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Cheng Z, Li Y, Li M, Huang J, Huang J, Liang Y, Lu S, Liang C, Xing T, Su K, Wen G, Zeng W, Huang L. Correlation between posterior paraspinal muscle atrophy and lumbar intervertebral disc degeneration in patients with chronic low back pain. INTERNATIONAL ORTHOPAEDICS 2023; 47:793-801. [PMID: 36352306 DOI: 10.1007/s00264-022-05621-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/24/2022] [Accepted: 10/24/2022] [Indexed: 11/11/2022]
Abstract
BACKGROUND Although enormous studies have been devoted to solving the problem of intervertebral disc degeneration/herniation, little attention is paid to the effect of paraspinal muscles on it. We aimed to investigate the correlation between paraspinal muscle atrophy and lumbar disc degeneration to recognize paraspinal muscle atrophy and its importance to the spine. PATIENTS AND METHODS A total of 107 patients were enrolled in the study (65 females, 42 males; age 50.87 ± 15.391 years old). Cross-sectional area, functional cross-sectional area, and fatty infiltration of the posterior paraspinal muscles were measured at the level of L4/5, and the degree of facet joint degeneration was evaluated at the levels of L3/4, L4/5, and L5/S1 by MRI. After controlling the confounding factors by multiple linear regression, the correlations among paraspinal muscle atrophy, disc degeneration, and facet joint degeneration were analyzed. Meanwhile, Pearson/Spearson rank analysis was used to analyze the correlation between clinical symptoms (VAS and ODI) and paraspinal muscle atrophy. RESULTS There was a strong correlation between paraspinal muscle atrophy and disc degeneration after controlling the confounding factors (p < 0.05, R > 0.5). There was a weak correlation between paraspinal muscle atrophy and facet joint degeneration (p < 0.05, R < 0.5). There was a significant correlation between facet joint degeneration and intervertebral disc degeneration (p < 0.05, R > 0.7). The fatty infiltration of paraspinal muscle was weakly correlated with ODI (p < 0.05, R < 0.3), but VAS was not. CONCLUSIONS The degree of paraspinal muscle atrophy increased with lumbar disc degeneration and facet joint degeneration and fatty infiltration of multifidus was more susceptible to weight.
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Affiliation(s)
- Ziying Cheng
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Yuxi Li
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Ming Li
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Junshen Huang
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Jiajun Huang
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Yuwei Liang
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Shixin Lu
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Changchun Liang
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Tong Xing
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Kaihui Su
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Guoming Wen
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China
| | - Weike Zeng
- Department of Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, Guangdong Province, China.
| | - Lin Huang
- Department of Orthopedics, Guangdong Province, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, 510120, China. .,Department of Orthopedics, First Hospital of Nanchang, Nanchang, Jiangxi Province, China.
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Velnar T, Gradisnik L. Endplate role in the degenerative disc disease: A brief review. World J Clin Cases 2023; 11:17-29. [PMID: 36687189 PMCID: PMC9846967 DOI: 10.12998/wjcc.v11.i1.17] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/03/2022] [Revised: 10/19/2022] [Accepted: 12/16/2022] [Indexed: 01/04/2023] Open
Abstract
The degenerative disease of the intervertebral disc is nowadays an important health problem, which has still not been understood and solved adequately. The vertebral endplate is regarded as one of the vital elements in the structure of the intervertebral disc. Its constituent cells, the chondrocytes in the endplate, may also be involved in the process of the intervertebral disc degeneration and their role is central both under physiological and pathological conditions. They main functions include a role in homeostasis of the extracellular environment of the intervertebral disc, metabolic support and nutrition of the discal nucleus and annulus beneath and the preservation of the extracellular matrix. Therefore, it is understandable that the cells in the endplate have been in the centre of research from several viewpoints, such as development, degeneration and growth, reparation and remodelling, as well as treatment strategies. In this article, we briefly review the importance of vertebral endplate, which are often overlooked, in the intervertebral disc degeneration.
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Affiliation(s)
- Tomaz Velnar
- Department of Neurosurgery, University Medical Centre Ljubljana, Ljubljana 1000, Slovenia
- Alma Mater Europaea Maribor, Maribor 2000, Slovenia
| | - Lidija Gradisnik
- Alma Mater Europaea Maribor, Maribor 2000, Slovenia
- Institute of Biomedical Sciences, University of Maribor, University of Maribor, Maribor 2000, Slovenia
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Page MI, Easley JT, Bonilla AF, Patel VV, Puttlitz CM. Biomechanical evaluation of a novel repair strategy for intervertebral disc herniation in an ovine lumbar spine model. Front Bioeng Biotechnol 2022; 10:1018257. [DOI: 10.3389/fbioe.2022.1018257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Accepted: 10/13/2022] [Indexed: 11/13/2022] Open
Abstract
Following herniation of the intervertebral disc, there is a need for advanced surgical strategies to protect the diseased tissue from further herniation and to minimize further degeneration. Accordingly, a novel tissue engineered implant for annulus fibrosus (AF) repair was fabricated via three-dimensional fiber deposition and evaluated in a large animal model. Specifically, lumbar spine kinetics were assessed for eight (n = 8) cadaveric ovine lumbar spines in three pure moment loading settings (flexion-extension, lateral bending, and axial rotation) and three clinical conditions (intact, with a defect in the AF, and with the defect treated using the AF repair implant). In ex vivo testing, seven of the fifteen evaluated biomechanical measures were significantly altered by the defect. In each of these cases, the treated spine more closely approximated the intact biomechanics and four of these cases were also significantly different to the defect. The same spinal kinetics were also assessed in a preliminary in vivo study of three (n = 3) ovine lumbar spines 12 weeks post-implantation. Similar to the ex vivo results, functional efficacy of the treatment was demonstrated as compared to the defect model at 12 weeks post-implantation. These promising results motivate a future large animal study cohort which will establish statistical power of these results further elucidate the observed outcomes, and provide a platform for clinical translation of this novel AF repair patch strategy. Ultimately, the developed approach to AF repair holds the potential to maintain the long-term biomechanical function of the spine and prevent symptomatic re-herniation.
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Kasamkattil J, Gryadunova A, Martin I, Barbero A, Schären S, Krupkova O, Mehrkens A. Spheroid-Based Tissue Engineering Strategies for Regeneration of the Intervertebral Disc. Int J Mol Sci 2022; 23:2530. [PMID: 35269672 PMCID: PMC8910276 DOI: 10.3390/ijms23052530] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 02/21/2022] [Accepted: 02/23/2022] [Indexed: 12/12/2022] Open
Abstract
Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.
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Affiliation(s)
- Jesil Kasamkattil
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
| | - Anna Gryadunova
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
- World-Class Research Center “Digital Biodesign and Personalized Healthcare”, Sechenov First Moscow State Medical University, 119435 Moscow, Russia
| | - Ivan Martin
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
| | - Andrea Barbero
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
| | - Stefan Schären
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
| | - Olga Krupkova
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
- Department of Biomedicine, University Hospital Basel, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; (I.M.); (A.B.)
- Lepage Research Institute, University of Prešov, 17. Novembra 1, 081 16 Prešov, Slovakia
| | - Arne Mehrkens
- Spine Surgery, University Hospital Basel, Spitalstrasse 21, 4031 Basel, Switzerland; (J.K.); (A.G.); (S.S.); (A.M.)
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Mainardi A, Cambria E, Occhetta P, Martin I, Barbero A, Schären S, Mehrkens A, Krupkova O. Intervertebral Disc-on-a-Chip as Advanced In Vitro Model for Mechanobiology Research and Drug Testing: A Review and Perspective. Front Bioeng Biotechnol 2022; 9:826867. [PMID: 35155416 PMCID: PMC8832503 DOI: 10.3389/fbioe.2021.826867] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Accepted: 12/20/2021] [Indexed: 12/14/2022] Open
Abstract
Discogenic back pain is one of the most diffused musculoskeletal pathologies and a hurdle to a good quality of life for millions of people. Existing therapeutic options are exclusively directed at reducing symptoms, not at targeting the underlying, still poorly understood, degenerative processes. Common intervertebral disc (IVD) disease models still do not fully replicate the course of degenerative IVD disease. Advanced disease models that incorporate mechanical loading are needed to investigate pathological causes and processes, as well as to identify therapeutic targets. Organs-on-chip (OoC) are microfluidic-based devices that aim at recapitulating tissue functions in vitro by introducing key features of the tissue microenvironment (e.g., 3D architecture, soluble signals and mechanical conditioning). In this review we analyze and depict existing OoC platforms used to investigate pathological alterations of IVD cells/tissues and discuss their benefits and limitations. Starting from the consideration that mechanobiology plays a pivotal role in both IVD homeostasis and degeneration, we then focus on OoC settings enabling to recapitulate physiological or aberrant mechanical loading, in conjunction with other relevant features (such as inflammation). Finally, we propose our view on design criteria for IVD-on-a-chip systems, offering a future perspective to model IVD mechanobiology.
