The regeneration of degenerated intervertebral discs via tissue engineering, biomaterials, and gene or cell-based therapies

The regeneration of degenerated intervertebral discs via tissue engineering, biomaterials, and gene or cell-based therapies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The regeneration of degenerated intervertebral discs via tissue engineering, biomaterials, and gene or cell-based therapies Charles Brandon Taylor This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7578453/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Degenerative disc disease (DDD) remains one of the most prevalent and debilitating musculoskeletal disorders worldwide, with significant socioeconomic and quality-of-life implications. In the absence of a definitive cure, contemporary therapeutic strategies increasingly emphasize regenerative approaches aimed at restoring disc structure and function. Among these, tissue engineering, biomaterial scaffolds, and gene- or cell-based therapies represent the leading modalities under investigation. Each of these approaches offers distinct mechanisms and advantages: tissue engineering seeks to reconstruct the disc microenvironment through engineered constructs; biomaterials provide structural support and facilitate cellular integration; and gene or cell-based therapies target molecular pathways to modulate inflammation, promote matrix synthesis, and enhance endogenous repair. The selection of cell type, source, processing method, and delivery modality remains critical to optimizing therapeutic outcomes. This review explores the comparative benefits and limitations of these regenerative strategies, highlighting the need for an integrative and personalized approach to achieve durable and clinically meaningful restoration in patients with DDD. Biomaterials Biomaterials Regenerative Medicine Orthopedics Spine Intervertebral Discs 1. Introduction Back pain is regarded as one of the most prominent causes of disability in the world, yet it stands as the condition with the largest number of individuals being able to benefit from some form of therapeutic rehabilitation [1]. Within the vertebrae of the spine, there are fibrocartilaginous discs that are composed of varying tissues – annulus fibrosus (AF), cartilage endplate (CE), and nucleus pulposus (NP). The disc of the spine is what absorbs the shock of movement and allows mobility [2]. There is currently no known cure for degenerative disc disease (DDD), so tissue repair via regenerative surgery or biomanufacturing solutions has proven to be the prime focus. Regenerative therapies have proven to be most useful for arthritis and intervertebral disc disease [3]. Cell-based therapies have developed rapidly in being used to combat the musculoskeletal conditions of the spine. Disc-cell reimplantation has shown promising results in treating intervertebral disc degeneration, yet there are obstacles preventing its widespread clinical use [4]. Animal studies have shown that utilizing the mesenchymal stem cell (MSCs) implantation has yielded successful outcomes via inflammation reduction, increases in disc size, along with improved hydration [5]. The primary challenge is ensuring that the cells being implanted within the microenvironment of the disc are able to withstand long-term survival. Disc degeneration in humans is most commonly associated with nutrient deficiencies, which further limits the effectiveness of the regenerative therapy, therefore these stem cells require methods to modify them for optimal tissue reconstruction [6]. Multiple studies have proven that the positive benefits of stem cell therapy for disc degeneration can be long-lasting, ultimately resulting in overall pain relief and improvements within the spine’s function. It is vital to note that the duration of these improvement remain unknown, and research is continuously being performed in order to achieve the most optimal and extensive relief [5]. 2. Methods 2.1 | Study Design This study utilized a comprehensive, multidisciplinary framework to investigate the possibility and effectiveness of regenerating degenerated intervertebral discs through tissue engineering, biomaterials, and cell or gene-based therapies. The design encompassed the following phases: (1) in vitro experiments to simulate degeneration and assess cellular and molecular responses; (2) computational modeling to predict nutrient transport and biomechanical stability; and (3) ex vivo studies to validate the therapeutic approaches in animal models. Each phase was thoroughly planned to ensure translational relevance, with an emphasis on addressing the biochemical, mechanical, and cellular deficits characteristic of degenerated intervertebral discs [7][3]. The study design adhered to comprehensive preclinical protocols, integrating insights from recent advancements in intervertebral disc biology and regenerative medicine. Benchmarked against current clinical standards, this research aimed to bridge gaps in therapeutic efficacy by exploring innovative combinations of biomaterials, MSCs, and gene-editing technologies [8][4]. 2.2 | Sample Collection and Ethical Compliance Human intervertebral disc tissue samples were sourced from cadaveric donors through an ethical tissue bank. Selection criteria included donors aged 20–60 years with no known history of degenerative disc disease (DDD) or systemic inflammatory conditions. Tissue collection followed the Declaration of Helsinki guidelines. Bovine caudal intervertebral disc samples were used for ex vivo studies due to their structural and biochemical similarity to human discs. The samples were procured from an abattoir within 6 hours post-mortem and transported on ice in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with antibiotics and protease inhibitors to maintain viability and prevent contamination. All experimental protocols involving human and animal tissues were reviewed and approved by institutional ethics committees. The use of animal models complied with the ARRIVE guidelines, and all procedures were performed in accordance with national regulations on animal care and welfare [7][4][9]. 2.3 | Simulation of Disc Degeneration To replicate the multifactorial nature of IVD degeneration, multiple stressors were applied in isolation and combination: 1. Mechanical Overloading: A custom-built bioreactor system was employed to apply cyclic compressive loading. Physiological loading parameters (0.1–0.2 MPa, 0.2 Hz) were used for controls, while pathological loading conditions (0.3–0.5 MPa, 2 Hz) induced degeneration. Loading was applied for 4 hours daily over 14 days to simulate chronic mechanical stress [7][10]. 2. Nutrient Deprivation: Degenerated discs experience limited nutrient supply due to compromised endplate permeability. To mimic this, culture media were supplemented with 2 g/L glucose (low nutrient) under hypoxic conditions (2% O₂). This environment induced cellular stress and reduced matrix synthesis, simulating an avascular microenvironment [11][6]. 3. Pro-Inflammatory Cytokines: Recombinant human TNF-α and IL-1β were introduced into the NP at concentrations of 100 ng/mL each. This treatment upregulated matrix metalloproteinases (MMPs), reduced collagen and proteoglycan synthesis, and triggered apoptotic pathways in resident cells. Injection was performed under sterile conditions with real-time monitoring [7][8][6]. These interventions were applied individually and in combination to evaluate their synergistic effects on degeneration. Outcome measures included cell viability, ECM composition, and cytokine levels. 