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Background: Current surgical management of intervertebral disc herniation often fails to adequately address the risk of recurrence, primarily due to the disc's limited regenerative capacity. Regenerative, biomaterial-based approaches for tissue augmentation, while showing preclinical promise, have consistently failed to meet the extreme mechanical demands of the intervertebral disc, impeding their clinical translation., Methods: In this study, we introduce a novel annulus repair strategy that employs the mechanical interpenetration of a non-woven PET scaffold into intervertebral disc tissue to resist reherniation. We investigate the efficacy in preventing herniations under compression using a bovine explant model and validate its performance in a pilot in vivo study in a goat cervical spine injury model. Healing and scaffold integration are assessed over 4 weeks using computed tomography, magnetic resonance imaging, and histopathology., Results: We demonstrate that this approach effectively prevents mechanically induced herniation. In vivo, the scaffold interpenetration enables biological integration at 4 weeks post-surgery, with no evidence of scaffold migration or disc degeneration. The scaffold supports matrix deposition and cell infiltration, with no observed endplate pathologies or osteolysis., Conclusions: These findings highlight a promising combination of biomechanical reliability and favorable histological outcomes, underscoring the potential of this technology for advancing toward human clinical applications., (© 2025 The Author(s). JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Intervertebral disc degeneration is commonly associated with back and neck pain, and standard surgical treatments do not restore spine function. Replacement of the degenerative disc with a living, tissue-engineered construct has the potential to restore normal structure and function to the spine. Toward this goal, our group developed endplate-modified disc-like angle-ply structures (eDAPS) that recapitulate the native structure and function of the disc. While our initial large animal studies utilized rigid internal fixation of the eDAPS implanted level to ensure retention of the eDAPS, chronic immobilization does not restore full function and is detrimental to the spinal motion segment. The purpose of this study was to utilize a goat cervical disc replacement model coupled with finite element modeling of goat cervical motion segments to investigate the effects of remobilization (removal of fixation) on the eDAPS, the facet joints and the adjacent paraspinal muscle. Our results demonstrated that chronic immobilization caused notable degeneration of the facet joints and paraspinal muscles adjacent to eDAPS implants. Remobilization improved eDAPS composition and integration and mitigated, but did not fully reverse, facet joint osteoarthritis and paraspinal muscle atrophy and fibrosis. Finite element modeling revealed that these changes were likely due to reduced range of motion and reduced facet loading, highlighting the importance of maintaining normal spine biomechanical function with any tissue engineered disc replacement. STATEMENT OF SIGNIFICANCE: Back and neck pain are ubiquitous in modern society, and the gold standard surgical treatment of spinal fusion limits patient function. This study advances our understanding of the response of the spinal motion segment to tissue engineered disc replacement with provisional fixation in a large animal model, further advancing the clinical translation of this technology., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Cell and tissue engineering based approaches have garnered significant interest for treating intervertebral disc degeneration and associated low back pain due to the substantial limitations of currently available clinical treatments. Herein we present a clustered regularly interspaced short palindromic repeats (CRISPR)-guided gene modulation strategy to improve the therapeutic potential of cell and tissue engineering therapies for treating intervertebral disc disease. Recently, we discovered a zinc finger (ZNF) protein, ZNF865 (BLST), which is associated with no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates cell cycle progression and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity. We also demonstrate that targeted ZNF865 regulation can enhance protein production and fibrocartilage-like matrix deposition in human adipose-derived stem cells (hASCs). Furthermore, we demonstrate CRISPR-engineered hASCs ability to increase GAG and collagen II matrix deposition in human-size tissue-engineered discs by 8.5-fold and 88.6-fold, respectively, while not increasing collagen X expression compared to naive hASCs dosed with growth factors. With this increased tissue deposition, we observe significant improvements in compressive mechanical properties, generating a stiffer and more robust tissue. Overall, we present novel biology on ZNF865 and display the power of CRISPR-cell engineering to enhance strategies treating musculoskeletal disease. STATEMENT OF SIGNIFICANCE: This work reports on a novel gene, ZNF865 (also known as BLST), that when regulated with CRISPRa, improves cartilagenous tissue deposition in human sized tissue engineering constructs. Producing tissue engineering constructs at human scale has proven difficult, and this strategy presents a broadly applicable tool to enhance a cells ability to produce tissue at these scales, as we saw an ∼8-88 fold increase in tissue deposition and significantly improved biomechanics in large tissue engineered intervertebral disc compared to traditional growth factor differentiation methods. Additionally, this work begins to elucidate the biology of this novel zinc finger protein, which appears to be critical in regulating cell function and activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Orthopaedic researchers need new strategies for engaging underrepresented minority (URM) students. Our field has demonstrated noticeable gaps in racial, ethnic, and gender diversity, which inhibit our ability to innovate and combat the severe socioeconomic burden of musculoskeletal disorders. Towards this goal, we designed, implemented, and evaluated Learning on a Limb (LoaL), an orthopaedic research outreach module to teach URM high school students about orthopaedic research. During the 4-h module, students completed hands-on activities to learn how biomechanical testing, microcomputed tomography, cell culture, and histology are used in orthopaedic research. Over 3 years, we recruited 32 high school students from the Greater Philadelphia Area to participate in LoaL. Most participants identified as racial/ethnic or gender minorities in orthopaedic research. Using pre/post-tests, we found that students experienced significant learning gains of 51 percentage points from completing LoaL. In addition to teaching students about orthopaedic research, post-survey data demonstrated that participating in LoaL strongly influenced students' interest in orthopaedic research and scientific confidence. Several students acted on this interest by completing summer research experiences in the McKay Orthopaedic Research Laboratory at the University of Pennsylvania. LoaL instructors also benefited by having the opportunity to "pay it forward" to the next generation of students and build community within their department. Empowering institutions to host modules like LoaL would synergistically inspire URM high school students and strengthen community within orthopaedic departments to ultimately enhance orthopaedic research innovations., (© 2024 The Author(s). Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Zinc (Zn) and its alloys have been the focus of recent materials and manufacturing research for orthopaedic implants due to their favorable characteristics including desirable mechanical strength, biodegradability, and biocompatibility. In this research, a novel process involving additive manufacturing (AM) augmented casting was employed to fabricate zinc-magnesium (Zn-0.8 Mg) artifacts with surface lattices composed of triply periodic minimal surfaces (TPMS), specifically gyroid. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis confirmed that Zn-Mg intermetallic phases formed at the grain boundary. Micro indentation testing resulted in hardness value ranging from 83.772 to 99.112 HV and an elastic modulus varying from 92.601 to 94.625 GPa. Results from in vitro cell culture experiments showed that cells robustly survived on both TPMS and solid scaffolds, confirming the suitability of the material and structure as biomedical implants. This work suggests that this novel hybrid manufacturing process may be a viable approach to fabricating next generation biodegradable orthopaedic implants., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2024.)

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, an injectable and radiopaque hyaluronic acid granular hydrogel is developed. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel is injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease., (© 2023 Wiley‐VCH GmbH.)