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Affiliation(s)
- Andrea Mainardi
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
- Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
- Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland
| | - Elena Cambria
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States
| | - Paola Occhetta
- Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy
| | - Ivan Martin
- Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
- Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland
| | - Andrea Barbero
- Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
| | - Stefan Schären
- Spine Surgery, University Hospital Basel, Basel, Switzerland
| | - Arne Mehrkens
- Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
- Spine Surgery, University Hospital Basel, Basel, Switzerland
| | - Olga Krupkova
- Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
- Spine Surgery, University Hospital Basel, Basel, Switzerland
- Lepage Research Institute, University of Prešov, Prešov, Slovakia
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Wang H, Li F, Ban W, Zhang J, Zhang G. Human Bone Marrow Mesenchymal Stromal Cell-Derived Extracellular Vesicles Promote Proliferation of Degenerated Nucleus Pulposus Cells and the Synthesis of Extracellular Matrix Through the SOX4/Wnt/β-Catenin Axis. Front Physiol 2021; 12:723220. [PMID: 34777000 PMCID: PMC8581610 DOI: 10.3389/fphys.2021.723220] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Accepted: 09/09/2021] [Indexed: 01/04/2023] Open
Abstract
Objective: Intervertebral disk degeneration (IDD) is a major cause of pain in the back, neck, and radiculus. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are therapeutic in musculoskeletal degenerative diseases such as IDD. This study explored the effect and functional mechanism of human bone MSCs (hBMSCs)-derived EVs in proliferation and apoptosis of degenerated nucleus pulposus cells (DNPCs) and extracellular matrix (ECM) synthesis. Methods: Extracellular vesicles were isolated from hBMSCs and identified. DNPCs were induced by TNF-α. EVs were incubated with DNPCs for 24h. Internalization of EVs by DNPCs, DNPCs proliferation, apoptosis, and expressions of ECM synthetic genes, degrading genes and miR-129-5p were assessed. Downstream target genes of miR-129-5p were predicted. Target relation between miR-129-5p and SRY-box transcription factor 4 (SOX4) was verified. DNPCs proliferation, apoptosis, and ECM synthesis were measured after treatment with EVs and miR-129-5p inhibitor or SOX4 overexpression. Expressions of SOX4 and Wnt/β-catenin pathway-related proteins were determined. Results: hBMSC-EVs promoted DNPCs proliferation, inhibited apoptosis, increased expressions of ECM synthetic genes, and reduced expressions of ECM degrading genes. hBMSC-EVs carried miR-129-5p into DNPCs. Silencing miR-129-5p in EVs partially inverted the effect of EVs on DNPCs proliferation and ECM synthesis. miR-129-5p targeted SOX4. SOX4 overexpression annulled the effect of EVs on DNPCs proliferation and ECM synthesis. Expressions of Wnt1 and β-catenin were decreased in EVs-treated DNPCs, while silencing miR-129-5p in EVs promoted expressions of Wnt1 and β-catenin. Conclusion: hBMSC-EVs promoted DNPCs proliferation and ECM synthesis by carrying miR-129-5p into DNPCs to target SOX4 and deactivating the Wnt/β-catenin axis.
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Affiliation(s)
- Haoyu Wang
- Department of Orthopedics, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China
| | - Fei Li
- Department of Orthopedics, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China
| | - Wenrui Ban
- Department of Orthopedics, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China
| | - Jing Zhang
- Department of Orthopedics, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China
| | - Guiqi Zhang
- Department of Spinal Surgery, Dalian Municipal Central Hospital, Dalian, China
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Activation of Hypoxia-Inducible Factor-1α Signaling Pathway Has the Protective Effect of Intervertebral Disc Degeneration. Int J Mol Sci 2021; 22:ijms222111355. [PMID: 34768786 PMCID: PMC8583205 DOI: 10.3390/ijms222111355] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 10/16/2021] [Accepted: 10/18/2021] [Indexed: 02/07/2023] Open
Abstract
Intervertebral discs (IVDs) have poor nutrient diffusion, because the nucleus pulposus (NP) lacks direct vascular supply and likely generates adenosine triphosphate by anaerobic glycolysis. Regulation of glycolysis is mediated by hypoxia-inducible factor-1α (HIF-1α), a transcription factor that responds to local oxygen tension. Constitutively active HIF-1α (CA HIF-1α) was created by point mutation and determined the protective role of HIF-1α in IVD degeneration. Under fluoroscopy, rat caudal IVD segments were stabbed by a needle puncture, and pcDNA3- HIF-1α wild-type (WT) or pcDNA3-CA HIF-1α was transfected into NP cell lines. The constitutive activity of CA HIF-1α was analyzed using a luciferase assay after cell lysis. Next, IVD tissue samples were retrieved from five patients with degenerative lumbar spinal stenosis at the time of surgery, and NP cells were cultured. NP cells were transfected with CA HIF-1α, and relevant gene expression was measured. HIF-1α protein levels in the nucleus were significantly higher, and transcriptional activity was 10.3-fold higher in NP cells with CA HIF-1α than in those with HIF-1α WT. Gene transfer of CA HIF-1α into NP cells enhanced the expression of Glut-1, Glut-3, aggrecan, type II collagen, and Sox9. Moreover, CA HIF-1α reduced the apoptosis of NP cells induced by the Fas ligand. The HIF-1α and collagen 2 expression levels were notably increased in the NP cells of the CA HIF-1α transfected segments in histology and immunohistochemistry study. Collectively, these results suggest that activation of HIF-1α signaling pathway may play a protective role against IVD degeneration and could be used as a future therapeutic agent.
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Production and Characterization of Recombinant Collagen-Binding Resilin Nanocomposite for Regenerative Medicine Applications. REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE 2019. [DOI: 10.1007/s40883-019-00092-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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Hattapoğlu E, Batmaz İ, Dilek B, Karakoç M, Em S, Çevik R. Efficiency of pulsed electromagnetic fields on pain, disability, anxiety, depression, and quality of life in patients with cervical disc herniation: a randomized controlled study. Turk J Med Sci 2019; 49:1095-1101. [PMID: 31385489 PMCID: PMC7018371 DOI: 10.3906/sag-1901-65] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Background/aim In this study, it was aimed to investigate the effects ofpulsed electromagnetic field(PEMF) therapy on pain, disability, psychological state, and quality of life in cervical disc herniation. Materials and methods Patients were randomly divided into two groups, including Group 1, which received a therapy consisting of transcutaneous electrical nerve stimulation (TENS), hot pack (HP), and PEMF, and Group 2, which received a magnetic field (sham magnetic field) without current flow in addition to TENS and HP therapy. Pain was assessed by a visual analog scale (VAS, 0–10 cm). The other outcome measures were function (Neck Pain and Disability Scale), anxiety-depressive mood (Hospital Anxiety and Depression Scale), and quality of life (Nottingham Health Profile). All evaluations were performed at baseline, in the 3rd week, and in the 12th week after treatment. Results A significant improvement was found in the neck pain, disability, depression, anxiety, and quality of life scores of both groups after treatment when compared to those before treatment. However, in the comparison between changes within groups, significant improvements were determined only in the VAS and Nottingham Health Profile sleep subparameter in the 12th week after treatment compared to those before treatment. Conclusion PEMF therapy in cervical disc herniation can be used safely in routine treatment in addition to conventional physical therapy modalities.