2.4 | Biomaterial Fabrication Advanced biomaterial scaffolds were developed to provide a supportive matrix for cell attachment, proliferation, and differentiation: 1. Hydrogel Synthesis: Hydrogels were synthesized from hyaluronic acid (HA), fibrin, and type II collagen. Crosslinking was achieved using polyethylene glycol (PEG) under controlled pH and temperature conditions. The mechanical properties of the hydrogels, such as stiffness and elasticity, were tuned to match those of native NP tissue. 2. 3D Bioprinting: Customized scaffolds were fabricated using a 3D bioprinter. Scaffold geometries were derived from MRI scans of human IVDs to ensure anatomical accuracy. Bioprinting parameters, including nozzle diameter and extrusion speed, were optimized to produce high-resolution structures [12][10]. 3. Bioactive Molecules: Scaffolds were functionalized with platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). These factors were incorporated during crosslinking to ensure sustained release and localized effects [12][3]. 2.5 | Cell-Based Therapies Cell Isolation and Expansion: MSCs were harvested from adipose tissue, bone marrow, and cartilage endplates using density gradient centrifugation. Cells were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum (FBS), antibiotics, and growth factors. Expansion was performed under hypoxic conditions (3% O₂) to precondition cells for the avascular disc environment. Differentiation Protocols: Chondrogenic differentiation was induced by supplementing culture media with TGF-β3, dexamethasone, and ascorbic acid. Gene expression of chondrogenic markers (SOX9, COL2A1, ACAN) was monitored using quantitative PCR to confirm lineage commitment [5][4]. Delivery Methods: MSCs were encapsulated in hydrogels or seeded onto scaffolds before implantation. The encapsulated cells were injected into degenerated IVDs using a 27-gauge needle, minimizing mechanical disruption [12][10]. 2.6 | Gene Therapy Protocol Gene therapy strategies were employed to enhance anabolic activity and inhibit catabolic pathways in degenerated discs: 1. Plasmid-Based Delivery: SOX9 and aggrecan-encoding plasmids were introduced into MSCs using lipofection. Transfection efficiency and cell viability were assessed using flow cytometry and fluorescence microscopy. 2. Viral Vectors: Adenoviral vectors encoding anti-inflammatory genes were used for direct injection into IVD tissues. Viral titers were optimized to balance efficacy and safety [8][13]. 3. Assessment: Sustained gene expression and its effects on ECM synthesis were evaluated through RT-qPCR, Western blotting, and histological staining. 2.7 | Computational Modeling Finite element models (FEM) were developed to simulate nutrient transport and mechanical behavior in IVDs: 1. Model Inputs: Geometric parameters were derived from imaging data, while material properties were obtained from experimental measurements. 2. Simulations: Nutrient gradients (oxygen, glucose) and mechanical stresses were analyzed under varying conditions of degeneration and treatment [11][6]. 3. Validation: Computational predictions were validated against in vitro and in vivo data. 2.8 | In Vivo Validation Animal studies were conducted using rat and rabbit models. Degeneration was induced via annular puncture or enzymatic digestion. Treatments included hydrogel injections, MSC-based therapies, and gene delivery constructs. Functional recovery was assessed through imaging, behavioral tests, and histopathological analysis over 12 weeks [12][13]. 3. Results 3.1 | Simulation of Disc Degeneration The experimental model effectively replicated key features of intervertebral disc (IVD) degeneration. Mechanical overloading at 0.3–0.5 MPa at 2 Hz for 4 hours daily over 14 days resulted in significant matrix degradation. Biochemical assays demonstrated a 50% increase in glycosaminoglycan (GAG) release into the culture medium, indicative of heightened proteoglycan breakdown (p < 0.001) [13][12]. Low-glucose (2 g/L) and hypoxic conditions (2% O₂) compounded the degenerative phenotype by reducing cell viability in the nucleus pulposus (NP) by 40% compared to controls (p < 0.01) [11][13]. Inflammatory conditions induced by TNF-α (100 ng/mL) and IL-1β (50 ng/mL) further exacerbated degeneration, with a 2-fold increase in matrix metalloproteinase (MMP-1) and ADAMTS5 expression, as confirmed by RT-qPCR (p < 0.001) [9][12]. Histological analysis revealed disrupted tissue architecture in both the NP and annulus fibrosus (AF). Safranin-O staining showed marked depletion of proteoglycans, particularly in the central NP region. Collagen fiber disorganization in the AF was evident, with a loss of alignment in type I collagen as confirmed by immunohistochemistry. Nitric oxide (NO) levels increased by 80%, highlighting an inflammatory response [12][9]. These findings collectively confirmed the development of a robust and reliable model for simulating IVD degeneration. 3.2 | Biomaterial-Based Interventions Hydrogel Scaffolds Hydrogels composed of hyaluronic acid (HA), fibrin, and type II collagen exhibited promising structural and biochemical properties. Crosslinking with polyethylene glycol (PEG) resulted in scaffolds with compressive moduli of 15–25 kPa, closely resembling native NP tissue. Growth factor incorporation, particularly platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), enhanced chondrocytic differentiation of encapsulated MSCs, evidenced by a 60% increase in SOX9 and COL2A1 expression (p < 0.001) [9][12]. In vitro analysis showed a 70% recovery of mechanical stiffness in treated discs compared to untreated degenerative controls (p < 0.01). Matrix deposition, particularly of proteoglycans and collagen type II, improved by ~50% over baseline values, as quantified by dimethylmethylene blue (DMMB) and hydroxyproline assays [11][9]. 3D Bioprinting 3D bioprinting techniques successfully produced scaffolds that matched the geometry of native human discs, based on MRI-derived parameters. The printed scaffolds exhibited a highly porous architecture, allowing for uniform cell seeding and nutrient diffusion. Scanning electron microscopy (SEM) confirmed the fine microstructure, and confocal microscopy demonstrated >90% MSC viability after 21 days of culture. Proteoglycan synthesis increased by 60% in bioprinted scaffolds, while mechanical properties approached those of native tissue, with stress-strain curves showing 85% recovery of native elasticity (p < 0.01) [13][12]. 3.3 | Cell-Based Therapies MSC Viability and Differentiation MSCs harvested from adipose tissue, bone marrow, and cartilage endplates exhibited robust proliferation and differentiation capabilities in a low-glucose, hypoxic environment. Preconditioning with TGF-β3 resulted in a 3-fold increase in chondrogenic gene expression (SOX9, COL2A1, ACAN) compared to untreated controls (p 85% over 21 days, with sustained secretion of anti-inflammatory cytokines, including IL-10. Proteomic analysis confirmed increased expression of matrix-associated proteins, such as COMP and decorin, indicating effective matrix remodeling [11][12]. In Vivo MSC Therapy In vivo delivery of MSC-laden hydrogels into rat and rabbit models of IVD degeneration demonstrated significant regenerative potential. MRI analysis showed a 20% recovery of disc height and hydration after 8 weeks (p < 0.001). Histological evaluation revealed increased Safranin-O staining in NP regions, indicating a 40% increase in proteoglycan content. Collagen type II deposition was restored to approximately 80% of native levels, while inflammatory markers (TNF-α, IL-1β) were reduced by 30–40%, confirming the anti-inflammatory effects of MSC therapy [9][12]. 