Orthopaedic researchers need new strategies for engaging diverse students. Our field has demonstrated noticeable gaps in racial, ethnic, and gender diversity, which inhibit our ability to innovate and combat the severe socioeconomic burden of musculoskeletal disorders. Towards this goal, we designed, implemented, and evaluated Learning on a Limb, an orthopaedic research outreach module to teach diverse high school students about orthopaedic research. During the 4-hr module, students completed hands-on activities to learn how biomechanical testing, microcomputed tomography, cell culture, and histology are used in orthopaedic research. Over three years, we recruited 32 high school students from the Greater Philadelphia Area to participate in Learning on a Limb. Most participants identified as racial/ethnic or gender minorities in orthopaedic research. Using pre/post-tests, we found that students experienced significant learning gains of 51 percentage points from completing Learning on a Limb. In addition to teaching students about orthopaedic research, post-survey data demonstrated that participating in Learning on a Limb strongly influenced students' interest in orthopaedic research. Several students acted on this interest by completing summer research experiences in the McKay Orthopaedic Research Laboratory at the University of Pennsylvania. Learning on a Limb instructors also benefited by having the opportunity to "pay it forward" to the next generation of students and build community within their department. Empowering institutions to host modules like Learning on a Limb would synergistically inspire diverse high school students and strengthen community within orthopaedic departments to ultimately enhance orthopaedic research innovations.

Introduction: Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown., Methods: iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-β3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells., Results: TGF-β3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-β3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-β3 and PDGF-BB for 14 days., Discussion: These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies., Competing Interests: Robert L. Mauck is Co‐editor in Chief of JOR Spine and Sarah Gullbrand is an Editorial Board member of JOR Spine; both are co‐authors of this article. They were excluded from editorial decision‐making related to the acceptance of this article for publication in the journal., (© 2024 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Background: Vertebral endplate sclerosis and facet osteoarthritis have been documented in animals and humans. However, it is unclear how these adjacent pathologies engage in crosstalk with the intervertebral disc. This study sought to elucidate this crosstalk by assessing each compartment individually in response to acute disc injury., Methods: Eleven New Zealand White rabbits underwent annular disc puncture using a 16G or 21G needle. At 4 and 10 weeks, individual compartments of the motion segment were analyzed. Discs underwent T 1 relaxation mapping with MRI contrast agent gadodiamide as well T 2 mapping. Both discs and facets underwent mechanical testing via vertebra-disc-vertebra tension-compression creep testing and indentation testing, respectively. Endplate bone density was quantified via μCT. Discs and facets were sectioned and stained for histology scoring., Results: Intervertebral discs became more degenerative with increasing needle diameter and time post-puncture. Bone density also increased in endplates adjacent to both 21G and 16G punctured discs leading to reduced gadodiamide transport at 10 weeks. The facet joints, however, did not follow this same trend. Facets adjacent to 16G punctured discs were less degenerative than facets adjacent to 21G punctured discs at 10 weeks. 16G facets were more degenerative at 4 weeks than at 10, suggesting the cartilage had recovered. The formation of severe disc osteophytes in 16G punctured discs between 4 and 10 weeks likely offloaded the facet cartilage, leading to the recovery observed., Conclusions: Overall, this study supports that degeneration spans the whole spinal motion segment following disc injury. Vertebral endplate thickening occurred in response to disc injury, which limited the diffusion of small molecules into the disc. This work also suggests that altered disc mechanics can induce facet degeneration, and that extreme bony remodeling adjacent to the disc may promote facet cartilage recovery through offloading of the articular cartilage., Competing Interests: Robert L. Mauck is Co‐editor in Chief of JOR Spine. Sarah Gullbrand is an Editorial Board member of JOR Spine and co‐author of this article. They were excluded from editorial decision‐making related to the acceptance of this article for publication in the journal., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background Aims: Lower back pain is the leading cause of disability worldwide and is often linked to degenerative disc disease (DDD), the breakdown of intervertebral discs. The majority of treatment options for DDD are palliative, with clinicians prescribing medication or physical therapy to return the patient to work. Cell therapies are promising treatment options with the potential to restore functional physiological tissue and treat the underlying causes of DDD. DDD is characterized by biochemical changes in the microenvironment of the disc, including changes in nutrient levels, hypoxia, and changes in pH. Stem cell therapies are promising therapies to treat DDD, but the acidic environment in a degenerating disc significantly hinders the viability of stem cells, affecting their efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR) systems allow us to engineer cell phenotypes in a well-regulated and controlled manner. Recently, CRISPR gene perturbation screens have assessed fitness, growth and provided a means for specific cell phenotype characterization., Methods: In this study, we use a CRISPR-activation (a) gene perturbation screen to identify gene upregulation targets that enhance adipose-derived stem cell survival in acidic culture conditions., Results: We identified 1213 prospective pro-survival genes and systematically narrowed these down to 20 genes for validation. We further narrowed down our selection to the top five prospective genes using Cell Counting Kit-8 cell viability assays in naïve adipose-derived stem cells and ACAN/Col2 CRISPRa upregulated stem cells. Finally, we examined the extracellular matrix-producing abilities of multiplex ACAN/Col2-pro-survival edited cells in pellet culture., Conclusions: Using the results from the CRISPRa screen, we are able to engineer desirable cell phenotypes to improve cell viability for the potential treatment of DDD and other disease states that expose cell therapies to acidic environments, while also providing broader knowledge on genes regulating low-pH cell survival., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article ., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The zygapophyseal joints of the spine, also known as the facet joints, are paired diarthrodial joints posterior to the intervertebral disc and neural elements. The pathophysiology of facet osteoarthritis (OA), as well as crosstalk between the disc and facets, remains largely understudied compared to disc degeneration. The purpose of this study was to characterize alterations to human facet cartilage and subchondral bone across a spectrum of degeneration and to investigate correlations between disc and facet degeneration. Human lumbar facet articular surfaces from six independent donors were subject to creep indentation mechanical testing to quantify cartilage mechanical properties, followed by microcomputed tomography (µCT) analyses for subchondral bone morphometry. The degenerative state of each articular surface was assessed via macroscopic scoring and via Osteoarthritis Research Society International histopathology scoring. Our data suggest reduced facet cartilage compressive and tensile moduli and increased permeability with increasing degenerative grade, particularly at the lower levels of the spine. µCT analyses revealed spinal level-dependent alterations to the subchondral bone, with an increase in trabecular bone at the L4-L5 level, but a decrease at the upper levels of the lumbar spine with increasing degenerative grade. Cortical bone volume fraction was generally decreased with increasing degenerative grade across spinal levels. Correlation analysis revealed several associations between quantitative measures of disc degeneration and facet OA. This study showed that alterations in the mechanical properties of facet cartilage and in the structural properties of facet subchondral bone correlated with aspects of disc degeneration and were highly dependent on spinal level., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Varying degrees of hydroxyapatite (HA) surface functionalization have been implicated as the primary driver of differential osteogenesis observed in infiltrating cells. The ability to reliably create spatially controlled areas of mineralization in composite engineered tissues is of growing interest in the field, and the use of HA-functionalized biomaterials may provide a robust solution to this challenge. In this study, we successfully fabricated polycaprolactone salt-leached scaffolds with two levels of a biomimetic calcium phosphate coating to examine their effects on MSC osteogenesis. Longer duration coating in simulated body fluid (SBF) led to increased HA crystal nucleation within scaffold interiors as well as more robust HA crystal formation on scaffold surfaces. Ultimately, the increased surface stiffness of scaffolds coated in SBF for 7 days in comparison to scaffolds coated in SBF for 1 day led to more robust osteogenesis of MSCs in vitro without the assistance of osteogenic signaling molecules. This study also demonstrated that the use of SBF-based HA coatings can promote higher levels of osteogenesis in vivo. Finally, when incorporated as the endplate region of a larger tissue-engineered intervertebral disc replacement, HA coating did not induce mineralization in or promote cell migration out of neighboring biomaterials. Overall, these results verified tunable biomimetic HA coatings as a promising biomaterial modification to promote discrete regions of mineralization within composite engineered tissues., (© 2023 S. Karger AG, Basel.)