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Affiliation(s)
- Erkam Hattapoğlu
- Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Dicle University, Diyarbakır, Turkey
| | - İbrahim Batmaz
- Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Dicle University, Diyarbakır, Turkey
| | - Banu Dilek
- Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Dokuz Eylül University, İzmir, Turkey
| | - Mehmet Karakoç
- Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Dicle University, Diyarbakır, Turkey
| | - Serda Em
- Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Dicle University, Diyarbakır, Turkey
| | - Remzi Çevik
- Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Dicle University, Diyarbakır, Turkey
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Ligorio C, Zhou M, Wychowaniec JK, Zhu X, Bartlam C, Miller AF, Vijayaraghavan A, Hoyland JA, Saiani A. Graphene oxide containing self-assembling peptide hybrid hydrogels as a potential 3D injectable cell delivery platform for intervertebral disc repair applications. Acta Biomater 2019; 92:92-103. [PMID: 31091473 PMCID: PMC6582688 DOI: 10.1016/j.actbio.2019.05.004] [Citation(s) in RCA: 85] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 05/02/2019] [Accepted: 05/03/2019] [Indexed: 12/23/2022]
Abstract
Cell-based therapies have shown significant promise in tissue engineering with one key challenge being the delivery and retention of cells. As a result, significant efforts have been made in the past decade to design injectable biomaterials to host and deliver cells at injury sites. Intervertebral disc (IVD) degeneration, a major cause of back pain, is a particularly relevant example where a minimally-invasive cellular therapy could bring significant benefits specifically at the early stages of the disease, when a cell-driven process starts in the gelatinous core of the IVD, the nucleus pulposus (NP). In this present study we explore the use of graphene oxide (GO) as nano-filler for the reinforcement of FEFKFEFK (β-sheet forming self-assembling peptide) hydrogels. Our results confirm the presence of strong interactions between FEFKFEFK and GO flakes with the peptide coating and forming short thin fibrils on the surface of the flakes. These strong interactions were found to affect the bulk properties of hybrid hydrogels. At pH 4 electrostatic interactions between the peptide fibres and the peptide-coated GO flakes are thought to govern the final bulk properties of the hydrogels while at pH 7, after conditioning with cell culture media, electrostatic interactions are removed leaving the hydrophobic interactions to govern hydrogel final properties. The GO-F820 hybrid hydrogel, with mechanical properties similar to the NP, was shown to promote high cell viability and retained cell metabolic activity in 3D over the 7 days of culture and therefore shown to harbour significant potential as an injectable hydrogel scaffold for the in-vivo delivery of NP cells. STATEMENT OF SIGNIFICANCE: Short self-assembling peptide hydrogels (SAPHs) have attracted significant interest in recent years as they can mimic the natural extra-cellular matrix, holding significant promise for the ab initio design of cells' microenvironments. Recently the design of hybrid hydrogels for biomedical applications has been explored through the incorporation of specific nanofillers. In this study we exploited graphene oxide (GO) as nanofiller to design hybrid injectable 3Dscaffolds for the delivery of nucleus pulposus cells (NPCs) for intervertebral disc regeneration. Our work clearly shows the presence of strong interactions between peptide and GO, mimicking the mechanical properties of the NP tissue and promoting high cell viability and metabolic activity. These hybrid hydrogels therefore harbour significant potential as injectable scaffolds for the in vivo delivery of NPCs.
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Affiliation(s)
- Cosimo Ligorio
- School of Materials, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; Manchester Institute of Biotechnology (MIB), The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Mi Zhou
- School of Materials, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; Manchester Institute of Biotechnology (MIB), The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Jacek K Wychowaniec
- School of Materials, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; Manchester Institute of Biotechnology (MIB), The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Xinyi Zhu
- Manchester Institute of Biotechnology (MIB), The University of Manchester, Oxford Road, Manchester M13 9PL, UK; School of Chemical Engineering and Analytical Sciences, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Cian Bartlam
- School of Materials, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Aline F Miller
- Manchester Institute of Biotechnology (MIB), The University of Manchester, Oxford Road, Manchester M13 9PL, UK; School of Chemical Engineering and Analytical Sciences, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Aravind Vijayaraghavan
- School of Materials, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; National Graphene Institute (NGI), The University of Manchester, Oxford Road, Manchester M13 9PL, UK
| | - Judith A Hoyland
- Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; NIHR Manchester Biomedical Research Centre, Manchester University NHS Foundation Trust, Manchester Academic Health Science Centre, Grafton St, M13 9WU Manchester, UK
| | - Alberto Saiani
- School of Materials, Faculty of Science and Engineering, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; Manchester Institute of Biotechnology (MIB), The University of Manchester, Oxford Road, Manchester M13 9PL, UK.
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Tendulkar G, Ehnert S, Sreekumar V, Chen T, Kaps HP, Golombek S, Wendel HP, Nüssler AK, Avci-Adali M. Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs-The Potential Role in Increasing Anabolic Response. Int J Mol Sci 2019; 20:E1716. [PMID: 30959917 PMCID: PMC6479841 DOI: 10.3390/ijms20071716] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Revised: 03/28/2019] [Accepted: 04/03/2019] [Indexed: 12/25/2022] Open
Abstract
Musculoskeletal disorders, such as osteoarthritis and intervertebral disc degeneration are causes of morbidity, which concomitantly burdens the health and social care systems worldwide, with massive costs. Link N peptide has recently been described as a novel anabolic stimulator for intervertebral disc repair. In this study, we analyzed the influence on anabolic response, by delivering synthetic Link N encoding mRNA into primary human chondrocytes and mesenchymal stromal cells (SCP1 cells), Furthermore, both cell types were seeded on knitted titanium scaffolds, and the influence of Link N peptide mRNA for possible tissue engineering applications was investigated. Synthetic modified Link N mRNA was efficiently delivered into both cell types and cell transfection resulted in an enhanced expression of aggrecan, Sox 9, and type II collagen with a decreased expression of type X collagen. Interestingly, despite increased expression of BMP2 and BMP7, BMP signaling was repressed and TGFβ signaling was boosted by Link N transfection in mesenchymal stromal cells, suggesting possible regulatory mechanisms. Thus, the exogenous delivery of Link N peptide mRNA into cells augmented an anabolic response and thereby increased extracellular matrix synthesis. Considering these findings, we suppose that the cultivation of cells on knitted titanium scaffolds and the exogenous delivery of Link N peptide mRNA into cells could mechanically support the stability of tissue-engineered constructs and improve the synthesis of extracellular matrix by seeded cells. This method can provide a potent strategy for articular cartilage and intervertebral disc regeneration.
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Affiliation(s)
- Gauri Tendulkar
- Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
| | - Sabrina Ehnert
- Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
| | - Vrinda Sreekumar
- Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
| | - Tao Chen
- Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
| | - Hans-Peter Kaps
- Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
| | - Sonia Golombek
- Department of Thoracic and Cardiovascular Surgery, University Hospital Tübingen, Calwerstraße 7/1, 72076 Tübingen, Germany.
| | - Hans-Peter Wendel
- Department of Thoracic and Cardiovascular Surgery, University Hospital Tübingen, Calwerstraße 7/1, 72076 Tübingen, Germany.
| | - Andreas K Nüssler
- Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, Schnarrenbergstraße 95, 72076 Tübingen, Germany.
| | - Meltem Avci-Adali
- Department of Thoracic and Cardiovascular Surgery, University Hospital Tübingen, Calwerstraße 7/1, 72076 Tübingen, Germany.
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12
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Otsuki S, Alvarez-Garcia O, Lotz MK, Neo M. Role of heparan sulfate 6-0 endosulfatases in intervertebral disc homeostasis. Histol Histopathol 2019; 34:1051-1060. [PMID: 30924907 DOI: 10.14670/hh-18-107] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The expression of heparan sulfate endosulfatases (Sulfs) was investigated in the intervertebral disc (IVD) to clarify their role in IVD homeostasis. Sulf-1 and -2 expression were elucidated in normal and degenerated human IVD. Age-related effects on Sulf expression, type II collagen levels, and structural changes were analyzed in IVDs of wild-type (WT) and Sulf-1 knockout (Sulf-1⁻/⁻) mice. The effect of recombinant Sulf-1 (100 ng/ml) and Sulf-1 knockdown on heparan sulfate proteoglycan and collagen expression in ATDC5 cells were examined. Finally, the effect of Sulf-1 on transforming growth factor (TGF) β1-induced signaling was evaluated. Results show that Sulf-1 and -2 levels were higher in degenerated human IVDs. In WT mice, Sulf-1 and -2 expression generally declined as the animals aged. In particular, Sulf-1 in the nucleus pulposus was higher compared with Sulf-2 at the age of 1 and 6 months and significantly declined with aging. Sulf-1⁻/⁻ mice showed more severe IVD pathology than WT mice, with lower type II collagen levels in nucleus pulposus, and degeneration with type I collagen in annulus fibrosus. In vitro, Sulf-1 induced type II collagen expression and significantly increased TGF-β1-induced Smad2/3 phosphorylation in ATDC5 cells. In conclusion, Sulf-1 might play a critical role from development to maintenance of IVD homeostasis by regulating collagen expression.
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Affiliation(s)
- Shuhei Otsuki
- Department of Orthopedic Surgery, Osaka Medical College, Japan.
| | | | - Martin K Lotz
- Department of Molecular Medicine, The Scripps Research Institute, USA
| | - Masashi Neo
- Department of Orthopedic Surgery, Osaka Medical College, Japan
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13
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Rider SM, Mizuno S, Kang JD. Molecular Mechanisms of Intervertebral Disc Degeneration. Spine Surg Relat Res 2019; 3:1-11. [PMID: 31435545 PMCID: PMC6690117 DOI: 10.22603/ssrr.2017-0095] [Citation(s) in RCA: 57] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 01/24/2018] [Indexed: 12/25/2022] Open
Abstract
Intervertebral disc degeneration is a well-known cause of disability, the result of which includes neck and back pain with associated mobility limitations. The purpose of this article is to provide an overview of the known molecular mechanisms through which intervertebral disc degeneration occurs as a result of complex interactions of exogenous and endogenous stressors. This review will focus on some of the identified molecular changes leading to the deterioration of the extracellular matrix of both the annulus fibrosus and nucleus pulposus. In addition, we will provide a summation of our current knowledge supporting the role of associated DNA and intracellular damage, cellular senescence's catabolic effects, oxidative stress, and the cell's inappropriate response to damage in contributing to intervertebral disc degeneration. Our current understanding of the molecular mechanisms through which intervertebral disc degeneration occurs provides us with abundant insight into how physical and chemical changes exacerbate the degenerative process of the entire spine. Furthermore, we will describe some of the related molecular targets and therapies that may contribute to intervertebral repair and regeneration.
