rate of monocytes/macrophages during adherent in-vitro culture. Flow cytometry analysis showed that GM-CSF alone and GM-CSF pretreatment with LPS+IFN-γ or IL-4 +IL-10 priming increased CD86 immunopositivity by 2-fold (p = 0.6); and CD206 immunopositivity remained unchanged. GM-CSF pretreatment and subsequent priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1ß compared to control (p = 0.012) and IL-4 +IL-10-primed (p = 0.0047) macrophages. GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by 6-fold (p < 0.05). There were no differences in TGFß-1 secretion among control, LPS+IFN-γ or IL-4 + IL-10 primed macrophages (p = 0.85). All groups contained an average of 643 ± 51.5 pg/mL of TGFß1. Among the culture conditions evaluated, IL-4 +IL-10 priming for 24 h after 6 days of adherent culture yielded macrophages that were the least inflammatory compared to GM-CSF pretreated and LPS+IFN-γ or IL-4 +IL-10-primed macrophages. These results provide a basis for subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches for enhancing tissue healing.

Nonviral overexpression of Scleraxis or Mohawk drives reprogramming of degenerate human annulus fibrosus cells from a diseased to a healthy phenotype.

Background: Intervertebral disc (IVD) degeneration is a major contributor to low back pain (LBP), yet there are no clinical therapies targeting the underlying pathology. The annulus fibrosus (AF) plays a critical role in maintaining IVD structure/function and undergoes degenerative changes such as matrix catabolism and inflammation. Thus, therapies targeting the AF are crucial to fully restore IVD function. Previously, we have shown nonviral delivery of transcription factors to push diseased nucleus pulposus cells to a healthy phenotype. As a next step in a proof-of-concept study, we report the use of Scleraxis (SCX) and Mohawk (MKX), which are critical for the development, maintenance, and regeneration of the AF and may have therapeutic potential to induce a healthy, pro-anabolic phenotype in diseased AF cells. Methods: MKX and SCX plasmids were delivered via electroporation into diseased human AF cells from autopsy specimens and patients undergoing surgery for LBP. Transfected cells were cultured over 14 days and assessed for cell morphology, viability, density, gene expression of key phenotypic, inflammatory, matrix, pain markers, and collagen accumulation. Results: AF cells demonstrated a fibroblastic phenotype posttreatment. Moreover, transfection of SCX and MKX resulted in significant upregulation of the respective genes, as well as SOX9. Transfected autopsy cells demonstrated upregulation of core extracellular matrix markers; however, this was observed to a lesser effect in surgical cells. Matrix-degrading enzymes and inflammatory cytokines were downregulated, suggesting a push toward a pro-anabolic, anti-inflammatory phenotype. Similarly, pain markers were downregulated over time in autopsy cells. At the protein level, collagen content was increased in both MKX and SCX transfected cells compared to controls. Conclusions: This exploratory study demonstrates the potential of MKX or SCX to drive reprogramming in mild to moderately degenerate AF cells from autopsy and severely degenerate AF cells from surgical patients toward a healthy phenotype and may be a potential nonviral gene therapy for LBP.

Pericellular heparan sulfate proteoglycans: Role in regulating the biosynthetic response of nucleus pulposus cells to osmotic loading.

Background: Daily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading. Methods: Bovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily iso-osmotic, hyper-osmotic, or hypo-osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified. Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6-O-sulfertransferase 1 (HS6ST1) enzyme. Results: Osmotic loading significantly influenced sGAG/DNA accumulation with a hyper-osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso-osmotic conditions. Heparanase-III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading. Conclusion: Results suggest that hyper-osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration-associated modifications to pericellular HSPGs may contribute to the altered mechanobiology observed in disease.

In vivo Mouse Intervertebral Disc Degeneration Models and Their Utility as Translational Models of Clinical Discogenic Back Pain: A Comparative Review.