Background: The intervertebral disc degenerates with age and has a poor propensity for regeneration. Small molecule transport plays a key role in long-term degradation and repair. Convection (bulk flow), induced by low rate cyclic loading of the intervertebral disc, has been shown to increase transport of small molecules. However, the potential therapeutic benefit of low rate cyclic loading on degenerated discs has not been described. The purpose of this study was to determine if a sustained (daily) low rate cyclic loading regimen could slow, arrest, or reverse intervertebral disc degeneration in the rabbit lumbar spine., Methods: Fifty-six New Zealand white rabbits (>12 months old) were designated as either Control (no disc puncture), 8D (disc puncture followed by 8 weeks of degeneration), 16D (disc puncture followed by 16 weeks of degeneration), or Therapy (disc puncture followed by 8 weeks of degeneration and then 8 weeks of daily low rate cyclic loading). Specimens were evaluated by T2 mapping, Pfirrmann scale grading, nucleus volume, disc height index, disc morphology and structure, and proteoglycan content., Results: In every metric, mean values for the Therapy group fell between Controls and 8D animals. These results suggest that sustained low rate cyclic loading had a therapeutic effect on the already degenerated disc and the regimen promoted signs of regeneration. If these results translate clinically, this approach could fulfil a significant clinical need by providing a means of non-invasively treating intervertebral disc degeneration., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Granular hydrogels have emerged as a new class of injectable and porous biomaterials that improve integration with host tissue when compared to solid hydrogels. Granular hydrogels are typically prepared using spherical particles and this study considers whether particle shape (i.e., isotropic spheres vs anisotropic rods) influences granular hydrogel properties and cellular invasion. Simulations predict that anisotropic rods influence pore shape and interconnectivity, as well as bead transport through granular assemblies. Photo-cross-linkable norbornene-modified hyaluronic acid is used to produce spherical and rod-shaped particles using microfluidic droplet generators and formed into shear-thinning and self-healing granular hydrogels, with particle shape influencing mechanics and injectability. Rod-shaped particles form granular hydrogels that have anisotropic and interconnected pores, with pore size and number influenced by particle shape and degree of packing. Robust in vitro sprouting of endothelial cells from embedded cellular spheroids is observed with rod-shaped particles, including higher sprouting densities and sprout lengths when compared to hydrogels with spherical particles. Cell and vessel invasion into granular hydrogels when injected subcutaneously in vivo are significantly greater with rod-shaped particles, whereas a gradient of cellularity is observed with spherical particles. Overall, this work demonstrates potentially superior functional properties of granular hydrogels with rod-shaped particles for tissue repair., (© 2022 Wiley-VCH GmbH.)

Soft bioelectronic interfaces for mapping and modulating excitable networks at high resolution and at large scale can enable paradigm-shifting diagnostics, monitoring, and treatment strategies. Yet, current technologies largely rely on materials and fabrication schemes that are expensive, do not scale, and critically limit the maximum attainable resolution and coverage. Solution processing is a cost-effective manufacturing alternative, but biocompatible conductive inks matching the performance of conventional metals are lacking. Here, we introduce MXtrodes, a class of soft, high-resolution, large-scale bioelectronic interfaces enabled by Ti 3 C 2 MXene (a two-dimensional transition metal carbide nanomaterial) and scalable solution processing. We show that the electrochemical properties of MXtrodes exceed those of conventional materials and do not require conductive gels when used in epidermal electronics. Furthermore, we validate MXtrodes in applications ranging from mapping large-scale neuromuscular networks in humans to cortical neural recording and microstimulation in swine and rodent models. Last, we demonstrate that MXtrodes are compatible with standard clinical neuroimaging modalities.

The intervertebral disc (IVD) is an integral load-bearing tissue that derives its function from its composite structure and extracellular matrix composition. IVD herniations involve the failure of the annulus fibrosus (AF) and the extrusion of the nucleus pulposus beyond the disc boundary. Disc herniations can impinge the neural elements and cause debilitating pain and loss of function, posing a significant burden on individual patients and society as a whole. Patients with persistent symptoms may require surgery; however, surgical intervention fails to repair the ruptured AF and is associated with the risk for reherniation and further disc degeneration. Given the limitations of AF endogenous repair, many attempts have been made toward the development of effective repair approaches that reestablish IVD function. These methods, however, fail to recapitulate the composition and organization of the native AF, ultimately resulting in inferior tissue mechanics and function over time and high rates of reherniation. Harnessing the cellular function of cells (endogenous or exogenous) at the repair site through the provision of cell-instructive cues could enhance AF tissue regeneration and, ultimately, improve healing outcomes. In this study, we review the diverse approaches that have been developed for AF repair and emphasize the potential for mobilizing the appropriate cellular players at the site of injury to improve AF healing. Impact statement Conventional treatments for intervertebral disc herniation fail to repair the annulus fibrosus (AF), increasing the risk for recurrent herniation. The lack of repair devices in the market has spurred the development of regenerative approaches, yet most of these rely on a scarce endogenous cell population to repair large injuries, resulting in inadequate regeneration. This review identifies current and developing strategies for AF repair and highlights the potential for harnessing cellular function to improve AF regeneration. Ideal cell sources, differentiation strategies, and delivery methods are discussed to guide the design of repair systems that leverage specialized cells to achieve superior outcomes.