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Affiliation(s)
- Sean M Rider
- Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Shuichi Mizuno
- Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - James D Kang
- Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA
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14
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van Uden S, Silva-Correia J, Oliveira JM, Reis RL. Current strategies for treatment of intervertebral disc degeneration: substitution and regeneration possibilities. Biomater Res 2017; 21:22. [PMID: 29085662 PMCID: PMC5651638 DOI: 10.1186/s40824-017-0106-6] [Citation(s) in RCA: 79] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2017] [Accepted: 10/05/2017] [Indexed: 02/06/2023] Open
Abstract
Background Intervertebral disc degeneration has an annual worldwide socioeconomic impact masked as low back pain of over 70 billion euros. This disease has a high prevalence over the working age class, which raises the socioeconomic impact over the years. Acute physical trauma or prolonged intervertebral disc mistreatment triggers a biochemical negative tendency of catabolic-anabolic balance that progress to a chronic degeneration disease. Current biomedical treatments are not only ineffective in the long-run, but can also cause degeneration to spread to adjacent intervertebral discs. Regenerative strategies are desperately needed in the clinics, such as: minimal invasive nucleus pulposus or annulus fibrosus treatments, total disc replacement, and cartilaginous endplates decalcification. Main body Herein, it is reviewed the state-of-the-art of intervertebral disc regeneration strategies from the perspective of cells, scaffolds, or constructs, including both popular and unique tissue engineering approaches. The premises for cell type and origin selection or even absence of cells is being explored. Choice of several raw materials and scaffold fabrication methods are evaluated. Extensive studies have been developed for fully regeneration of the annulus fibrosus and nucleus pulposus, together or separately, with a long set of different rationales already reported. Recent works show promising biomaterials and processing methods applied to intervertebral disc substitutive or regenerative strategies. Facing the abundance of studies presented in the literature aiming intervertebral disc regeneration it is interesting to observe how cartilaginous endplates have been extensively neglected, being this a major source of nutrients and water supply for the whole disc. Conclusion Several innovative avenues for tackling intervertebral disc degeneration are being reported – from acellular to cellular approaches, but the cartilaginous endplates regeneration strategies remain unaddressed. Interestingly, patient-specific approaches show great promise in respecting patient anatomy and thus allow quicker translation to the clinics in the near future.
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Affiliation(s)
- Sebastião van Uden
- 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR Gandra, Portugal.,ICVS/3B's-PT Government Associate Laboratory, Guimarães, Braga Portugal.,Present Address: Bioengineering Laboratories Srl, Viale Brianza 8, Meda, Italy.,Present Address: Politecnico di Milano, Piazza Leonardo da Vinci, 32 Milan, Italy
| | - Joana Silva-Correia
- 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR Gandra, Portugal.,ICVS/3B's-PT Government Associate Laboratory, Guimarães, Braga Portugal
| | - Joaquim Miguel Oliveira
- 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR Gandra, Portugal.,ICVS/3B's-PT Government Associate Laboratory, Guimarães, Braga Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco Guimarães, Portugal
| | - Rui Luís Reis
- 3B's Research Group-Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco GMR Gandra, Portugal.,ICVS/3B's-PT Government Associate Laboratory, Guimarães, Braga Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco Guimarães, Portugal
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15
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Liang L, Li X, Li D, Jiang W, Wang H, Chen J, Sun Z, Zhang N, Zhu Y. The characteristics of stem cells in human degenerative intervertebral disc. Medicine (Baltimore) 2017; 96:e7178. [PMID: 28640098 PMCID: PMC5484206 DOI: 10.1097/md.0000000000007178] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/08/2016] [Revised: 05/18/2017] [Accepted: 05/22/2017] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND The aim of this study is to identify which possessed the best stem-cell-like characteristics in 3 kinds of cell in human degenerative intervertebral disc: NPSCs (nucleus pulposus-derived stem cells), AFSCs (annulus fibrosus-derived stem cells), or CESCs (cartilage endplate-derived stem cells). METHODS We separated the disc samples obtained from 15 surgically treated patients with disc degenerative diseases into nucleus pulposus, annulus fibrosus, and cartilage endplate. After cultivating, we used the cell counting kit-8 to analysis the cell activity of 3 kinds of disc tissue-derived stem cell separately; different stem cells were defined with multilineage (osteogenic, chondrogenic, and adipogenic) differentiation. We extracted the total RNA and detected the expression of different lineage differentiation-related genes with the real-time polymerase chain reaction (RT-PCR). RESULTS Cell morphology of NPSCs, AFSCs, and CESCs did not show significant difference. Cell proliferation capacity of NPSCs and AFSCs was stronger than that of CESCs. The differentiation outcomes showed that osteocyte-like cells were stained red by Alizarin red S, chondrocyte-like cells blue by toluidine blue, and adipocyte-like red by oil red O. The RT-PCR reflected that the expression of different lineage differentiation-related genes of AFSCs was stronger than NPSCs and CESCs. CONCLUSION In conclusion, we found that the cell morphology was not significantly different among NPSCs, AFSCs, and CESCs. Both differentiation and RT-PCR tests demonstrated that AFSCs had the best stem-cell-like characteristics in the human degenerative intervertebral disc.
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Affiliation(s)
- Lin Liang
- Department of Orthopaedics, Shangyu People's Hospital, Shaoxing
| | - Xuefeng Li
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou
| | - Dapeng Li
- Department of Orthopaedics, The Affiliated Hospital of Jiangsu University, Zhenjiang, China
| | - Weimin Jiang
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou
| | - Heng Wang
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou
| | - Jie Chen
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou
| | - Zhiyong Sun
- Department of Orthopaedics, The First Affiliated Hospital of Soochow University, Suzhou
| | - Niannian Zhang
- Department of Orthopaedics, Shangyu People's Hospital, Shaoxing
| | - Yangyi Zhu
- Department of Orthopaedics, Shangyu People's Hospital, Shaoxing
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16
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Hoffman H, Choi AW, Chang V, Kimball J, S. Verkman A, Virani R, Kim B, Niu T, Lu DC. Aquaporin-1 Expression in Herniated Human Lumbar Intervertebral Discs. Global Spine J 2017; 7:133-140. [PMID: 28507882 PMCID: PMC5415154 DOI: 10.1177/2192568217694007] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
STUDY DESIGN Case series. OBJECTIVE Intervertebral disc (IVD) degeneration is the cause of spondylosis. The pathogenesis is poorly understood, but disc dehydration often plays a role. In this study, we aim to identify and quantify aquaporin-1 (AQP1) in ex vivo human degenerated IVDs obtained intraoperatively and to investigate the relationship between AQP1 levels and magnetic resonance imaging (MRI) T2 intensity of the disc. METHODS Ex vivo samples of nucleus pulposus (NP) tissue from lumbar IVDs were obtained from 18 consecutive patients who underwent surgery for disc herniation at L4/5 and L5/S1 level. Immunohistochemistry was performed to determine the presence of AQP1 expression, and this was quantified by Western blot analysis. AQP1 expression was compared to preoperative IVD signal intensity on T2-weighted MRI. RESULTS NP tissue was obtained from 18 patients (9 for L4/5 level and 9 for L5/S1 level). AQP1 expression was detected in all samples by Western blot and immunohistochemistry. AQP1 expression had a linear correlation with the preoperative IVD signal intensity on T2-weighted MRI at L4/5 level (R2 = 0.90) and at L5/S1 level (R2 = 0.92). AQP1 expression was 52.2 ± 59.0 at L5/S1 level and 15.9 ± 20.6 at L4/5 (P = .10). CONCLUSIONS Our results show that AQP1 can be detected in IVD obtained from live human subjects. Increased AQP1 expression is associated with greater disc hydration as measured by signal intensity on T2-weighted MRI. AQP1 may have a role in the dehydration associated with disc degeneration.
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Affiliation(s)
- Haydn Hoffman
- University of California, Los Angeles, CA, USA,University of California, San Francisco, CA, USA
| | - Aaron W. Choi
- University of California, Los Angeles, CA, USA,*Contributed equally to this article
| | | | - Jon Kimball
- University of California, Los Angeles, CA, USA
| | | | | | - Brian Kim
- University of California, Los Angeles, CA, USA
| | - Tianyi Niu
- University of California, Los Angeles, CA, USA
| | - Daniel C. Lu
- University of California, Los Angeles, CA, USA,Daniel C. Lu, Department of Neurosurgery, University of California, Los Angeles, 300 Stein Plaza, Ste 536, Los Angeles, CA 90095-6901, USA.