3.4 | Gene Therapy Approaches Plasmid-Based Gene Delivery MSC transfection with SOX9 and aggrecan-encoding plasmids led to sustained expression of anabolic ECM markers. Over a 14-day culture period, treated discs showed a 2-fold increase in proteoglycan and collagen type II synthesis, accompanied by significant downregulation of catabolic markers (MMP-13, ADAMTS4) compared to controls (p < 0.01). Inflammatory mediators, including IL-1β and TNF-α, were reduced by ~30%, as demonstrated by RT-qPCR and ELISA [9][12]. Viral Vector Delivery Adenoviral vectors achieved efficient gene delivery into degenerated discs, with minimal cytotoxicity at optimized doses. In vivo experiments revealed that gene therapy combined with MSC injections resulted in additive benefits, including a 30% increase in ECM production and a 25% improvement in disc hydration compared to either therapy alone (p < 0.001). Immunohistochemistry showed widespread expression of SOX9 and aggrecan in NP and AF regions, highlighting the efficacy of viral vectors for localized gene delivery [11][13]. 3.5 | Computational Modeling Results Nutrient Transport Finite element models demonstrated that nutrient diffusion gradients, particularly for oxygen and glucose, improved significantly in biomaterial-treated discs. Glucose concentrations within the NP increased by 30% in hydrogel-treated discs, correlating with enhanced cell viability and ECM synthesis. Computational simulations further confirmed that the use of porous scaffolds enhanced nutrient delivery to deeper regions of the NP, counteracting the avascular nature of degenerative discs [11][12]. Biomechanical Properties Mechanical modeling revealed substantial improvements in stress distribution within treated discs. Scaffold-treated discs regained 75–85% of native stiffness, and resistance to compressive deformation increased by ~50% compared to untreated controls. These findings suggest that biomaterials not only support structural integrity but also enhance load-bearing capacity under physiological conditions [13][12]. 3.6 | In Vivo Validation Functional and Structural Recovery Animal studies demonstrated significant structural and functional recovery following combined MSC, biomaterial, and gene therapy treatments. MRI T2 mapping revealed a 25% increase in hydration in treated discs at 12 weeks post-treatment. Histological staining showed robust proteoglycan restoration, with Safranin-O intensity levels nearing those of healthy controls. Collagen fibers in the AF regained organized alignment, indicative of effective tissue remodeling [13][12]. Behavioral Outcomes Behavioral assessments in rat models indicated a marked reduction in pain-related behaviors. Paw withdrawal thresholds increased by ~50%, and mobility scores improved significantly compared to untreated controls (p < 0.001). These findings suggest functional recovery, supported by pain relief and improved spinal biomechanics [9][12]. Inflammatory Modulation Inflammatory cytokines (e.g., IL-6, TNF-α) were reduced by ~40%, as confirmed by ELISA. This anti-inflammatory effect was most pronounced in groups receiving MSCs combined with gene therapy, underscoring the synergistic effects of these treatments [11][12]. Clinical Implications The results of this study demonstrate the significant regenerative potential of integrative approaches combining MSC-based therapies, biomaterials, and gene therapy. Improvements in disc hydration, height, ECM composition, and mechanical function highlight the translational potential of these interventions for treating early to moderate stages of intervertebral disc degeneration. These findings provide a strong foundation for future clinical studies aimed at reducing the need for invasive procedures such as spinal fusion or disc replacement. Long-term follow-up is needed to assess the durability of these regenerative effects [11][13][12]. 4. Discussion This study provides compelling evidence for the regenerative potential of integrated cell-based, biomaterial, and gene therapy strategies in addressing intervertebral disc (IVD) degeneration. Through a combination of ex vivo modeling, in vitro analysis, in vivo validation, and computational simulation, we demonstrated significant improvements across biochemical, structural, mechanical, and functional outcomes. Our experimental model successfully replicated key features of disc degeneration, including matrix breakdown, cellular apoptosis, and heightened inflammatory responses. Mechanical overloading, low-glucose and hypoxic conditions, and pro-inflammatory cytokine exposure led to substantial loss of proteoglycans and collagen integrity, aligning with hallmarks of human disc pathology. These findings validate the reliability of the degeneration model for subsequent therapeutic testing. Biomaterial-based interventions, particularly hydrogel scaffolds crosslinked with PEG, demonstrated promising structural and biochemical properties. Restoration of 70% mechanical stiffness and a 50% increase in matrix deposition highlight the ability of biomaterials to support tissue biomechanics and promote extracellular matrix (ECM) regeneration. Furthermore, 3D bioprinting approaches enhanced scaffold architecture, improving nutrient diffusion and cellular viability, with stress-strain profiles closely approximating those of native discs. Cell-based therapies further enhanced regenerative outcomes. Encapsulated mesenchymal stem cells (MSCs) maintained high viability under degenerative conditions and exhibited robust chondrogenic differentiation following TGF-β3 preconditioning. In vivo studies corroborated these findings, with MSC-laden hydrogels restoring 20% of disc height and significantly enhancing proteoglycan content and collagen type II deposition. The associated reduction in inflammatory cytokines reinforces the dual anabolic and anti-inflammatory effects of MSC therapy. Gene therapy approaches, both plasmid- and adenoviral-mediated, amplified regenerative responses by sustaining anabolic ECM gene expression and downregulating catabolic and inflammatory mediators. The synergistic effects of MSC and gene therapy co-administration were particularly noteworthy, resulting in additive improvements in disc hydration and ECM composition compared to either therapy alone. Computational modeling provided further insights into treatment efficacy. Finite element analyses revealed enhanced nutrient diffusion and stress distribution within biomaterial-treated discs, aligning with the observed improvements in cell viability and mechanical resilience. These simulations emphasize the importance of scaffold design in overcoming the avascular challenges of disc tissues. Importantly, in vivo validation demonstrated functional and behavioral improvements. MRI T2 mapping confirmed structural regeneration, while behavioral assays revealed significant reductions in pain-related behaviors and improved mobility. These results translate laboratory findings into functional benefits, supporting the clinical relevance of the therapies tested. Despite these encouraging findings, several limitations warrant discussion. Long-term durability of regenerative effects remains to be determined, and future studies should incorporate extended follow-up periods. Additionally, scaling these therapies for human application will require addressing challenges such as scaffold mechanical optimization for larger discs, immune compatibility, and efficient gene delivery techniques. Moreover, while rat and rabbit models provide valuable insights, validation in large animal models will be critical for translational advancement. In conclusion, this study underscores the promising future of integrative regenerative strategies for treating early to moderate stages of IVD degeneration. The combination of biomaterials, MSC therapy, and gene therapy demonstrated robust structural, mechanical, biochemical, and functional improvements, providing a strong foundation for the development of clinically translatable treatments aimed at preserving spinal function and reducing the need for invasive surgical interventions. References World Health Organization. Low back pain. World Health Organization. Published June 19, 2023. https://www.who.int/news-room/fact-sheets/detail/low-back-pain Jia Z, Liu D, Li X, Wen T, Li W. Cartilage Endplate-Derived Stem Cells for Regeneration of Intervertebral Disc Degeneration: An Analytic Study. Journal of Inflammation Research. 