Low back pain is a leading cause of disability worldwide and studies have demonstrated intervertebral disc (IVD) degeneration as a major risk factor. While many in vitro models have been developed and used to study IVD pathophysiology and therapeutic strategies, the etiology of IVD degeneration is a complex multifactorial process involving crosstalk of nearby tissues and systemic effects. Thus, the use of appropriate in vivo models is necessary to fully understand the associated molecular, structural, and functional changes and how they relate to pain. Mouse models have been widely adopted due to accessibility and ease of genetic manipulation compared to other animal models. Despite their small size, mice lumbar discs demonstrate significant similarities to the human IVD in terms of geometry, structure, and mechanical properties. While several different mouse models of IVD degeneration exist, greater standardization of the methods for inducing degeneration and the development of a consistent set of output measurements could allow mouse models to become a stronger tool for clinical translation. This article reviews current mouse models of IVD degeneration in the context of clinical translation and highlights a critical set of output measurements for studying disease pathology or screening regenerative therapies with an emphasis on pain phenotyping. First, we summarized and categorized these models into genetic, age-related, and mechanically induced. Then, the outcome parameters assessed in these models are compared including, molecular, cellular, functional/structural, and pain assessments for both evoked and spontaneous pain. These comparisons highlight a set of potential key parameters that can be used to validate the model and inform its utility to screen potential therapies for IVD degeneration and their translation to the human condition. As treatment of symptomatic pain is important, this review provides an emphasis on critical pain-like behavior assessments in mice and explores current behavioral assessments relevant to discogenic back pain. Overall, the specific research question was determined to be essential to identify the relevant model with histological staining, imaging, extracellular matrix composition, mechanics, and pain as critical parameters for assessing degeneration and regenerative strategies.

Controversies in spine research: Organ culture versus in vivo models for studies of the intervertebral disc.

Intervertebral disc degeneration is a common cause of low back pain, the leading cause of disability worldwide. Appropriate preclinical models for intervertebral disc research are essential to achieving a better understanding of underlying pathophysiology and for the development, evaluation, and translation of more effective treatments. To this end, in vivo animal and ex vivo organ culture models are both widely used by spine researchers; however, the relative strengths and weaknesses of these two approaches are a source of ongoing controversy. In this article, members from the Spine and Preclinical Models Sections of the Orthopedic Research Society, including experts in both basic and translational spine research, present contrasting arguments in support of in vivo animal models versus ex vivo organ culture models for studies of the disc, supported by a comprehensive review of the relevant literature. The objective is to provide a deeper understanding of the respective advantages and limitations of these approaches, and advance the field toward a consensus with respect to appropriate model selection and implementation. We conclude that complementary use of several model types and leveraging the unique advantages of each is likely to result in the highest impact research in most instances.

Investigating the role of culture conditions on hypertrophic differentiation in human cartilage endplate cells.

Cartilage endplate degeneration/calcification has been linked to the onset and progression of intervertebral disc degeneration and there is a critical need to understand mechanisms, such as hypertrophic differentiation, of cartilage endplate degeneration/calcification to inform treatment strategies for discogenic back pain. In vitro cell culture conditions capable of inducing hypertrophic differentiation are used to study pathophysiological mechanisms in articular chondrocytes, but culture conditions capable of inducing a hypertrophic cartilage endplate cell phenotype have yet to be explored. The goal of this study was to investigate the role of culture conditions capable of inducing hypertrophic differentiation in articular chondrocytes on hypertrophic differentiation in human cartilage endplate cells. Isolated human cartilage endplate cells were cultured as pellets for 21 days at either 5% O2 (physiologic for cartilage) or 20.7% O2 (hyperoxic) and treated with 10% fetal bovine serum or Wnt agonist, two stimuli used to induce hypertrophic differentiation in articular chondrocytes. Cartilage endplate cells did not exhibit a hypertrophic cell morphology in response to fetal bovine serum or Wnt agonist but did display other hallmarks of chondrocyte hypertrophy and degeneration such as hypertrophic gene and protein expression, and a decrease in healthy proteoglycans and an increase in fibrous collagen accumulation. These findings demonstrate that cartilage endplate cells take on a degenerative phenotype in response to hypertrophic stimuli in vitro, but do not undergo classical changes in morphology associated with hypertrophic differentiation regardless of oxygen levels, highlighting potential differences in the response of cartilage endplate cells versus articular chondrocytes to the same stimuli.