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17
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Krupkova O, Hlavna M, Amir Tahmasseb J, Zvick J, Kunz D, Ito K, Ferguson SJ, Wuertz-Kozak K. An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate. Int J Mol Sci 2016; 17:ijms17101640. [PMID: 27689996 PMCID: PMC5085673 DOI: 10.3390/ijms17101640] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2016] [Revised: 09/15/2016] [Accepted: 09/19/2016] [Indexed: 12/18/2022] Open
Abstract
Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1-20 U/mL) or by the diffusion of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) (both 100 ng/mL) from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA) and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM) was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1β and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinases (MMP1, MMP3, and MMP13). The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease.
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Affiliation(s)
- Olga Krupkova
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
| | - Marian Hlavna
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
| | - Julie Amir Tahmasseb
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
| | - Joel Zvick
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
| | - Dominik Kunz
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
- Health Department, ZHAW-Zurich University of Applied Sciences, Technikumstrasse 71, CH-8401 Winterthur, Switzerland.
| | - Keita Ito
- Department of Biomedical Engineering, Eindhoven University of Technology, Postbus 513, 5600 MB Eindhoven, The Netherlands.
| | - Stephen J Ferguson
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
- Competence Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
| | - Karin Wuertz-Kozak
- Institute for Biomechanics, ETH Zurich, Hoenggerbergring 64, CH-8093 Zurich, Switzerland.
- Competence Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
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18
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Tao H, Lin Y, Zhang G, Gu R, Chen B. Experimental observation of human bone marrow mesenchymal stem cell transplantation into rabbit intervertebral discs. Biomed Rep 2016; 5:357-360. [PMID: 27588177 DOI: 10.3892/br.2016.731] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2016] [Accepted: 07/13/2016] [Indexed: 11/06/2022] Open
Abstract
Allogeneic bone marrow mesenchymal stem cell (BMSC) transplantation has been investigated worldwide. However, few reports have addressed the survival status of human BMSCs in the intervertebral discs (IVDs) in vivo following transplantation. The current study aimed to observe the survival status of human BMSCs in rabbit IVDs. The IVDs of 15 New Zealand white rabbits were divided into three groups: Punctured blank control group (L1-2); punctured physiological saline control group (L2-3); and punctured human BMSCs transfected with green fluorescent protein (GFP) group (L3-4, L4-5 and L5-6). One, 2, 4, 6 and 8 weeks after transplantation the IVDs were removed and a fluorescence microscope was used to observe the density of GFP-positive human BMSCs. The results indicated that in the sections of specimens removed at 1, 2, 4, 6 and 8 weeks post-transplantation, no GFP-positive cells were observed in the control groups, whereas GFP-positive cells were apparent in the nucleus pulposus at all periods in the GFP-labeled human BMSCs group, and the cell density at 6 and 8 weeks was significantly less than that at 1, 2 and 4 weeks post-transplantation (P<0.001). Thus, it was identified that human BMSCs were able to survive in the rabbit IVDs for 8 weeks.
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Affiliation(s)
- Hao Tao
- Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China
| | - Yazhou Lin
- Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China
| | - Guoqing Zhang
- Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China
| | - Rui Gu
- Department of Spinal and Neural Function Reconstruction, China Rehabilitation Research Center, Beijing 100068, P.R. China
| | - Bohua Chen
- Department of Orthopedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong 266003, P.R. China
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19
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The Natural Polyphenol Epigallocatechin Gallate Protects Intervertebral Disc Cells from Oxidative Stress. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2016; 2016:7031397. [PMID: 27119009 PMCID: PMC4826942 DOI: 10.1155/2016/7031397] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/20/2015] [Revised: 01/30/2016] [Accepted: 02/16/2016] [Indexed: 12/19/2022]
Abstract
Oxidative stress-related phenotypic changes and a decline in the number of viable cells are crucial contributors to intervertebral disc degeneration. The polyphenol epigallocatechin 3-gallate (EGCG) can interfere with painful disc degeneration by reducing inflammation, catabolism, and pain. In this study, we hypothesized that EGCG furthermore protects against senescence and/or cell death, induced by oxidative stress. Sublethal and lethal oxidative stress were induced in primary human intervertebral disc cells with H2O2 (total n = 36). Under sublethal conditions, the effects of EGCG on p53-p21 activation, proliferative capacity, and accumulation of senescence-associated β-galactosidase were tested. Further, the effects of EGCG on mitochondria depolarization and cell viability were analyzed in lethal oxidative stress. The inhibitor LY249002 was applied to investigate the PI3K/Akt pathway. EGCG inhibited accumulation of senescence-associated β-galactosidase but did not affect the loss of proliferative capacity, suggesting that EGCG did not fully neutralize exogenous radicals. Furthermore, EGCG increased the survival of IVD cells in lethal oxidative stress via activation of prosurvival PI3K/Akt and protection of mitochondria. We demonstrated that EGCG not only inhibits inflammation but also can enhance the survival of disc cells in oxidative stress, which makes it a suitable candidate for the development of novel therapies targeting disc degeneration.
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20
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Ahn J, Park EM, Kim BJ, Kim JS, Choi B, Lee SH, Han I. Transplantation of human Wharton's jelly-derived mesenchymal stem cells highly expressing TGFβ receptors in a rabbit model of disc degeneration. Stem Cell Res Ther 2015; 6:190. [PMID: 26432097 PMCID: PMC4592544 DOI: 10.1186/s13287-015-0183-1] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2015] [Revised: 03/17/2015] [Accepted: 09/11/2015] [Indexed: 12/17/2022] Open
Abstract
Introduction Mesenchymal stem cells (MSCs) are widely considered to hold promise for the treatment of intervertebral disc (IVD) degeneration. However, variation in the therapeutic efficacy of MSCs is a major problem and the derivation of MSCs for use in IVD regeneration has not been optimized. Additionally, no data are available on the efficacy of Wharton’s Jelly-derived MSC (WJ-MSC) transplantation in an animal model of IVD degeneration. Methods This study evaluated the effectiveness of a cross-linked hyaluronic acid (XHA) scaffold loaded with human WJ-MSCs, according to their expression levels of transforming growth factor-β receptor I/activin-like kinase receptor 5 (TβRI/ALK5) and TβRII, for IVD regeneration in a rabbit model. We compared the degree of IVD regeneration between rabbits transplanted with a XHA scaffold loaded with WJ-MSCs highly and lowly expressing TβRI/ALK5 and TβRII (MSC-highTR and MSC-lowTR, respectively) using magnetic resonance imaging (MRI) and histological analysis. Results At 12 weeks after transplantation, T2-weighted MRI analysis showed significant restoration of the disc water content in rabbits treated with a MSC-highTR-loaded XHA scaffold in comparison to rabbits treated with the scaffold alone or a MSC-lowTR-loaded XHA scaffold. In addition, morphological and histological analyses revealed that IVD regeneration was highest in rabbits transplanted with a MSC-highTR-loaded XHA scaffold. Conclusion Taken together, our results suggest that a MSC-highTR-loaded XHA scaffold supports IVD regeneration more effectively than a MSC-lowTR-loaded XHA scaffold. This study supports the potential clinical use of MSC-highTR-loaded XHA scaffolds to halt IVD degeneration or to enhance IVD regeneration. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0183-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Jongchan Ahn
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
| | - Eun-Mi Park
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
| | - Byeong Ju Kim
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
| | - Jin-Soo Kim
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
| | - Bogyu Choi
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
| | - Soo-Hong Lee
- Department of Biomedical Science, CHA University, 335 Pangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400, South Korea.
| | - Inbo Han
- Department of Neurosurgery, CHA University, CHA Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, 13496, South Korea.
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Electroacupuncture stimulates remodeling of extracellular matrix by inhibiting apoptosis in a rabbit model of disc degeneration. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2015; 2015:386012. [PMID: 25763091 PMCID: PMC4339975 DOI: 10.1155/2015/386012] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/26/2014] [Revised: 12/18/2014] [Accepted: 12/23/2014] [Indexed: 12/13/2022]
Abstract
The present study was designed to determine whether EA stimulates remodeling of extracellular matrix by inhibiting apoptosis in degenerated disc. 40 rabbits were randomly assigned to one of the four groups. Animal model was established by a loading device. Magnetic resonance imaging and Pfirrmann's classification were obtained to evaluate both the model and the EA treatment on disc degeneration. The ultrastructure of discs was observed by TEM. Apoptosis involvement was determined with TUNEL staining and western blot for the protein expression of Bax and Bcl-2. The results indicated that EA intervention decreased the MRI grades. TEM analysis showed an apparent remodeling and rearrangement of disc ECM after EA intervention for 28 days. The number of TUNEL-positive cells in the EA group was significantly lower than that in the compression group. The protein expression demonstrated an antiapoptosis effect mediated by EA. Increased expression of Bcl-2 proteins and reduced Bax protein expression were detected after 28 days treatment. It was concluded that antiapoptosis pathway probably participates in the mechanism of EA stimulating the remodeling of ECM in disc degeneration.