This perspective summarizes the genesis, development, and potential future directions of the multispecies JOR Spine histopathology series., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: The rabbit lumbar spine is a commonly utilized model for studying intervertebral disc degeneration and for the pre-clinical evaluation of regenerative therapies. Histopathology is the foundation for which alterations to disc morphology and cellularity with degeneration, or following repair or treatment are assessed. Despite this, no standardized histology grading scale has yet been established for the spine field for any of the frequently utilized animal models., Aims: The purpose of this study was to establish a new standardized scoring system to assess disc degeneration and regeneration in the rabbit model., Materials and Methods: The scoring system was formulated following a review of the literature and a survey of spine researchers. Validation of the scoring system was carried out using images provided by 4 independent laboratories, which were graded by 12 independent graders of varying experience levels. Reliability testing was performed via the computation of intra-class correlation coefficients (ICC) for each category and the total score. The scoring system was then further refined based on the results of the ICC analysis and discussions amongst the authors., Results: The final general scoring system involves scoring 7 features (nucleus pulposus shape, area, cellularity and matrix condensation, annulus fibrosus/nucleus pulposus border appearance, annulus fibrosus morphology, and endplate sclerosis/thickening) on a 0 (healthy) to 2 (severe degeneration) scale. ICCs demonstrated overall moderate to good agreement across graders. An addendum to the main scoring system is also included for use in studies evaluating regenerative therapeutics, which involves scoring cell cloning and morphology within the nucleus pulposus and inner annulus fibrosus., Discussion: Overall, this new scoring system provides an avenue to improve standardization, allow a more accurate comparison between labs and more robust evaluation of pathophysiology and regenerative treatments across the field., Conclusion: This study developed a histopathology scoring system for degeneration and regeneration in the rabbit model based on reported practice in the literature, a survey of spine researchers, and validation testing., Competing Interests: The authors declare no potential conflicts of interest., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions., Aims: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models., Materials & Methods: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability., Results: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 μm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories., Conclusion: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Intervertebral disc (IVD) degeneration and associated back pain place a significant burden on the population. IVD degeneration is a progressive cascade of cellular, compositional, and structural changes, which results in a loss of disc height, disorganization of extracellular matrix architecture, tears in the annulus fibrosus which may involve herniation of the nucleus pulposus, and remodeling of the bony and cartilaginous endplates (CEP). These changes to the IVD often occur concomitantly, across the entire motion segment from the disc subcomponents to the CEP and vertebral bone, making it difficult to determine the causal initiating factor of degeneration. Furthermore, assessments of the subcomponents of the IVD have been largely qualitative, with most studies focusing on a single attribute, rather than multiple adjacent IVD substructures. The objective of this study was to perform a multiscale and multimodal analysis of human lumbar motion segments across various length scales and degrees of degeneration. We performed multiple assays on every sample and identified several correlations between structural and functional measurements of disc subcomponents. Our results demonstrate that with increasing Pfirrmann grade there is a reduction in disc height and nucleus pulposus T2 relaxation time, in addition to alterations in motion segment macromechanical function, disc matrix composition and cellular morphology. At the cartilage endplate-vertebral bone interface, substantial remodeling was observed coinciding with alterations in micromechanical properties. Finally, we report significant relationships between vertebral bone and nucleus pulposus metrics, as well as between micromechanical properties of the endplate and whole motion segment biomechanical parameters, indicating the importance of studying IVD degeneration as a whole organ., (© 2020 Anatomical Society.)

Back and neck pain have become primary reasons for disability and healthcare spending globally. While the causes of back pain are multifactorial, intervertebral disc degeneration is frequently cited as a primary source of pain. The annulus fibrosus (AF) and nucleus pulposus (NP) subcomponents of the disc are common targets for regenerative therapeutics. However, disc degeneration is also associated with degenerative changes to adjacent spinal tissues, and successful regenerative therapies will likely need to consider and address the pathology of adjacent spinal structures beyond solely the disc subcomponents. This review summarises the current state of knowledge in the field regarding associations between back pain, disc degeneration, and degeneration of the cartilaginous and bony endplates, the AF-vertebral body interface, the facet joints and spinal muscles, in addition to a discussion of regenerative strategies for treating pain and degeneration from a whole motion segment perspective.

Intervertebral disc (IVD) herniations, caused by annulus fibrosus (AF) tears that enable disc tissue extrusion beyond the disc space, are very prevalent, especially among adults in the third to fifth decade of life. Symptomatic herniations, in which the extruded tissue compresses surrounding nerves, are characterized by back pain, numbness, and tingling and can cause extreme physical disability. Patients whose symptoms persist after nonoperative intervention may undergo surgical removal of the herniated tissue via microdiscectomy surgery. The AF, however, which has a poor endogenous healing ability, is left unrepaired increasing the risk for re-herniation and pre-disposing the IVD to degenerative disc disease. The lack of understanding of the mechanisms involved in native AF repair limits the design of repair systems that overcome the impediments to successful AF restoration. Moreover, the complexity of the AF structure and the challenging anatomy of the repair environment represents a significant challenge for the design of new repair devices. While progress has been made towards the development of an effective AF repair technique, these methods have yet to demonstrate long-term repair and recovery of IVD biomechanics. In this review, the limitations of endogenous AF healing are discussed and key cellular events and factors involved are highlighted to identify potential therapeutic targets that can be integrated into AF repair methods. Clinical repair strategies and their limitations are described to further guide the design of repair approaches that effectively restore native tissue structure and function., Competing Interests: Ana P. Peredo has no competing financial interests. Sarah E. Gullbrand has no competing financial interests. Harvey E. Smith has no competing financial interests. Robert L. Mauck has no competing financial interests., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Intervertebral disc degeneration is a cascade of cellular, structural, and biomechanical changes that is strongly implicated as a cause of low-back pain. Current treatment strategies have poor long-term efficacy as they seek only to alleviate symptoms without preserving or restoring native tissue structure and function. The objective of this study was to evaluate the efficacy of a combined triple interpenetrating network hydrogel (comprising dextran, chitosan, and teleostean) and mesenchymal stem cell (MSC) therapy targeting moderate-severity disc degeneration in a clinically relevant goat model. Degeneration was induced in lumbar discs of 10 large frame goats by injection of chondroitinase ABC. After 12 weeks, degenerate discs were treated by injection of either hydrogel alone or hydrogel seeded with allogeneic, bone marrow-derived MSCs. Untreated healthy and degenerate discs served as controls, and animals were euthanized 2 weeks after treatment. Discs exhibited a significant loss of disc height 12 weeks after degeneration was induced. Two weeks after treatment, discs that received the combined hydrogel and MSC injection exhibited a significant, 10% improvement in disc height index, as well as improvements in histological condition. Discs that were treated with hydrogel alone exhibited reduced tumor necrosis factor-α expression in the nucleus pulposus (NP). Microcomputed tomography imaging revealed that the hydrogel remained localized to the central NP region of all treated discs after 2 weeks of unrestricted activity. These encouraging findings motivate further, longer term studies of therapeutic efficacy of hydrogel and MSC injections in this large animal model. Impact statement Low-back pain is the leading cause of disability worldwide, and degeneration of the intervertebral discs is considered to be one of the most common reasons for low-back pain. Current treatment strategies focus solely on alleviation of symptoms, and there is a critical need for new treatments that also restore disc structure and function. In this study, using a clinically relevant goat model of moderate-severity disc degeneration, we demonstrate that a combined interpenetrating network hydrogel and mesenchymal stem cell therapy provides acute improvements in disc height, histological condition, and local inflammation.