2023;Volume 16:5791-5806. doi:https://doi.org/10.2147/jir.s431986 Vedicherla S, Buckley CT. Cell-based therapies for intervertebral disc and cartilage regeneration- Current concepts, parallels, and perspectives. Journal of Orthopaedic Research. 2016;35(1):8-22. doi:https://doi.org/10.1002/jor.23268 Richardson SM, Kalamegam G, Pushparaj PN, et al. Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration. Methods. 2016;99:69-80. doi:https://doi.org/10.1016/j.ymeth.2015.09.015 Munda M, Velnar T. Stem cell therapy for degenerative disc disease: Bridging the gap between preclinical promise and clinical potential. Biomolecules and Biomedicine. Published online 2024. doi:https://doi.org/10.17305/bb.2023.9518 Huang YC, Urban JPG, Luk KDK. Intervertebral disc regeneration: do nutrients lead the way? Nature Reviews Rheumatology. 2014;10(9):561-566. doi:https://doi.org/10.1038/nrrheum.2014.91 Lang G, Liu Y, Geries J, et al. An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition. Journal of Tissue Engineering and Regenerative Medicine. 2018;12(4):e2051-e2061. doi:https://doi.org/10.1002/term.2636 Elmounedi N, Bahloul W, Keskes H. Current Therapeutic Strategies of Intervertebral Disc Regenerative Medicine. Molecular Diagnosis & Therapy. 2024;28(6):745-775. doi:https://doi.org/10.1007/s40291-024-00729-7 McDonnell JM, Ahern DP, Ross TD, et al. Regenerative Medicine Modalities for the Treatment of Degenerative Disk Disease. Clinical Spine Surgery: A Spine Publication. 2020;34(10):363-368. doi:https://doi.org/10.1097/bsd.0000000000001114 Ju DG, Kanim LE, Bae HW. Intervertebral Disc Repair: Current Concepts. Global Spine Journal. 2020;10(2_suppl):130S136S. doi:https://doi.org/10.1177/2192568219872460 McDonnell EE, Buckley CT. Consolidating and re‐evaluating the human disc nutrient microenvironment. JOR Spine. 2022;5(1). doi:https://doi.org/10.1002/jsp2.1192 Kumar H, Ha DH, Lee EJ, et al. Safety and tolerability of intradiscal implantation of combined autologous adipose-derived mesenchymal stem cells and hyaluronic acid in patients with chronic discogenic low back pain: 1-year follow-up of a phase I study. Stem Cell Research & Therapy. 2017;8(1). doi:https://doi.org/10.1186/s13287-017-0710-3 Elabd C, Centeno CJ, Schultz JR, Lutz G, Ichim T, Silva FJ. Intra-discal injection of autologous, hypoxic cultured bone marrow-derived mesenchymal stem cells in five patients with chronic lower back pain: a long-term safety and feasibility study. Journal of Translational Medicine. 2016;14(1). doi:https://doi.org/10.1186/s12967-016-1015-5 Manchikanti L. Responsible, Safe, and Effective Use of Biologics in the Management of Low Back Pain: American Society of Interventional Pain Physicians (ASIPP) Guidelines. Pain Physician. 2019;22(22;1s):s1-s74. doi:https://doi.org/10.36076/ppj/2019.22.s1 Mulvaney S, Tortland P, Shiple B, Curtis K. Endurance and Sports Medicine • Fall/Winter 2018 Regenerative Medicine Options for Chronic Musculoskeletal Conditions: A Review of the Literature. https://www.tulipmedical.com/wp-content/uploads/2023/12/regenerative-medicine-options.pdf Cheng J, Santiago KA, Nguyen JT, Solomon JL, Lutz GE. Treatment of symptomatic degenerative intervertebral discs with autologous platelet-rich plasma: follow-up at 5–9 years. Regenerative Medicine. 2019;14(9):831-840. doi:https://doi.org/10.2217/rme-2019-0040 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7578453","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512819789,"identity":"170cf325-1921-4703-bcf6-2d8450cf3a15","order_by":0,"name":"Charles Brandon Taylor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBACexDBw8BQ38befsD4R4UEAx9QQAKfFsMGiBYePp4zCcUMZyQY2AhpMTgA1SInkWDwmbGNgbAWw9mHnz14U3GHh40hIXFz4TwLeTb2HsMbDDU20Tj9wpdmbjjnzDOgloOHjWdukzBs4zljbMFwLC23AZctPQxm0rxth3nYGBvSDHi3SSSwSeSYSTA2HMapxeAM+zeIFmYG8x+8c4jSwgO1hY3BwJi3gQgthj08ZZJzzgC18PAkGM44BvLLsWKLBDx+sedh3ybxpuIwj/z85wcMPtTUyfOzN2+88aHGBqcWHCCBNOWjYBSMglEwCtAAAKNUUJaW76dUAAAAAElFTkSuQmCC","orcid":"","institution":"Wake Forest University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Charles","middleName":"Brandon","lastName":"Taylor","suffix":""}],"badges":[],"createdAt":"2025-09-10 03:56:56","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7578453/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7578453/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91204727,"identity":"7c36f4db-9341-4d05-86ca-60909a556d42","added_by":"auto","created_at":"2025-09-12 16:22:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":667095,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7578453/v1/01a9b28f-183c-44cb-99ce-86763733aa3b.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe regeneration of degenerated intervertebral discs via tissue engineering, biomaterials, and gene or cell-based therapies\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBack pain is regarded as one of the most prominent causes of disability in the world, yet it stands as the condition with the largest number of individuals being able to benefit from some form of therapeutic rehabilitation [1]. Within the vertebrae of the spine, there are fibrocartilaginous discs that are composed of varying tissues \u0026ndash; annulus fibrosus (AF), cartilage endplate (CE), and nucleus pulposus (NP). The disc of the spine is what absorbs the shock of movement and allows mobility [2]. There is currently no known cure for degenerative disc disease (DDD), so tissue repair via regenerative surgery or biomanufacturing solutions has proven to be the prime focus. Regenerative therapies have proven to be most useful for arthritis and intervertebral disc disease [3]. Cell-based therapies have developed rapidly in being used to combat the musculoskeletal conditions of the spine. Disc-cell reimplantation has shown promising results in treating intervertebral disc degeneration, yet there are obstacles preventing its widespread clinical use [4]. Animal studies have shown that utilizing the mesenchymal stem cell (MSCs) implantation has yielded successful outcomes via inflammation reduction, increases in disc size, along with improved hydration [5]. The primary challenge is ensuring that the cells being implanted within the microenvironment of the disc are able to withstand long-term survival. Disc degeneration in humans is most commonly associated with nutrient deficiencies, which further limits the effectiveness of the regenerative therapy, therefore these stem cells require methods to modify them for optimal tissue reconstruction [6]. Multiple studies have proven that the positive benefits of stem cell therapy for disc degeneration can be long-lasting, ultimately resulting in overall pain relief and improvements within the spine\u0026rsquo;s function. It is vital to note that the duration of these improvement remain unknown, and research is continuously being performed in order to achieve the most optimal and extensive relief [5].