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22
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Yang Q, Zhao YH, Xia Q, Xu BS, Ma XL, Liu Y, Hu YC, Li HF, Miao J, Wang T, Ma JX, Sun XL. Novel cartilage-derived biomimetic scaffold for human nucleus pulposus regeneration: a promising therapeutic strategy for symptomatic degenerative disc diseases. Orthop Surg 2013; 5:60-3. [PMID: 23420750 DOI: 10.1111/os.12020] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/2012] [Accepted: 10/30/2012] [Indexed: 12/30/2022] Open
Abstract
Because current therapies have not always been successful and effective, the possibility of regenerating the nucleus pulposus (NP) through a tissue-engineered construct offers a novel therapeutic possibility for symptomatic degenerative disc diseases (DDDs). However, more research is necessary to identify the optimal scaffold, cell type and mixture of signal factors needed for NP regeneration. Numerous possible scaffolds for NP regeneration have been investigated; they have many shortcomings in common. Various biological scaffolds derived from decellularized tissue and organs have been successfully used in tissue engineering and received approval for use in humans. Regretfully, harvesting of human NP is difficult and only small amounts can be obtained. The macromolecules of cartilage, which include collagen and proteoglycan aggrecan, are similar to those of the extracellular matrix of immature NP. Recent studies have shown that adipose-derived stem cells (ADSC) can be induced to develop NP-like phenotypes when stimulated by appropriate signals. We thus reasonably postulated that an ideal NP scaffold for tissue engineering could be fabricated from decellularized cartilage matrix (DCM). Furthermore, a combination of ADSCs and DCM-derived biomimetic scaffolds would be advantageous in NP tissue engineering and, in the long run, could become an effective treatment option for symptomatic DDD.
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Affiliation(s)
- Qiang Yang
- Department of Spine Surgery, Tianjin Hospital, Tianjin, China
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23
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Issy AC, Castania V, Castania M, Salmon CEG, Nogueira-Barbosa MH, Bel ED, Defino HLA. Experimental model of intervertebral disc degeneration by needle puncture in Wistar rats. Braz J Med Biol Res 2013; 46:235-44. [PMID: 23532265 PMCID: PMC3854370 DOI: 10.1590/1414-431x20122429] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2012] [Accepted: 10/16/2012] [Indexed: 01/08/2023] Open
Abstract
Animal models of intervertebral disc degeneration play an important role in
clarifying the physiopathological mechanisms and testing novel therapeutic
strategies. The objective of the present study is to describe a simple animal
model of disc degeneration involving Wistar rats to be used for research
studies. Disc degeneration was confirmed and classified by radiography, magnetic
resonance and histological evaluation. Adult male Wistar rats were anesthetized
and submitted to percutaneous disc puncture with a 20-gauge needle on levels 6-7
and 8-9 of the coccygeal vertebrae. The needle was inserted into the discs
guided by fluoroscopy and its tip was positioned crossing the nucleus pulposus
up to the contralateral annulus fibrosus, rotated 360° twice, and held for 30 s.
To grade the severity of intervertebral disc degeneration, we measured the
intervertebral disc height from radiographic images 7 and 30 days after the
injury, and the signal intensity T2-weighted magnetic resonance imaging.
Histological analysis was performed with hematoxylin-eosin and collagen fiber
orientation using picrosirius red staining and polarized light microscopy.
Imaging and histological score analyses revealed significant disc degeneration
both 7 and 30 days after the lesion, without deaths or systemic complications.
Interobserver histological evaluation showed significant agreement. There was a
significant positive correlation between histological score and intervertebral
disc height 7 and 30 days after the lesion. We conclude that the tail disc
puncture method using Wistar rats is a simple, cost-effective and reproducible
model for inducing disc degeneration.
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Affiliation(s)
- A C Issy
- Departamento de Morfologia, Fisiologia e Patologia Básica, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil
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Sivakamasundari V, Lufkin T. Stemming the Degeneration: IVD Stem Cells and Stem Cell Regenerative Therapy for Degenerative Disc Disease. ACTA ACUST UNITED AC 2013; 2013. [PMID: 23951558 DOI: 10.5171/2013.724547] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The intervertebral disc (IVD) is immensely important for the integrity of vertebral column function. The highly specialized IVD functions to confer flexibility and tensile strength to the spine and endures various types of biomechanical force. Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain and includes the more severe degenerative lumbar scoliosis, disc herniation and spinal stenosis. DDD is a multifactorial disorder whereby an imbalance of anabolic and catabolic factors, or alterations to cellular composition, or biophysical stimuli and genetic background can all play a role in its genesis. However, our comprehension of IVD formation and theetiology of disc degeneration (DD) are far from being complete, hampering efforts to formulate appropriate therapies to tackle DD. Knowledge of the stem cells and various techniques to manipulate and direct them to particular fates have been promising in adopting a stem-cell based regenerative approach to DD. Moreover, new evidence on the residence of stem/progenitor cells within particular IVD niches has emerged holding promise for future therapeutic applications. Existing issues pertaining to current therapeutic approaches are also covered in this review.
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Liu ZH, Sun Z, Wang HQ, Ge J, Jiang TS, Chen YF, Ma Y, Wang C, Hu S, Samartzis D, Luo ZJ. FasL expression on human nucleus pulposus cells contributes to the immune privilege of intervertebral disc by interacting with immunocytes. Int J Med Sci 2013; 10:1053-60. [PMID: 23801893 PMCID: PMC3691805 DOI: 10.7150/ijms.6223] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2013] [Accepted: 06/17/2013] [Indexed: 01/23/2023] Open
Abstract
The mechanisms of immune privilege in human nucleus pulposus (NP) remain unclear. Accumulating evidence indicates that Fas ligand (FasL) might play an important role in the immune privilege of the disc. We aimed for addressing the role of FasL expression in human intervertebral disc degeneration (IDD) and immune privilege in terms of the interaction between NP cells and immunocytes via the FasL-Fas machinery. We collected NP specimens from 20 patients with IDD as degenerative group and 8 normal cadaveric donors as control. FasL expression was detected by qRT-PCR, western blotting and flow cytometry (FCM). We also collected macrophages and CD8(+) T cells from the peripheral blood of patients with IDD for co-cultures with NP cells. And macrophages and CD8(+) T cells were harvested for apoptosis analysis by FCM after 2 days of co-cultures. We found that FasL expression in mRNA, protein and cellular resolutions demonstrated a significant decrease in degenerative group compared with normal control (p<0.05). FCM analysis found that human NP cells with increased FasL expression resulted in significantly increased apoptosis ratio of macrophages and CD8(+) T cells. Our study demonstrated that FasL expression tends to decrease in degenerated discs and FasL plays an important role in human disc immune privilege, which might provide a novel target for the treatment strategies for IDD.
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Affiliation(s)
- Zhi-Heng Liu
- Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, 127 Changle Western Road, Xi'an, P. R. China
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26
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Chen YF, Zhang YZ, Zhang WL, Luan GN, Liu ZH, Gao Y, Wan ZY, Sun Z, Zhu S, Samartzis D, Wang CM, Wang HQ, Luo ZJ. Insights into the hallmarks of human nucleus pulposus cells with particular reference to cell viability, phagocytic potential and long process formation. Int J Med Sci 2013; 10:1805-16. [PMID: 24324357 PMCID: PMC3856371 DOI: 10.7150/ijms.6530] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/22/2013] [Accepted: 10/14/2013] [Indexed: 11/05/2022] Open
Abstract
OBJECTIVE As a main cellular component within the disc, nucleus pulposus (NP) cells play important roles in disc physiology. However, little is known on the biologic hallmarks of human NP cells. Therefore, the present study aimed to address the features of human NP cells. METHODS Human NP samples were collected from normal cadavers, patients with scoliosis and disc degeneration as normal, disease control and degenerative NP, respectively. The NP samples were studied using transmission electron microscopy and TUNEL assay. Pre-digested NP samples were studied using flow cytometry with PI/Annexin V staining. RESULTS Both control and degenerative human NP consisted of mainly viable cells with a variety of morphology. Both necrosis and apoptosis were noted in human NP as forms of cell death with increased apoptosis in degenerative NP, which was further confirmed by the TUNEL assay. Phagocytic NP cells had the hallmarks of both stationary macrophages with lysosomes and NP cells with the endoplasmic reticulum. Annulus fibrosus cells have similar morphologic characteristics with NP cells in terms of cell nest, phagocytosis and intracellular organs. Moreover, NP cells with long processes existed in degenerative and scoliotic NP rather than normal NP. When cultured in glucose-free medium, NP cells developed long and thin processes. CONCLUSION Human degenerative NP consists of primarily viable cells. We present direct and in vivo evidence that both human annulus fibrosus and NP cells have phagocytic potential. Moreover, NP cells with long processes exist in both scoliotic and degenerative NP with lack of glucose as one of the possible underlying mechanisms.