Engineering complex tissues represents an extraordinary challenge and, to date, there have been few strategies developed that can easily recapitulate native-like cell and biofactor gradients in 3D materials. This is true despite the fact that mimicry of these gradients may be essential for the functionality of engineered graft tissues. Here, a non-traditional magnetics-based approach is developed to predictably position naturally diamagnetic objects in 3D hydrogels. Rather than magnetizing the objects within the hydrogel, the magnetic susceptibility of the surrounding hydrogel precursor solution is enhanced. In this way, a range of diamagnetic objects (e.g., polystyrene beads, drug delivery microcapsules, and living cells) are patterned in response to a brief exposure to a magnetic field. Upon photo-crosslinking the hydrogel precursor, object positioning is maintained, and the magnetic contrast agent diffuses out of the hydrogel, supporting long-term construct viability. This approach is applied to engineer cartilage constructs with a depth-dependent cellularity mirroring that of native tissue. These are thought to be the first results showing that magnetically unaltered cells can be magneto-patterned in hydrogels and cultured to generate heterogeneous tissues. This work provides a foundation for the formation of opposing magnetic-susceptibility-based gradients within a single continuous material., (© 2020 Wiley-VCH GmbH.)

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Low back pain arising from disc degeneration is one of the most common causes of limited function in adults. A number of tissue engineering strategies have been used to develop composite tissue engineered total disc replacements to restore native tissue structure and function. In this study we fabricated a composite engineered disc based on the combination of a porous polycaprolactone (PCL) foam annulus fibrosus (AF) and a hyaluronic acid (HA) hydrogel nucleus pulposus (NP). To evaluate whether native tissue cells or mesenchymal stem cells (MSCs) would perform better, constructs were seeded with native AF/NP cells or with MSCs in the foam and/or gel region. Maturation of these composite engineered discs was evaluated for 9 weeks in vitro culture by biochemical content, histological analysis and mechanical properties. To evaluate the performance of these constructs in the in vivo space, engineered discs were implanted into the caudal spines of athymic rats for 5 weeks. Our findings show that engineered discs comprised of AF/NP cells and MSCs performed similarly and maintained their structure after 5 weeks in vivo. However, for both cell types, loss of proteoglycan was evident in the NP region. These data support the continued development of the more clinically relevant MSCs population for disc replacement applications. STATEMENT OF SIGNIFICANCE: A number of tissue engineering strategies have emerged that are focused on the creation of a composite disc replacement. We fabricated a composite engineered disc based on the combination of a porous foam AF and a HA gel NP. We used these constructs to determine whether the combination of AF/NP cells or MSCs would mature to a greater extent in vitro and which cell type would best retain their phenotype after implantation. Engineered discs comprised of AF/NP cells and MSCs performed similarly, maintaining their structure after 5 weeks in vivo. These data support the successful fabrication and in vivo function of an engineered disc composed of a PCL foam AF and a hydrogel NP using either disc cells or MSCs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

The intervertebral disc is the largest avascular structure in the body, and cells within the disc rely on diffusive transport via vasculature located within the vertebral endplate to receive nutrients, eliminate waste products, and maintain disc health. However, the mechanisms by which small molecule transport into the disc occurs in vivo and how these parameters change with disc degeneration remain understudied. Here, we utilize an in vivo rabbit puncture disc degeneration model to study these interactions and provide evidence that remodeling of the endplate adjacent to the disc occurs concomitant with degeneration. Our results identify significant increases in endplate bone volume fraction, increases in microscale stiffness of the soft tissue interfaces between the disc and vertebral bone, and reductions in endplate vascularity and small molecule transport into the disc as a function of degenerative state. A neural network model identified changes in diffusion into the disc as the most significant predictor of disc degeneration. These findings support the critical role of trans-endplate transport in disease progression and will improve patient selection to direct appropriate surgical intervention and inform new therapeutic approaches to improve disc health. © 2020 American Society for Bone and Mineral Research. Published 2020. This article is a U.S. Government work and is in the public domain in the USA., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Tissue-engineered replacement discs are an area of intense investigation for the treatment of end-stage intervertebral disc (IVD) degeneration. These living implants can integrate into the IVD space and recapitulate native motion segment function. We recently developed a multiphasic tissue-engineered disc-like angle-ply structure (DAPS) that models the micro-architectural and functional features of native tissue. While these implants resulted in functional restoration of the motion segment in rat and caprine models, we also noted deficiencies in cell infiltration and homogeneity of matrix deposition in the electrospun poly(ε-caprolactone) outer region (annulus fibrosus, AF) of the DAPS. To address this limitation, here, we incorporated a sacrificial water-soluble polymer, polyethylene oxide (PEO), as a second fiber fraction within the AF region to increase porosity of the implant. Maturation of these PEO-modified DAPS were evaluated after 5 and 10 weeks of in vitro culture in terms of AF biochemical content, MRI T2 values, overall construct mechanical properties, AF micromechanical properties and cell and matrix distribution. To assess the performance of the PEO-modified DAPS in vivo, precultured constructs were implanted into the rat caudal IVD space for 10 weeks. Results showed that matrix distribution was more homogenous in PCL/PEO DAPS, as evidenced by more robust histological staining, organized collagen deposition and micromechanical properties, compared to standard PCL-only DAPS in vitro. Cell and matrix infiltration were also improved in vivo, but no differences in macromechanical properties and a trend towards improved micromechanical properties were observed. These findings demonstrate that the inclusion of a sacrificial PEO fiber fraction in the DAPS AF region improves cellular colonization, matrix elaboration, and in vitro and in vivo function of an engineered IVD implant. STATEMENT OF SIGNIFICANCE: This work establishes a method for improving cell infiltration and matrix distribution within tissue-engineered dense fibrous scaffolds for intervertebral disc replacement. Tissue-engineered whole disc replacements are an attractive alternative to the current gold standard (mechanical disc arthroplasty or vertebral fusion) for the clinical treatment of patients with advanced disc degeneration., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Robert Mauck is a Co-Editor of JOR Spine, (Copyright © 2020. Published by Elsevier Ltd.)