Characterization of the human intervertebral disc cartilage endplate at the molecular, cell, and tissue levels.

Given the importance of the cartilage endplate (CEP) in low back pain (LBP), there is a need to characterize the human CEP at the molecular, cell, and tissue levels to inform treatment strategies that target it. The goal of this study was to characterize the structure, matrix composition, and cell phenotype of the human CEP compared with adjacent tissues within the intervertebral joint: the nucleus pulposus (NP), annulus fibrosus (AF), and articular cartilage (AC). Isolated CEP, NP, AF, and AC tissues and cells were evaluated for cell morphology, matrix composition, collagen structure, glycosaminoglycan content, and gene and protein expression. The CEP contained elongated cells that mainly produce a collagen-rich interterritorial matrix and a proteoglycan-rich territorial matrix. The CEP contained significantly fewer glycosaminoglycans than the NP tissue. Significant differences in matrix and cell marker gene expression were observed between CEP and NP or AF, with the greatest differences between CEP and AC. We were able to distinguish NP from CEP cells using collagen-10 (COLX), highlighting COLX as a potential CEP marker. Our findings suggest that at the cell and tissue levels, the CEP demonstrates both similarities and differences when compared with NP, AF, and hyaline AC. This study highlights a unique structure, matrix composition, and cell phenotype for the human CEP and can help to inform regenerative strategies that target the intervertebral disc joint in chronic LBP.

Characterization of bovine and canine animal model cartilage endplates and comparison to human cartilage endplate structure, matrix composition, and cell phenotype.

There is a need to further explore mechanisms of cartilage endplate (CEP) degeneration, due to its role in the onset and progression of intervertebral disc degeneration and low back pain. Therefore, the goal of this study was to evaluate structure, matrix composition, and cell phenotype between the human and bovine or canine, both clinically relevant animal models currently used to study the intervertebral disc, CEP. This information may be used in addition to other relevant studies, to help determine optimal animal models for use in studying the role of the CEP in intervertebral disc degeneration and back pain. Endplate structure, matrix composition, cell morphology, and gene expression were evaluated using a picrosirius red/alcian blue and hematoxylin and eosin stain, a dimethylmethylene blue assay, and quantitative reverse transcription polymerase chain reaction. The bovine and canine CEPs were thinner with more rounded cells and thicker bony endplates. The canine CEP contained significantly more sulfated glycosaminoglycans. The bovine CEP demonstrated higher expression of ACAN, COL1, and COL2 and lower expression of T, FBLN1, and collagen X (COLX) compared to the human CEP. The canine CEP had higher COL2 and lower COL1, KRT19, MKX, FBLN1, COLX expression compared to human. These similarities and differences between human and bovine or canine CEP are important to consider when evaluating which animal model is most optimal to use in future studies, interpreting research findings using these animal models and assessing translatability to the human condition.

Non-viral Gene Delivery Methods for Bone and Joints.

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.

Nonviral Transfection With Brachyury Reprograms Human Intervertebral Disc Cells to a Pro-Anabolic Anti-Catabolic/Inflammatory Phenotype: A Proof of Concept Study.