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Affiliation(s)
- Yu-Fei Chen
- 1. Department of Orthopaedics, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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27
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Nosikova YS, Santerre JP, Grynpas M, Gibson G, Kandel RA. Characterization of the annulus fibrosus-vertebral body interface: identification of new structural features. J Anat 2012; 221:577-89. [PMID: 22747710 DOI: 10.1111/j.1469-7580.2012.01537.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Current surgical treatments for degenerative intervertebral disc disease do not restore full normal spinal movement. Tissue engineering a functional disc replacement may be one way to circumvent this limitation, but will require an integration of the different tissues making up the disc for this approach to be successful. Hence, an in-depth characterization of the native tissue interfaces, including annulus insertion into bone is necessary, as knowledge of this interface is limited. The objective of this study was to characterize the annulus fibrosus-vertebral bone (AF-VB) interface in immature (6-9 months old) and mature (18-24 months old) bovine discs, as well as to define these structures for normal adult human (22 and 45 years old) discs. Histological assessment showed that collagen fibers in the inner annulus, which are predominantly type II collagen, all appear to insert into the mineralized endplate zone. In contrast, some of the collagen fibers of the outer annulus, predominantly type I collagen, insert into this endplate, while other fibers curve laterally, at an ∼ 90° angle, to the outer aspect of the bone, and merge with the periosteum. This is seen in both human and bovine discs. Where the AF inserts into the calcified zone of the AF-VB interface, it passes through a chondroid region, rich in type II collagen and proteoglycans. Annulus cells (elongated cells that are not surrounded by proteoglycans) are present at this interface. This cartilage zone is evident in both human and bovine discs. Type X collagen and alkaline phosphatase are localized to the interface region. Age-associated differences in bovine spines are observed when examining the interface thickness and the matrix composition of the cartilaginous endplate, as well as the thickness of the mineralized endplate. These findings will assist with the design of the AF-VB interface in the tissue engineered disc.
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Affiliation(s)
- Y S Nosikova
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, ON, Canada
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28
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Strassburg S, Hodson NW, Hill PI, Richardson SM, Hoyland JA. Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells. PLoS One 2012; 7:e33739. [PMID: 22438989 PMCID: PMC3305345 DOI: 10.1371/journal.pone.0033739] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2011] [Accepted: 02/16/2012] [Indexed: 02/07/2023] Open
Abstract
Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.
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Affiliation(s)
- Sandra Strassburg
- Regenerative Medicine, School of Biomedicine, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, United Kingdom
| | - Nigel W. Hodson
- Regenerative Medicine, School of Biomedicine, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, United Kingdom
| | - Patrick I. Hill
- School of Chemical Engineering, Faculty of Engineering and Physical Sciences, The University of Manchester, Manchester, United Kingdom
| | - Stephen M. Richardson
- Regenerative Medicine, School of Biomedicine, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, United Kingdom
| | - Judith A. Hoyland
- Regenerative Medicine, School of Biomedicine, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, United Kingdom
- * E-mail:
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29
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Ciapetti G, Granchi D, Devescovi V, Leonardi E, Greggi T, Di Silvestre M, Baldini N. Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs. EUROPEAN SPINE JOURNAL : OFFICIAL PUBLICATION OF THE EUROPEAN SPINE SOCIETY, THE EUROPEAN SPINAL DEFORMITY SOCIETY, AND THE EUROPEAN SECTION OF THE CERVICAL SPINE RESEARCH SOCIETY 2012; 21 Suppl 1:S10-9. [PMID: 22395304 DOI: 10.1007/s00586-012-2234-y] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 02/13/2012] [Accepted: 02/19/2012] [Indexed: 01/07/2023]
Abstract
PURPOSE Disc degeneration, and associated low back pain, are a primary cause of disability. Disc degeneration is characterized by dysfunctional cells and loss of proteoglycans: since intervertebral tissue has a limited capacity to regenerate, this process is at present considered irreversible. Recently, cell therapy has been suggested to provide more successful treatment of IVD degeneration. To understand the potential of cells to restore IVD structure/function, tissue samples from degenerated IVD versus healthy discs have been compared. METHODS Discal tissue from 27 patients (40.17 ± 11 years) undergoing surgery for degenerative disc disease (DDD), DDD + herniation and congenital scoliosis, as controls, was investigated. Cells and matrix in the nucleus pulposus (NP) and annulus fibrosus (AF) were characterized by histology. AF- and NP-derived cells were isolated, expanded and characterized for senescence and gene expression. Three-dimensional NP pellets were cultured and stained for glycosaminoglycan formation. RESULTS Phenotypical markers of degeneration, such as cell clusters, chondrons, and collagen disorganization were seen in the degenerate samples. In severe degeneration, granulation tissue and peripheral vascularization were observed. No correlation was found between the Pfirrmann clinical score and the extent of degeneration. CONCLUSION The tissue disorganization in degenerate discs and the paucity of cells out of cluster/chondron association, make the IVD-derived cells an unreliable option for disc regeneration.
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Affiliation(s)
- Gabriela Ciapetti
- Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, 40136 Bologna, Italy.
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30
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Stem cells and gene therapy for cartilage repair. Stem Cells Int 2012; 2012:168385. [PMID: 22481959 PMCID: PMC3306906 DOI: 10.1155/2012/168385] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2011] [Accepted: 12/06/2011] [Indexed: 01/06/2023] Open
Abstract
Cartilage defects represent a common problem in orthopaedic practice. Predisposing factors include traumas, inflammatory conditions, and biomechanics alterations. Conservative management of cartilage defects often fails, and patients with this lesions may need surgical intervention. Several treatment strategies have been proposed, although only surgery has been proved to be predictably effective. Usually, in focal cartilage defects without a stable fibrocartilaginous repair tissue formed, surgeons try to promote a natural fibrocartilaginous response by using marrow stimulating techniques, such as microfracture, abrasion arthroplasty, and Pridie drilling, with the aim of reducing swelling and pain and improving joint function of the patients. These procedures have demonstrated to be clinically useful and are usually considered as first-line treatment for focal cartilage defects. However, fibrocartilage presents inferior mechanical and biochemical properties compared to normal hyaline articular cartilage, characterized by poor organization, significant amounts of collagen type I, and an increased susceptibility to injury, which ultimately leads to premature osteoarthritis (OA). Therefore, the aim of future therapeutic strategies for articular cartilage regeneration is to obtain a hyaline-like cartilage repair tissue by transplantation of tissues or cells. Further studies are required to clarify the role of gene therapy and mesenchimal stem cells for management of cartilage lesions.
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31
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Zweig T, Hemmeler C, Aghayev E, Melloh M, Etter C, Röder C. Influence of preoperative nucleus pulposus status and radiculopathy on outcomes in mono-segmental lumbar total disc replacement: results from a nationwide registry. BMC Musculoskelet Disord 2011; 12:275. [PMID: 22136141 PMCID: PMC3250959 DOI: 10.1186/1471-2474-12-275] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2011] [Accepted: 12/02/2011] [Indexed: 11/10/2022] Open
Abstract
Background Currently, herniated nucleus pulposus (HNP) with radiculopathy and other preconditions are regarded as relative or absolute contraindications for lumbar total disc replacement (TDR). In Switzerland it is left to the surgeon's discretion when to operate. The present study is based on the dataset of SWISSspine, a governmentally mandated health technology assessment registry. We hypothesized that preoperative nucleus pulposus status and presence or absence of radiculopathy has an influence on clinical outcomes in patients treated with mono-segmental lumbar TDR. Methods Between March 2005 and April 2009, 416 patients underwent mono-segmental lumbar TDR, which was documented in a prospective observational multicenter mode. The data collection consisted of perioperative and follow-up data (physician based) and clinical outcomes (NASS, EQ-5D). Patients were divided into four groups according to their preoperative status: 1) group degenerative disc disease ("DDD"): 160 patients without HNP and no radiculopathy, classic precondition for TDR; 2) group "HNP-No radiculopathy": 68 patients with HNP but without radiculopathy; 3) group "Stenosis": 73 patients without HNP but with radiculopathy, and 4) group "HNP-Radiculopathy": 132 patients with HNP and radiculopathy. The groups were compared regarding preoperative patient characteristics and pre- and postoperative VAS and EQ-5D scores using general linear modeling. Results Demographics in all four groups were comparable. Regarding the improvement of quality of life (EQ-5D) there were no differences across the four groups. For the two main groups DDD and HNP-Radiculopathy no differences were found in the adjusted postoperative back- and leg pain alleviation levels, in the stenosis group back- and leg pain relief were lower. Conclusions Despite higher preoperative leg pain levels, outcomes in lumbar TDR patients with HNP and radiculopathy were similar to outcomes in patients with the classic indication; this because patients with higher preoperative leg pain levels benefit from a relatively greater leg pain alleviation. The group with absence of HNP but presence of radiculopathy showed considerably less benefits from the operation, which is probably related to ongoing degenerative processes of the posterior segmental structures. This observational multicenter study suggests that the diagnoses HNP and radiculopathy, combined or alone, may not have to be considered as absolute or relative contraindications for mono-segmental lumbar TDR anymore, whereas patients without HNP but with radiculopathy seem to be suboptimal candidates for the procedure.