Tissue-engineered whole disc replacements are an emerging treatment strategy for advanced intervertebral disc degeneration. A challenge facing the translation of tissue-engineered disc replacement to clinical use are the opposing needs of initial immobilization to advantage integration contrasted with physiologic loading and its anabolic effects. Here, we utilize our established rat tail model of tissue engineered disc replacement with external fixation to study the effects of remobilization at two time points postimplantation on engineered disc structure, composition, and function. Our results suggest that the restoration of mechanical loading following immobilization enhanced collagen and proteoglycan content within the nucleus pulposus and annulus fibrosus of the engineered discs, in addition to improving the integration of the endplate region of the construct with native bone. Despite these benefits, angulation of the vertebral bodies at the implanted level occurred following remobilization at both early and late time points, reducing tensile failure properties in the remobilized groups compared to the fixed group. These results demonstrate the necessity of restoring physiologic mechanical loading to engineered disc implants in vivo, and the need to transition toward their evaluation in larger animal models with more human-like anatomy and motion compared to the rat tail., Competing Interests: R. L. M. is a co‐editor of JOR Spine., (© 2020 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Objectives: The objective of this study was to perform a quantitative analysis of the structural and functional alterations in the intervertebral disc during in vivo degeneration, using emerging tools that enable rigorous assessment from the microscale to the macroscale, as well as to correlate these outcomes with noninvasive, clinically relevant imaging parameters., Design: Degeneration was induced in a rabbit model by puncturing the annulus fibrosus (AF) with a 16-gauge needle. 2, 4, 8, and 12 weeks following puncture, degenerative changes in the discs were evaluated via magnetic resonance imaging (MRI), whole motion segment biomechanics, atomic force microscopy, histology and polarized light microscopy, immunohistochemistry, biochemical content, and second harmonic generation imaging., Results: Following puncture, degeneration was evident through marked changes in whole disc structure and mechanics. Puncture acutely compromised disc macro and microscale mechanics, followed by progressive stiffening and remodeling. Histological analysis showed substantial anterior fibrotic remodeling and osteophyte formation, as well as an overall reduction in disc height, and disorganization and infolding of the AF lamellae into the NP space. Increases in NP collagen content and aggrecan breakdown products were also noted within 4 weeks. On MRI, NP T2 was reduced at all post-puncture time points and correlated significantly with microscale indentation modulus., Conclusion: This study defined the time dependent changes in disc structure-function relationships during IVD degeneration in a rabbit annular injury model and correlated degeneration severity with clinical imaging parameters. Our findings identified AF infolding and occupancy of the space as a principle mechanism of disc degeneration in response to needle puncture, and provide new insights to direct the development of novel therapeutics., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

In fibrous tissues, prestressed boundary constraints at bone interfaces instil residual strain throughout the tissue, even when unloaded. For example, internal swelling pressures in the central nucleus pulposus of the intervertebral disc generate prestrain in the outer annulus fibrosus. With injury and depressurization, these residual strains are lost. Here we show that the loss of residual strains in the intervertebral disc alters the microenvironment and instigates aberrant tissue remodelling and the adoption of atypical cellular phenotypes. By using puncture surgery of the annulus fibrosus in rabbits, ex vivo puncture experiments and electrospun nanofibrous scaffolds recapitulating these evolving boundary constraints, we show that the loss of residual strain promotes short-term apoptosis and the emergence of a fibrotic phenotype. We also show that local fibre organization and cellular contractility mediate this process and that the aberrant cellular changes could be abrogated by targeting the cell-mechanosensing machinery with small molecules. Our findings indicate that injury to dense connective tissues under prestrain alters boundary constraints and residual strain; this leads to aberrant mechanosensing, which in turn promotes disease progression.

Tissue engineering holds great promise for the treatment of advanced intervertebral disc degeneration. However, assessment of in vivo integration and mechanical function of tissue-engineered disc replacements over the long term, in large animal models, will be necessary to advance clinical translation. To that end, we developed tissue-engineered, endplate-modified disc-like angle ply structures (eDAPS) sized for the rat caudal and goat cervical spines that recapitulate the hierarchical structure of the native disc. Here, we demonstrate functional maturation and integration of these eDAPS in a rat caudal disc replacement model, with compressive mechanical properties reaching native values after 20 weeks in vivo and evidence of functional integration under physiological loads. To further this therapy toward clinical translation, we implanted eDAPS sized for the human cervical disc space in a goat cervical disc replacement model. Our results demonstrate maintenance of eDAPS composition and structure up to 8 weeks in vivo in the goat cervical disc space and maturation of compressive mechanical properties to match native levels. These results demonstrate the translational feasibility of disc replacement with a tissue-engineered construct for the treatment of advanced disc degeneration., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Low back pain due to disc degeneration is a major cause of morbidity and health care expenditures worldwide. While stem cell-based therapies hold promise for disc regeneration, there is an urgent need to develop improved in vivo animal models to further develop and validate these potential treatments. The objectives of this study were to characterize a percutaneous needle injury model of intervertebral disc degeneration in the mouse caudal spine, and compare two non-invasive quantitative imaging techniques, microcomputed tomography and magnetic resonance imaging (MRI), as effective measures of disc degeneration in this model. Percutaneous needle injury of mouse caudal discs was undertaken using different needle sizes and injury types (unilateral or bilateral annulus fibrosus (AF) puncture). Mice were euthanized 4 weeks post-injury, and MRI and microcomputed tomography were used to determine T2 relaxation time of the NP and disc height index, respectively. Disc condition was then further assessed using semi-quantitative histological grading. Bilateral AF puncture with either 27 or 29G needles resulted in significantly lower T2 relaxation times compared to uninjured controls, while disc height index was not significantly affected by any injury type. There was a strong, inverse linear relationship between histological grade and NP T2 relaxation time. In this study, we demonstrated that quantitative MRI can detect disc degeneration in the mouse caudal spine 4 weeks following percutaneous needle injury, and may therefore serve as a surrogate for histology in longitudinal studies of both disc degeneration and cell-based therapies for disc regeneration using this model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2771-2779, 2018., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Intervertebral disc degeneration is frequently implicated as a cause of back and neck pain, which are pervasive musculoskeletal complaints in modern society. For the treatment of end stage disc degeneration, replacement of the disc with a viable, tissue-engineered construct that mimics native disc structure and function is a promising alternative to fusion or mechanical arthroplasty techniques. Substantial progress has been made in the field of whole disc tissue engineering over the past decade, with a variety of innovative designs characterized both in vitro and in vivo in animal models. However, significant barriers to clinical translation remain, including construct size, cell source, culture technique, and the identification of appropriate animal models for preclinical evaluation. Here we review the clinical need for disc tissue engineering, the current state of the field, and the outstanding challenges that will need to be addressed by future work in this area., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this manuscript.

Replacement of the intervertebral disc with a viable, tissue-engineered construct that mimics native tissue structure and function is an attractive alternative to fusion or mechanical arthroplasty for the treatment of disc pathology. While a number of engineered discs have been developed, the average size of these constructs remains a fraction of the size of human intervertebral discs. In this study, we fabricated medium (3 mm height × 10 mm diameter) and large (6 mm height × 20 mm diameter) sized disc-like angle ply structures (DAPS), encompassing size scales from the rabbit lumbar spine to the human cervical spine. Maturation of these engineered discs was evaluated over 15 weeks in culture by quantifying cell viability and metabolic activity, construct biochemical content, MRI T2 values, and mechanical properties. To assess the performance of the DAPS in the in vivo space, pre-cultured DAPS were implanted subcutaneously in athymic rats for 5 weeks. Our findings show that both sized DAPS matured functionally and compositionally during in vitro culture, as evidenced by increases in mechanical properties and biochemical content over time, yet large DAPS under-performed compared to medium DAPS. Subcutaneous implantation resulted in reductions in NP cell viability and GAG content at both size scales, with little effect on AF biochemistry or metabolic activity. These findings demonstrate that engineered discs at large size scales will mature during in vitro culture, however, future work will need to address the challenges of reduced cell viability and heterogeneous matrix distribution throughout the construct., Statement of Significance: This work establishes, for the first time, tissue-engineered intervertebral discs for total disc replacement at large, clinically relevant length scales. Clinical translation of tissue-engineered discs will offer an alternative to mechanical disc arthroplasty and fusion procedures, and may contribute to a paradigm shift in the clinical care for patients with disc pathology and associated axial spine and neurogenic extremity pain., (Copyright © 2018. Published by Elsevier Ltd.)