Intervertebral disc (IVD) degeneration is a major contributor to chronic low back pain and is characterized by decreases in cellularity and proteoglycan synthesis, upregulation of matrix degradation, and increases in pro-inflammatory factors with neurovascular invasion. Current treatments fail to target the underlying pathology or promote tissue repair and approaches such as viral transfection raise safety concerns due to mutagenesis and unwarranted immune responses. To avoid such concerns, nonviral transfection is a viable method of gene delivery into the host cell while bypassing the caveats of viral delivery. Brachyury is expressed in the developing notochord and is associated with an immature healthy nucleus pulposus (NP). We hypothesize that Brachyury can reprogram degenerate NP cells to a healthy pro-anabolic phenotype with increased proteoglycan content and decreased expression of catabolic, inflammatory, and neurovascular markers. NP cells obtained from human autopsy and surgical tissues were transfected with plasmids encoding for Brachyury or an empty vector control via bulk electroporation. Post transfection, cells were seeded in three-dimensional agarose constructs cultured over 4 weeks and analyzed for viability, gene expression, and proteoglycan. Results demonstrated successful transfection of both autopsy and surgical NP cells. We observed long-term Brachyury expression, significant increased expression of NP phenotypic markers FOXF1, KRT19, and chondrogenic marker SOX9 with decreases in inflammatory cytokines IL1-ß/IL6, NGF, and MMPs and significant increases in glycosaminoglycan accumulation. These results highlight nonviral transfection with developmental transcription factors, such as Brachyury, as a promising method to reprogram degenerate human disc cells toward a healthy NP phenotype. Clinical significance: This project proposes a novel translational approach for the treatment of intervertebral disc degeneration via direct reprogramming of diseased human patient-derived IVD cells to a healthy phenotype. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2389-2400, 2019.

Mast Cell/Proteinase Activated Receptor 2 (PAR2) Mediated Interactions in the Pathogenesis of Discogenic Back Pain.

Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs' role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in in vitro human and ex vivo bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in vitro in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a "proof of concept" study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.

The chondrodystrophic dog: A clinically relevant intermediate-sized animal model for the study of intervertebral disc-associated spinal pain.

Low back pain (LBP) is the leading cause of disability worldwide, with an estimated 80% of the American population suffering from a painful back condition at some point during their lives. The most common cause of LBP is intervertebral disc (IVD) degeneration (IVDD), a condition that can be difficult to treat, either surgically or medically, with current available therapies. Thus, understanding the pathological mechanisms of IVDD and developing novel treatments are critical for improving outcome and quality of life in people living with LBP. While experimental animal models provide valuable mechanistic insight, each model has limitations that complicate translation to the clinical setting. This review focuses on the chondrodystrophic canine clinical model of IVDD as a promising model to assess IVD-associated spinal pain and translational therapeutic strategies for LBP. The canine IVD, while smaller in size than human, goat, ovine, and bovine IVDs, is larger than most other small animal IVDD models and undergoes maturational changes similar to those of the human IVD. Furthermore, both dogs and humans develop painful IVDD as a spontaneous process, resulting in similar characteristic pathologies and clinical signs. Future exploration of the canine model as a model of IVD-associated spinal pain and biological treatments using the canine clinical model will further demonstrate its translational capabilities with the added ethical benefit of treating an existing veterinary patient population with IVDD.

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

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

Publication trends in spine research from 2007 to 2016: Comparison of the Orthopaedic Research Society Spine Section and the International Society for the Study of the Lumbar Spine.

This study investigated current trends in spine publications of the membership of Orthopaedic Research Society Spine Section (ORS3) and the more global and clinically focused International Society for the Study of the Lumbar Spine (ISSLS). The PubMed database was probed to quantify trends in the overall number of articles published, the number of journals these articles were published in, and the number of active scientists producing new manuscripts. We also evaluated trends in flagship spine journals (Spine, European Spine Journal, and The Spine Journal) and in the Journal of Orthopaedic Research. The total number of active ORS3 and ISSLS authors and articles published have increased over the last 10 years. These articles are being published in hundreds of distinct journals; the number of journals is also increasing. Members of both societies published their work in Spine more than any other journal. Yet, publications in Spine decreased over the last 5 years for both ORS3 and ISSLS members, while those in European Spine Journal, and The Spine Journal remained unchanged. Furthermore, members of both societies have published in Journal of Orthopaedic Research at a consistent level. The increasing number of manuscripts and journals reflects a characteristic intrinsic to science as a whole-the global scientific workforce and output are growing and new journals are being created to accommodate the demand. These data suggest that existing spine journals do not fully serve the diverse publication needs of ORS3 and ISSLS members and highlight an unmet need for consolidating the premiere basic and translational spine research in an open access spine-specific journal. This analysis was an important part of a decision process by the ORS to introduce JOR Spine.