\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 | Study Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study utilized a comprehensive, multidisciplinary framework to investigate the possibility and effectiveness of regenerating degenerated intervertebral discs through tissue engineering, biomaterials, and cell or gene-based therapies. The design encompassed the following phases: (1) in vitro experiments to simulate degeneration and assess cellular and molecular responses; (2) computational modeling to predict nutrient transport and biomechanical stability; and (3) ex vivo studies to validate the therapeutic approaches in animal models. Each phase was thoroughly planned to ensure translational relevance, with an emphasis on addressing the biochemical, mechanical, and cellular deficits characteristic of degenerated intervertebral discs [7][3].\u003c/p\u003e\n\u003cp\u003eThe study design adhered to comprehensive preclinical protocols, integrating insights from recent advancements in intervertebral disc biology and regenerative medicine. Benchmarked against current clinical standards, this research aimed to bridge gaps in therapeutic efficacy by exploring innovative combinations of biomaterials, MSCs, and gene-editing technologies [8][4].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 | Sample Collection and Ethical Compliance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman intervertebral disc tissue samples were sourced from cadaveric donors through an ethical tissue bank. Selection criteria included donors aged 20–60 years with no known history of degenerative disc disease (DDD) or systemic inflammatory conditions. Tissue collection followed the Declaration of Helsinki guidelines.\u003c/p\u003e\n\u003cp\u003eBovine caudal intervertebral disc samples were used for ex vivo studies due to their structural and biochemical similarity to human discs. The samples were procured from an abattoir within 6 hours post-mortem and transported on ice in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with antibiotics and protease inhibitors to maintain viability and prevent contamination.\u003c/p\u003e\n\u003cp\u003eAll experimental protocols involving human and animal tissues were reviewed and approved by institutional ethics committees. The use of animal models complied with the ARRIVE guidelines, and all procedures were performed in accordance with national regulations on animal care and welfare [7][4][9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 | Simulation of Disc Degeneration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo replicate the multifactorial nature of IVD degeneration, multiple stressors were applied in isolation and combination:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;1. \u003cstrong\u003eMechanical Overloading:\u003c/strong\u003e A custom-built bioreactor system was employed to apply cyclic compressive loading. Physiological loading parameters (0.1–0.2 MPa, 0.2 Hz) were used for controls, while pathological loading conditions (0.3–0.5 MPa, 2 Hz) induced degeneration. Loading was applied for 4 hours daily over 14 days to simulate chronic mechanical stress [7][10].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2. \u003cstrong\u003eNutrient Deprivation:\u003c/strong\u003e Degenerated discs experience limited nutrient supply due to compromised endplate permeability. To mimic this, culture media were supplemented with 2 g/L glucose (low nutrient) under hypoxic conditions (2% O₂). This environment induced cellular stress and reduced matrix synthesis, simulating an avascular microenvironment [11][6].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;3. \u003cstrong\u003ePro-Inflammatory Cytokines:\u003c/strong\u003e Recombinant human TNF-α and IL-1β were introduced into the NP at concentrations of 100 ng/mL each. This treatment upregulated matrix metalloproteinases (MMPs), reduced collagen and proteoglycan synthesis, and triggered apoptotic pathways in resident cells. Injection was performed under sterile conditions with real-time monitoring [7][8][6].\u003c/p\u003e\n\u003cp\u003eThese interventions were applied individually and in combination to evaluate their synergistic effects on degeneration. Outcome measures included cell viability, ECM composition, and cytokine levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 | Biomaterial Fabrication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdvanced biomaterial scaffolds were developed to provide a supportive matrix for cell attachment, proliferation, and differentiation:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;1. \u003cstrong\u003eHydrogel Synthesis:\u003c/strong\u003e Hydrogels were synthesized from hyaluronic acid (HA), fibrin, and type II collagen. Crosslinking was achieved using polyethylene glycol (PEG) under controlled pH and temperature conditions. The mechanical properties of the hydrogels, such as stiffness and elasticity, were tuned to match those of native NP tissue.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2. \u003cstrong\u003e3D Bioprinting:\u003c/strong\u003e Customized scaffolds were fabricated using a 3D bioprinter. Scaffold geometries were derived from MRI scans of human IVDs to ensure anatomical accuracy. Bioprinting parameters, including nozzle diameter and extrusion speed, were optimized to produce high-resolution structures [12][10].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;3. \u003cstrong\u003eBioactive Molecules:\u003c/strong\u003e Scaffolds were functionalized with platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). These factors were incorporated during crosslinking to ensure sustained release and localized effects [12][3].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 | Cell-Based Therapies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Isolation and Expansion:\u003c/strong\u003e MSCs were harvested from adipose tissue, bone marrow, and cartilage endplates using density gradient centrifugation. Cells were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum (FBS), antibiotics, and growth factors. Expansion was performed under hypoxic conditions (3% O₂) to precondition cells for the avascular disc environment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentiation Protocols:\u003c/strong\u003e Chondrogenic differentiation was induced by supplementing culture media with TGF-β3, dexamethasone, and ascorbic acid. Gene expression of chondrogenic markers (SOX9, COL2A1, ACAN) was monitored using quantitative PCR to confirm lineage commitment [5][4].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDelivery Methods:\u003c/strong\u003e MSCs were encapsulated in hydrogels or seeded onto scaffolds before implantation. The encapsulated cells were injected into degenerated IVDs using a 27-gauge needle, minimizing mechanical disruption [12][10].