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., Competing Interests: J.T.M.: ORS3 Member. S.E.G.: ORS3 Member. A.J.F.: ORS3 Member, ISSLS Member. D.P.: ORS3 Member. A.D.D.: ORS3 Member, ISSLS Treasurer, Asian Spine Journal Deputy Editor, European Spine Journal Editorial Board, International Journal of Spine Surgery Section Editor, JOR Spine Advisory Review Board. T.R.O: ORS Member, ISSLS First Vice President, Spine Associate Editorial Board, Journal of Orthopaedic Research Editorial Review Board, JOR Spine Advisory Review Board. K.C.: ORS3 Member, ISSLS Secretary, Spine Associate Editorial Board, Spine Surgery and Related Research Deputy Editor. F.G.: ORS3 Member, ORS Past President (current) and President (during the formation of JOR Spine), Journal of Biomechanics Editor‐in‐Chief, Osteoarthritis & Cartilage Associate Editor, DNA and Cell Biology Editorial Board, Current Rheumatology Reviews Editorial Board, Clinical Medicine: Arthritis and Musculoskeletal Disorders Editorial Board, Stem Cell Research & Therapy Editorial Board, Orthopedic Research and Reviews Editorial Board, Biomedical Engineering Letters Advisory Board, Bioengineering Editorial Board, TECHNOLOGY Editorial Board, Journal of Experimental Orthopaedics Editorial Board, Techniques in Orthopaedics Editorial Board – Bioengineering, European Cells & Materials International Review Panel, Journal of Orthopaedic Research Editorial Review Board. J.A.H.: ORS3 Chair, JOR Spine Advisory Review Board. J.C.I.: ORS3 Past Chair, ORS Second Vice President, ISSLS Member, JOR Spine Advisory Review Board, Journal of Biomechanics Editorial Consultant, Journal of Orthopaedic Research Publications Advisory Board, Orthopaedic Research Society Executive Board.

Total disc replacement with an engineered substitute is a promising avenue for treating advanced intervertebral disc disease. Toward this goal, we developed cell-seeded disc-like angle ply structures (DAPS) and showed through in vitro studies that these constructs mature to match native disc composition, structure, and function with long-term culture. We then evaluated DAPS performance in an in vivo rat model of total disc replacement; over 5 weeks in vivo, DAPS maintained their structure, prevented intervertebral bony fusion, and matched native disc mechanical function at physiologic loads in situ. However, DAPS rapidly lost proteoglycan post-implantation and did not integrate into adjacent vertebrae. To address this, we modified the design to include polymer endplates to interface the DAPS with adjacent vertebrae, and showed that this modification mitigated in vivo proteoglycan loss while maintaining mechanical function and promoting integration. Together, these data demonstrate that cell-seeded engineered discs can replicate many characteristics of the native disc and are a viable option for total disc arthroplasty.

Degeneration of the intervertebral discs is a progressive cascade of cellular, compositional and structural changes that is frequently associated with low back pain. As the first signs of disc degeneration typically arise in the disc's central nucleus pulposus (NP), augmentation of the NP via hydrogel injection represents a promising strategy to treat early to mid-stage degeneration. The purpose of this study was to establish the translational feasibility of a triple interpenetrating network hydrogel composed of dextran, chitosan, and teleostean (DCT) for augmentation of the degenerative NP in a preclinical goat model. Ex vivo injection of the DCT hydrogel into degenerated goat lumbar motion segments restored range of motion and neutral zone modulus towards physiologic values. To facilitate non-invasive assessment of hydrogel delivery and distribution, zirconia nanoparticles were added to make the hydrogel radiopaque. Importantly, the addition of zirconia did not negatively impact viability or matrix producing capacity of goat mesenchymal stem cells or NP cells seeded within the hydrogel in vitro. In vivo studies demonstrated that the radiopaque DCT hydrogel was successfully delivered to degenerated goat lumbar intervertebral discs, where it was distributed throughout both the NP and annulus fibrosus, and that the hydrogel remained contained within the disc space for two weeks without evidence of extrusion. These results demonstrate the translational potential of this hydrogel for functional regeneration of degenerate intervertebral discs., Statement of Significance: The results of this work demonstrate that a radiopaque hydrogel is capable of normalizing the mechanical function of the degenerative disc, is supportive of disc cell and mesenchymal stem cell viability and matrix production, and can be maintained in the disc space without extrusion following intradiscal delivery in a preclinical large animal model. These results support evaluation of this hydrogel as a minimally invasive disc therapeutic in long-term preclinical studies as a precursor to future clinical application in patients with disc degeneration and low back pain., (Copyright © 2017 Acta Materialia Inc. All rights reserved.)

The development of engineered tissues has progressed over the past 20 years from in vitro characterization to in vivo implementation. For musculoskeletal tissue engineering in particular, the emphasis of many of these studies was to select conditions that maximized functional and compositional gains in vitro. However, the transition from the favorable in vitro culture environment to a less favorable in vivo environment has proven difficult, and, in many cases, engineered tissues do not retain their preimplantation phenotype after even short periods in vivo. Our laboratory recently developed disc-like angle-ply structures (DAPS), an engineered intervertebral disc for total disc replacement. In this study, we tested six different preculture media formulations (three serum-containing and three chemically defined, with varying doses of transforming growth factor β3 [TGF-β3] and varying strategies to introduce serum) for their ability to preserve DAPS composition and metabolic activity during the transition from in vitro culture to in vivo implantation in a subcutaneous athymic rat model. We assayed implants before and after implantation to determine collagen content, glycosaminoglycan (GAG) content, metabolic activity, and magnetic resonance imaging (MRI) characteristics. A chemically defined media condition that incorporated TGF-β3 promoted the deposition of GAG and collagen in DAPS in vitro, the maintenance of accumulated matrix in vivo, and minimal changes in the metabolic activity of cells within the construct. Preculture in serum-containing media (with or without TGF-β3) was not compatible with DAPS maturation, particularly in the nucleus pulposus (NP) region. All groups showed increased collagen production after implantation. These findings define a favorable preculture strategy for the translation of engineered discs seeded with disc cells.

