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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.)

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.)

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.)

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.)

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.)

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.)