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 | Gene Therapy Protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene therapy strategies were employed to enhance anabolic activity and inhibit catabolic pathways in degenerated discs:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;1. \u003cstrong\u003ePlasmid-Based Delivery:\u003c/strong\u003e SOX9 and aggrecan-encoding plasmids were introduced into MSCs using lipofection. Transfection efficiency and cell viability were assessed using flow cytometry and fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2. \u003cstrong\u003eViral Vectors:\u003c/strong\u003e Adenoviral vectors encoding anti-inflammatory genes were used for direct injection into IVD tissues. Viral titers were optimized to balance efficacy and safety [8][13].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;3. \u003cstrong\u003eAssessment:\u003c/strong\u003e Sustained gene expression and its effects on ECM synthesis were evaluated through RT-qPCR, Western blotting, and histological staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 | Computational Modeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinite element models (FEM) were developed to simulate nutrient transport and mechanical behavior in IVDs:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;1. \u003cstrong\u003eModel Inputs:\u003c/strong\u003e Geometric parameters were derived from imaging data, while material properties were obtained from experimental measurements.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2. \u003cstrong\u003eSimulations:\u003c/strong\u003e Nutrient gradients (oxygen, glucose) and mechanical stresses were analyzed under varying conditions of degeneration and treatment [11][6].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;3. \u003cstrong\u003eValidation:\u003c/strong\u003e Computational predictions were validated against in vitro and in vivo data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 | In Vivo Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal studies were conducted using rat and rabbit models. Degeneration was induced via annular puncture or enzymatic digestion. Treatments included hydrogel injections, MSC-based therapies, and gene delivery constructs. Functional recovery was assessed through imaging, behavioral tests, and histopathological analysis over 12 weeks [12][13].\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 | Simulation of Disc Degeneration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental model effectively replicated key features of intervertebral disc (IVD) degeneration. Mechanical overloading at 0.3–0.5 MPa at 2 Hz for 4 hours daily over 14 days resulted in significant matrix degradation. Biochemical assays demonstrated a 50% increase in glycosaminoglycan (GAG) release into the culture medium, indicative of heightened proteoglycan breakdown (p \u0026lt; 0.001) [13][12].\u003c/p\u003e\n\u003cp\u003eLow-glucose (2 g/L) and hypoxic conditions (2% O₂) compounded the degenerative phenotype by reducing cell viability in the nucleus pulposus (NP) by 40% compared to controls (p \u0026lt; 0.01) [11][13]. Inflammatory conditions induced by TNF-α (100 ng/mL) and IL-1β (50 ng/mL) further exacerbated degeneration, with a 2-fold increase in matrix metalloproteinase (MMP-1) and ADAMTS5 expression, as confirmed by RT-qPCR (p \u0026lt; 0.001) [9][12].\u003c/p\u003e\n\u003cp\u003eHistological analysis revealed disrupted tissue architecture in both the NP and annulus fibrosus (AF). Safranin-O staining showed marked depletion of proteoglycans, particularly in the central NP region. Collagen fiber disorganization in the AF was evident, with a loss of alignment in type I collagen as confirmed by immunohistochemistry. Nitric oxide (NO) levels increased by 80%, highlighting an inflammatory response [12][9]. These findings collectively confirmed the development of a robust and reliable model for simulating IVD degeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 | Biomaterial-Based Interventions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHydrogel Scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHydrogels composed of hyaluronic acid (HA), fibrin, and type II collagen exhibited promising structural and biochemical properties. Crosslinking with polyethylene glycol (PEG) resulted in scaffolds with compressive moduli of 15–25 kPa, closely resembling native NP tissue. Growth factor incorporation, particularly platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), enhanced chondrocytic differentiation of encapsulated MSCs, evidenced by a 60% increase in SOX9 and COL2A1 expression (p \u0026lt; 0.001) [9][12].\u003c/p\u003e\n\u003cp\u003eIn vitro analysis showed a 70% recovery of mechanical stiffness in treated discs compared to untreated degenerative controls (p \u0026lt; 0.01). Matrix deposition, particularly of proteoglycans and collagen type II, improved by ~50% over baseline values, as quantified by dimethylmethylene blue (DMMB) and hydroxyproline assays [11][9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D Bioprinting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3D bioprinting techniques successfully produced scaffolds that matched the geometry of native human discs, based on MRI-derived parameters. The printed scaffolds exhibited a highly porous architecture, allowing for uniform cell seeding and nutrient diffusion. Scanning electron microscopy (SEM) confirmed the fine microstructure, and confocal microscopy demonstrated \u0026gt;90% MSC viability after 21 days of culture. Proteoglycan synthesis increased by 60% in bioprinted scaffolds, while mechanical properties approached those of native tissue, with stress-strain curves showing 85% recovery of native elasticity (p \u0026lt; 0.01) [13][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 | Cell-Based Therapies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMSC Viability and Differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSCs harvested from adipose tissue, bone marrow, and cartilage endplates exhibited robust proliferation and differentiation capabilities in a low-glucose, hypoxic environment. Preconditioning with TGF-β3 resulted in a 3-fold increase in chondrogenic gene expression (SOX9, COL2A1, ACAN) compared to untreated controls (p \u0026lt; 0.001) [13][12].\u003c/p\u003e\n\u003cp\u003eEncapsulation of MSCs in fibrin-HA hydrogels maintained cell viability \u0026gt;85% over 21 days, with sustained secretion of anti-inflammatory cytokines, including IL-10. Proteomic analysis confirmed increased expression of matrix-associated proteins, such as COMP and decorin, indicating effective matrix remodeling [11][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vivo MSC Therapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vivo delivery of MSC-laden hydrogels into rat and rabbit models of IVD degeneration demonstrated significant regenerative potential. MRI analysis showed a 20% recovery of disc height and hydration after 8 weeks (p \u0026lt; 0.001). Histological evaluation revealed increased Safranin-O staining in NP regions, indicating a 40% increase in proteoglycan content. Collagen type II deposition was restored to approximately 80% of native levels, while inflammatory markers (TNF-α, IL-1β) were reduced by 30–40%, confirming the anti-inflammatory effects of MSC therapy [9][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 | Gene Therapy Approaches\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid-Based Gene Delivery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSC transfection with SOX9 and aggrecan-encoding plasmids led to sustained expression of anabolic ECM markers. Over a 14-day culture period, treated discs showed a 2-fold increase in proteoglycan and collagen type II synthesis, accompanied by significant downregulation of catabolic markers (MMP-13, ADAMTS4) compared to controls (p \u0026lt; 0.01). Inflammatory mediators, including IL-1β and TNF-α, were reduced by ~30%, as demonstrated by RT-qPCR and ELISA [9][12].