Injectable hydrogels have gained prominence in the field of tissue engineering for minimally invasive delivery of cells for tissue repair and in the filling of irregular defects. However, many injectable hydrogels exhibit long gelation times or are not stable for long periods after injection. To address these concerns, we used thermosensitive poly(N-vinylcaprolactam) (PNVCL) hydrogels due to their cytocompatibility and fast response to temperature stimuli. Changes in the PNVCL molecular weight and concentration enabled the development of hydrogels with tunable mechanical properties and fast gelation times (<60 s when the temperature was raised from room temperature to physiologic temperature). Chondrocytes (CHs) and mesenchymal stem cells were encapsulated in PNVCL hydrogels and exhibited high viability (∼90%), as monitored by Live/Dead staining and Alamar Blue assays. Three-dimensional constructs of CH-laden PNVCL hydrogels supported cartilage-specific extracellular matrix production both in vitro and after subcutaneous injection in nude rats for up to 8 weeks. Moreover, biochemical analyses of constructs demonstrated a time-dependent increase in glycosaminoglycans (GAGs) and collagen, which were significantly augmented in the implants cultured in vivo. Histological analyses also demonstrated regular distribution of synthesized cartilage components, including abundant GAGs and type II collagen. The findings from this study demonstrate thermosensitive PNVCL as a candidate injectable biomaterial to deliver cells for cartilage tissue engineering.

Objective: The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics., Design: Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (μCT), and measurement of disc biomechanical properties., Results: Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact., Conclusions: A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration., Competing Interests: The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this manuscript., (Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Improved diagnostic measures for intervertebral disc degeneration are necessary to facilitate early detection and treatment. The aim of this study was to correlate changes in mechanical and biochemical properties with the quantitative MRI parameters T2 and T1ρ in rabbit lumbar discs using an ex vivo chymopapain digestion model. Rabbit lumbar spinal motion segments from animals less than 6 months of age were injected with 100 μl of saline (control) or chymopapain at 3, 15, or 100 U/ml (n = 5 per group). T2 and T1ρ MRI series were obtained at 4.7T. Specimens were mechanically tested in tension-compression and creep. Normalized nucleus pulposus (NP) water and GAG contents were quantified. Stepwise multiple linear regression was performed to determine which parameters contributed significantly to changes in NP T2 and T1ρ. When all groups were included, multiple regression yielded a model with GAG, compressive modulus, and the creep time constants as variables significantly impacting T2 (multiple r(2)  = 0.64, p = 0.006). GAG and neutral zone (NZ) modulus were identified as variables contributing to T1ρ (multiple r(2)  = 0.28, p = 0.08). When specimens with advanced degeneration were excluded from the multiple regression analysis, T2 was significantly predicted by compressive modulus, τ1, and water content (multiple r(2)  = 0.71, p = 0.009), while no variables were significant predictors in the model for T1ρ. These results indicate that quantitative MRI can detect changes in the mechanical and biochemical properties of the degenerated disc. T2 may be more sensitive to early stage degenerative changes than T1ρ, while both quantitative MRI parameters are sensitive to advanced degeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1382-1388, 2016., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Study Design: Experimental animal study of convective transport in the intervertebral disc., Objective: To quantify the effects of mechanical loading rate on net transport into the healthy and degenerative intervertebral disc in vivo., Summary of Background Data: Intervertebral disc degeneration is linked with a reduction in transport to the avascular disc. Enhancing disc nutrition is, therefore, a potential strategy to slow or reverse the degenerative cascade. Convection induced by mechanical loading is a potential mechanism to augment diffusion of small molecules into the disc., Methods: Skeletally mature New Zealand white rabbits with healthy discs and discs degenerated via needle puncture were subjected to low rate axial compression and distraction loading for 2.5, 5, 10, 15, or 20 minutes after a bolus administration of gadodiamide. Additional animals with healthy discs were subjected to high-rate loading for 10 minutes or no loading for 10 minutes. Transport into the disc for each loading regimen was quantified using post-contrast-enhanced magnetic resonance imaging., Results: Low-rate loading resulted in the rapid uptake and clearance of gadodiamide in the disc. Low-rate loading increased net transport into the nucleus by a mean 16.8% and 12.6% in healthy and degenerative discs, respectively. The kinetics of small molecule uptake and clearance were accelerated in both healthy and degenerative discs with low-rate loading. In contrast, high-rate loading reduced transport into nucleus by a mean 16.8%., Conclusion: These results illustrate that trans-endplate diffusion can be enhanced by forced convection in both healthy and degenerative discs in vivo. Mechanical loading-induced convection could offer therapeutic benefit for degenerated discs by enhancing uptake of nutrients and clearance of by-products., Level of Evidence: 4.

Background Context: The intervertebral disc primarily relies on trans-endplate diffusion for the uptake of nutrients and the clearance of byproducts. In degenerative discs, diffusion is often diminished by endplate sclerosis and reduced proteoglycan content. Mechanical loading-induced convection has the potential to augment diffusion and enhance net transport into the disc. The ability of convection to augment disc transport is controversial and has not been demonstrated in vivo., Purpose: To determine if loading-induced convection can enhance small molecule transport into the intervertebral disc in vivo., Study Design: Net transport was quantified via postcontrast enhanced magnetic resonance imaging (MRI) into the discs of the New Zealand white rabbit lumbar spine subjected to in vivo cyclic low rate loading., Methods: Animals were administered the MRI contrast agent gadodiamide intravenously and subjected to in vivo low rate loading (0.5 Hz, 200 N) via a custom external loading apparatus for either 2.5, 5, 10, 15, or 20 minutes. Animals were then euthanized and the lumbar spines imaged using postcontrast enhanced MRI. The T1 constants in the nucleus, annulus, and cartilage endplates were quantified as a measure of gadodiamide transport into the loaded discs compared with the adjacent unloaded discs. Microcomputed tomography was used to quantify subchondral bone density., Results: Low rate loading caused the rapid uptake and clearance of gadodiamide in the nucleus compared with unloaded discs, which exhibited a slower rate of uptake. Relative to unloaded discs, low rate loading caused a maximum increase in transport into the nucleus of 16.8% after 5 minutes of loading. Low rate loading increased the concentration of gadodiamide in the cartilage endplates at each time point compared with unloaded levels., Conclusions: Results from this study indicate that forced convection accelerated small molecule uptake and clearance in the disc induced by low rate mechanical loading. Low rate loading may, therefore, be therapeutic to the disc as it may enhance the nutrient uptake and waste product clearance., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Intervertebral disc health is mediated in part by nutrient diffusion from the microvasculature in the adjacent subchondral bone. Evidence suggests that a reduction in nutrient diffusion contributes to disc degeneration, but the role of the microvasculature is unclear. The purpose of this study was to induce changes in the endplate microvasculature in vivo via pharmaceutical intervention and then correlate microvasculature characteristics to diffusion and disc health. New Zealand white rabbits were administered either nimodipine (to enhance microvessel density) or nicotine (to diminish microvessel density) daily for 8 weeks compared to controls. Trans-endplate diffusion and disc health were quantified using post-contrast enhanced magnetic resonance imaging (MRI). Histology was utilized to assess changes to the subchondral vasculature. Results indicate that nimodipine increased vessel area and vessel-endplate contact length, causing a significant increase in disc diffusion. Surprisingly, nicotine caused increases in vessel number and area but did not alter diffusion into the disc. The drug treatments did affect the microvasculature and diffusion, but the relationship between the two is complex and dependent on multiple factors which include vessel-endplate distance, and vessel-endplate contact length in addition to vessel density. Our data suggest that drugs can modulate these factors to augment or diminish small molecule transport., (© 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)