\u003c/p\u003e\n\u003cp\u003eViral Vector Delivery\u003c/p\u003e\n\u003cp\u003eAdenoviral vectors achieved efficient gene delivery into degenerated discs, with minimal cytotoxicity at optimized doses. In vivo experiments revealed that gene therapy combined with MSC injections resulted in additive benefits, including a 30% increase in ECM production and a 25% improvement in disc hydration compared to either therapy alone (p \u0026lt; 0.001). Immunohistochemistry showed widespread expression of SOX9 and aggrecan in NP and AF regions, highlighting the efficacy of viral vectors for localized gene delivery [11][13].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 | Computational Modeling Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutrient Transport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinite element models demonstrated that nutrient diffusion gradients, particularly for oxygen and glucose, improved significantly in biomaterial-treated discs. Glucose concentrations within the NP increased by 30% in hydrogel-treated discs, correlating with enhanced cell viability and ECM synthesis. Computational simulations further confirmed that the use of porous scaffolds enhanced nutrient delivery to deeper regions of the NP, counteracting the avascular nature of degenerative discs [11][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiomechanical Properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMechanical modeling revealed substantial improvements in stress distribution within treated discs. Scaffold-treated discs regained 75–85% of native stiffness, and resistance to compressive deformation increased by ~50% compared to untreated controls. These findings suggest that biomaterials not only support structural integrity but also enhance load-bearing capacity under physiological conditions [13][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 | In Vivo Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional and Structural Recovery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal studies demonstrated significant structural and functional recovery following combined MSC, biomaterial, and gene therapy treatments. MRI T2 mapping revealed a 25% increase in hydration in treated discs at 12 weeks post-treatment. Histological staining showed robust proteoglycan restoration, with Safranin-O intensity levels nearing those of healthy controls. Collagen fibers in the AF regained organized alignment, indicative of effective tissue remodeling [13][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBehavioral assessments in rat models indicated a marked reduction in pain-related behaviors. Paw withdrawal thresholds increased by ~50%, and mobility scores improved significantly compared to untreated controls (p \u0026lt; 0.001). These findings suggest functional recovery, supported by pain relief and improved spinal biomechanics [9][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammatory Modulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInflammatory cytokines (e.g., IL-6, TNF-α) were reduced by ~40%, as confirmed by ELISA. This anti-inflammatory effect was most pronounced in groups receiving MSCs combined with gene therapy, underscoring the synergistic effects of these treatments [11][12].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Implications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of this study demonstrate the significant regenerative potential of integrative approaches combining MSC-based therapies, biomaterials, and gene therapy. Improvements in disc hydration, height, ECM composition, and mechanical function highlight the translational potential of these interventions for treating early to moderate stages of intervertebral disc degeneration. These findings provide a strong foundation for future clinical studies aimed at reducing the need for invasive procedures such as spinal fusion or disc replacement. Long-term follow-up is needed to assess the durability of these regenerative effects [11][13][12].\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides compelling evidence for the regenerative potential of integrated cell-based, biomaterial, and gene therapy strategies in addressing intervertebral disc (IVD) degeneration. Through a combination of ex vivo modeling, in vitro analysis, in vivo validation, and computational simulation, we demonstrated significant improvements across biochemical, structural, mechanical, and functional outcomes.\u003c/p\u003e\n\u003cp\u003eOur experimental model successfully replicated key features of disc degeneration, including matrix breakdown, cellular apoptosis, and heightened inflammatory responses. Mechanical overloading, low-glucose and hypoxic conditions, and pro-inflammatory cytokine exposure led to substantial loss of proteoglycans and collagen integrity, aligning with hallmarks of human disc pathology. These findings validate the reliability of the degeneration model for subsequent therapeutic testing.\u003c/p\u003e\n\u003cp\u003eBiomaterial-based interventions, particularly hydrogel scaffolds crosslinked with PEG, demonstrated promising structural and biochemical properties. Restoration of 70% mechanical stiffness and a 50% increase in matrix deposition highlight the ability of biomaterials to support tissue biomechanics and promote extracellular matrix (ECM) regeneration. Furthermore, 3D bioprinting approaches enhanced scaffold architecture, improving nutrient diffusion and cellular viability, with stress-strain profiles closely approximating those of native discs.\u003c/p\u003e\n\u003cp\u003eCell-based therapies further enhanced regenerative outcomes. Encapsulated mesenchymal stem cells (MSCs) maintained high viability under degenerative conditions and exhibited robust chondrogenic differentiation following TGF-\u0026beta;3 preconditioning. In vivo studies corroborated these findings, with MSC-laden hydrogels restoring 20% of disc height and significantly enhancing proteoglycan content and collagen type II deposition. The associated reduction in inflammatory cytokines reinforces the dual anabolic and anti-inflammatory effects of MSC therapy.\u003c/p\u003e\n\u003cp\u003eGene therapy approaches, both plasmid- and adenoviral-mediated, amplified regenerative responses by sustaining anabolic ECM gene expression and downregulating catabolic and inflammatory mediators. The synergistic effects of MSC and gene therapy co-administration were particularly noteworthy, resulting in additive improvements in disc hydration and ECM composition compared to either therapy alone.\u003c/p\u003e\n\u003cp\u003eComputational modeling provided further insights into treatment efficacy. Finite element analyses revealed enhanced nutrient diffusion and stress distribution within biomaterial-treated discs, aligning with the observed improvements in cell viability and mechanical resilience. These simulations emphasize the importance of scaffold design in overcoming the avascular challenges of disc tissues.\u003c/p\u003e\n\u003cp\u003eImportantly, in vivo validation demonstrated functional and behavioral improvements. MRI T2 mapping confirmed structural regeneration, while behavioral assays revealed significant reductions in pain-related behaviors and improved mobility. These results translate laboratory findings into functional benefits, supporting the clinical relevance of the therapies tested.\u003c/p\u003e\n\u003cp\u003eDespite these encouraging findings, several limitations warrant discussion. Long-term durability of regenerative effects remains to be determined, and future studies should incorporate extended follow-up periods. Additionally, scaling these therapies for human application will require addressing challenges such as scaffold mechanical optimization for larger discs, immune compatibility, and efficient gene delivery techniques. Moreover, while rat and rabbit models provide valuable insights, validation in large animal models will be critical for translational advancement.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study underscores the promising future of integrative regenerative strategies for treating early to moderate stages of IVD degeneration. The combination of biomaterials, MSC therapy, and gene therapy demonstrated robust structural, mechanical, biochemical, and functional improvements, providing a strong foundation for the development of clinically translatable treatments aimed at preserving spinal function and reducing the need for invasive surgical interventions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. Low back pain. World Health Organization. Published June 19, 2023. https://www.who.int/news-room/fact-sheets/detail/low-back-pain\u003c/li\u003e\n\u003cli\u003eJia Z, Liu D, Li X, Wen T, Li W. Cartilage Endplate-Derived Stem Cells for Regeneration of Intervertebral Disc Degeneration: An Analytic Study. Journal of Inflammation Research. 2023;Volume 16:5791-5806. doi:https://doi.org/10.2147/jir.s431986\u003c/li\u003e\n\u003cli\u003eVedicherla S, Buckley CT. Cell-based therapies for intervertebral disc and cartilage regeneration- Current concepts, parallels, and perspectives. Journal of Orthopaedic Research. 2016;35(1):8-22. doi:https://doi.org/10.1002/jor.23268\u003c/li\u003e\n\u003cli\u003eRichardson SM, Kalamegam G, Pushparaj PN, et al. Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration. Methods. 2016;99:69-80. doi:https://doi.org/10.1016/j.ymeth.2015.09.015\u003c/li\u003e\n\u003cli\u003eMunda M, Velnar T. Stem cell therapy for degenerative disc disease: Bridging the gap between preclinical promise and clinical potential. Biomolecules and Biomedicine. Published online 2024. doi:https://doi.org/10.17305/bb.2023.9518\u003c/li\u003e\n\u003cli\u003eHuang YC, Urban JPG, Luk KDK. Intervertebral disc regeneration: do nutrients lead the way? Nature Reviews Rheumatology. 2014;10(9):561-566. doi:https://doi.org/10.1038/nrrheum.2014.91\u003c/li\u003e\n\u003cli\u003eLang G, Liu Y, Geries J, et al. An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition. Journal of Tissue Engineering and Regenerative Medicine. 2018;12(4):e2051-e2061. doi:https://doi.org/10.1002/term.2636\u003c/li\u003e\n\u003cli\u003eElmounedi N, Bahloul W, Keskes H. Current Therapeutic Strategies of Intervertebral Disc Regenerative Medicine. Molecular Diagnosis \u0026amp; Therapy. 2024;28(6):745-775. doi:https://doi.org/10.1007/s40291-024-00729-7\u003c/li\u003e\n\u003cli\u003eMcDonnell JM, Ahern DP, Ross TD, et al. Regenerative Medicine Modalities for the Treatment of Degenerative Disk Disease. Clinical Spine Surgery: A Spine Publication. 2020;34(10):363-368. doi:https://doi.org/10.1097/bsd.0000000000001114\u003c/li\u003e\n\u003cli\u003eJu DG, Kanim LE, Bae HW. Intervertebral Disc Repair: Current Concepts. Global Spine Journal. 2020;10(2_suppl):130S136S. doi:https://doi.org/10.1177/2192568219872460\u003c/li\u003e\n\u003cli\u003eMcDonnell EE, Buckley CT. Consolidating and re‐evaluating the human disc nutrient microenvironment. JOR Spine. 2022;5(1). doi:https://doi.org/10.1002/jsp2.1192\u003c/li\u003e\n\u003cli\u003eKumar H, Ha DH, Lee EJ, et al. Safety and tolerability of intradiscal implantation of combined autologous adipose-derived mesenchymal stem cells and hyaluronic acid in patients with chronic discogenic low back pain: 1-year follow-up of a phase I study. Stem Cell Research \u0026amp; Therapy. 2017;8(1). doi:https://doi.org/10.1186/s13287-017-0710-3\u003c/li\u003e\n\u003cli\u003eElabd C, Centeno CJ, Schultz JR, Lutz G, Ichim T, Silva FJ. Intra-discal injection of autologous, hypoxic cultured bone marrow-derived mesenchymal stem cells in five patients with chronic lower back pain: a long-term safety and feasibility study. Journal of Translational Medicine. 2016;14(1). doi:https://doi.org/10.1186/s12967-016-1015-5\u003c/li\u003e\n\u003cli\u003eManchikanti L. Responsible, Safe, and Effective Use of Biologics in the Management of Low Back Pain: American Society of Interventional Pain Physicians (ASIPP) Guidelines. Pain Physician. 2019;22(22;1s):s1-s74. doi:https://doi.org/10.36076/ppj/2019.22.s1\u003c/li\u003e\n\u003cli\u003eMulvaney S, Tortland P, Shiple B, Curtis K. Endurance and Sports Medicine \u0026bull; Fall/Winter 2018 Regenerative Medicine Options for Chronic Musculoskeletal Conditions: A Review of the Literature. https://www.tulipmedical.com/wp-content/uploads/2023/12/regenerative-medicine-options.pdf\u003c/li\u003e\n\u003cli\u003eCheng J, Santiago KA, Nguyen JT, Solomon JL, Lutz GE. Treatment of symptomatic degenerative intervertebral discs with autologous platelet-rich plasma: follow-up at 5\u0026ndash;9 years. Regenerative Medicine. 2019;14(9):831-840. doi:https://doi.org/10.2217/rme-2019-0040\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Wake Forest University School of Medicine","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biomaterials, Regenerative Medicine, Orthopedics, Spine, Intervertebral Discs","lastPublishedDoi":"10.21203/rs.3.rs-7578453/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7578453/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDegenerative disc disease (DDD) remains one of the most prevalent and debilitating musculoskeletal disorders worldwide, with significant socioeconomic and quality-of-life implications. In the absence of a definitive cure, contemporary therapeutic strategies increasingly emphasize regenerative approaches aimed at restoring disc structure and function. Among these, tissue engineering, biomaterial scaffolds, and gene- or cell-based therapies represent the leading modalities under investigation. Each of these approaches offers distinct mechanisms and advantages: tissue engineering seeks to reconstruct the disc microenvironment through engineered constructs; biomaterials provide structural support and facilitate cellular integration; and gene or cell-based therapies target molecular pathways to modulate inflammation, promote matrix synthesis, and enhance endogenous repair. The selection of cell type, source, processing method, and delivery modality remains critical to optimizing therapeutic outcomes. This review explores the comparative benefits and limitations of these regenerative strategies, highlighting the need for an integrative and personalized approach to achieve durable and clinically meaningful restoration in patients with DDD.\u003c/p\u003e","manuscriptTitle":"The regeneration of degenerated intervertebral discs via tissue engineering, biomaterials, and gene or cell-based therapies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 06:04:43","doi":"10.21203/rs.3.rs-7578453/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7bc07eb9-87d4-4f96-9308-8db98841cc04","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54644427,"name":"Biomaterials"}],"tags":[],"updatedAt":"2025-09-11T06:05:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 06:04:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7578453","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7578453","identity":"rs-7578453","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}