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Affiliation(s)
- Thomas Zweig
- Institute for Evaluative Research in Medicine, University of Bern, Stauffacherstrasse 78, 3014 Bern, Switzerland
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32
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Park SH, Gil ES, Cho H, Mandal BB, Tien LW, Min BH, Kaplan DL. Intervertebral disk tissue engineering using biphasic silk composite scaffolds. Tissue Eng Part A 2011; 18:447-58. [PMID: 21919790 DOI: 10.1089/ten.tea.2011.0195] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Scaffolds composed of synthetic, natural, and hybrid materials have been investigated as options to restore intervertebral disk (IVD) tissue function. These systems fall short of the lamellar features of the native annulus fibrosus (AF) tissue or focus only on the nucleus pulposus (NP) tissue. However, successful regeneration of the entire IVD requires a combination approach to restore functions of both the AF and NP. To address this need, a biphasic biomaterial structure was generated by using silk protein for the AF and fibrin/hyaluronic acid (HA) gels for the NP. Two cell types, porcine AF cells and chondrocytes, were utilized. For the AF tissue, two types of scaffold morphologies, lamellar and porous, were studied with the porous system serving as a control. Toroidal scaffolds formed out of the lamellar, and porous silk materials were used to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm, and a height of 3 mm (the interlamellar distance in the lamellar scaffold was 150-250 μm, and the average pore sizes in the porous scaffolds were 100-250 μm). The scaffolds were seeded with porcine AF cells to form AF tissue, whereas porcine chondrocytes were encapsulated in fibrin/HA hydrogels for the NP tissue and embedded in the center of the toroidal disk. Histology, biochemical assays, and gene expression indicated that the lamellar scaffolds supported AF-like tissue over 2 weeks. Porcine chondrocytes formed the NP phenotype within the hydrogel after 4 weeks of culture with the AF tissue that had been previously cultured for 2 weeks, for a total of 6 weeks of cultivation. This biphasic scaffold simulating in combination of both AF and NP tissues was effective in the formation of the total IVD in vitro.
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Affiliation(s)
- Sang-Hyug Park
- Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA
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33
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Bertolo A, Mehr M, Aebli N, Baur M, Ferguson SJ, Stoyanov JV. Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells. EUROPEAN SPINE JOURNAL : OFFICIAL PUBLICATION OF THE EUROPEAN SPINE SOCIETY, THE EUROPEAN SPINAL DEFORMITY SOCIETY, AND THE EUROPEAN SECTION OF THE CERVICAL SPINE RESEARCH SOCIETY 2011; 21 Suppl 6:S826-38. [PMID: 21863459 DOI: 10.1007/s00586-011-1975-3] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Accepted: 08/04/2011] [Indexed: 12/27/2022]
Abstract
INTRODUCTION Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. MATERIALS AND METHODS Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. RESULTS MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. CONCLUSIONS Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.
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Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture. Spine J 2010; 10:32-41. [PMID: 19926342 PMCID: PMC2818300 DOI: 10.1016/j.spinee.2009.10.006] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/05/2009] [Revised: 09/28/2009] [Accepted: 10/13/2009] [Indexed: 02/03/2023]
Abstract
BACKGROUND CONTEXT The therapeutic strategies that have thus far been used for the treatment of intervertebral disc degeneration (IDD) have focused on relieving the symptoms, although reversal of the degeneration remains an important challenge for the effective treatment of IDD. Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins. PURPOSE The purpose of the study was to develop a lumbar disc degeneration model in mice and determine the effect of adenoviral GDF5 gene therapy. STUDY DESIGN The study design was to compare the degeneration changes of discs punctured by different-size needles to develop a mice lumbar disc degeneration model and to evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene. METHODS A lumbar disc degeneration model was developed by needle punctures to the discs in Balb/c mice. Afterward, a gene therapy treatment to disc degeneration was evaluated. Two of the mice lumbar discs were randomly chosen to be punctured by a 30-gauge needle and then injected with adenovirus that had been engineered to express either the luciferase gene (Ad-Luc) or the GDF5 gene (Ad-GDF5). Animals were analyzed by bioluminescent imaging, radiographic, and magnetic resonance imaging (MRI) scanning, then sacrificed at 1, 2, 4, or 8 weeks after operation, and subjected to histological and biochemical assays. RESULTS By the detection of T2-weighted MRI scanning and histological study, the degeneration was found in all of the discs punctured by different-size needles. But the development of the degeneration in the discs injured by the 30-gauge needle was more reliable and moderate compared with that in other groups. The detection of luciferase activity by bioluminescent imaging revealed that adenovirus survived and the introduced genes were expressed over 6 weeks after injection. There were no T2-weighted MRI signals in the mice injected with either Ad-Luc or Ad-GDF5 up to 4 weeks after operation. At 6 and 8 weeks, T2-weighted signals were detected in the Ad-GDF5 group but none in the Ad-Luc control group. The percent disc height index (%DHI) was significantly decreased (approximately 20%) by 1 week after injury in both groups, indicating the development of disc degeneration. At 2 weeks, the %DHI in the mice injected with Ad-GDF5 increased significantly compared with that of the mice injected with Ad-Luc; the increase was sustained for the rest of the experiment period. The disc histology treated with Ad-GDF5 was improved compared with that in the control group. Glycosaminoglycan (GAG) levels were significantly decreased in the Ad-Luc injection group since 2 weeks after injury, and the DNA content had diminished by 4 weeks after the operation. In contrast, in the discs injected with Ad-GDF5, there was no decrease in the GAG and DNA levels after injury throughout the 8-week treatment period. CONCLUSIONS Disc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment.
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Le Maitre CL, Fotheringham AP, Freemont AJ, Hoyland JA. Development of an in vitro model to test the efficacy of novel therapies for IVD degeneration. J Tissue Eng Regen Med 2009; 3:461-9. [PMID: 19444863 DOI: 10.1002/term.180] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Low back pain (LBP) is a major cause of disability worldwide that has been linked to intervertebral disc (IVD) degeneration. An improved understanding of the pathogenesis of disc degeneration is now developing, which is leading to the development of a number of possible future therapies targeted at the underlying pathology and regeneration strategies. Although results thus far are promising, the investigation of such therapies in an environment that mimics the mechanical environment of the human disc in vivo is problematic. The development of an in vitro model system that can maintain metabolically active IVD tissue within a loading environment pertaining to that of the human spine is crucial for testing the efficacy of future cell-based and tissue-engineering therapies for IVD degeneration. Here, using our novel loading rig, capable of mimicking the loading environment experienced within the human spine, we have cultured nucleus pulposus tissue explants, applied a daily hydrostatic loading regime for up to 2 weeks and investigated proteoglycan retention, metabolic activity and cellular phenotype. IVD tissue cultured under a loading environment pertaining to the in vivo loading environment maintained metabolic cell activity, proteoglycan content and cellular phenotype. Indeed, all parameters were improved in IVD tissue cultured with load compared to unloaded controls. Such a model is invaluable for investigations assessing the feasibility and efficacy of future therapeutic approaches to inhibiting degeneration or stimulating regeneration of the IVD, where the in vivo loading environment may be crucial to their success or failure.
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Affiliation(s)
- Christine L Le Maitre
- Biomedical Research Centre, Biosciences, Sheffield Hallam University, City Campus, Owen Building, Howard Street, Sheffield S1 1WB, UK
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Richardson SM, Doyle P, Minogue BM, Gnanalingham K, Hoyland JA. Increased expression of matrix metalloproteinase-10, nerve growth factor and substance P in the painful degenerate intervertebral disc. Arthritis Res Ther 2009; 11:R126. [PMID: 19695094 PMCID: PMC2745810 DOI: 10.1186/ar2793] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2009] [Revised: 07/27/2009] [Accepted: 08/20/2009] [Indexed: 11/25/2022] Open
Abstract
Introduction Matrix metalloproteinases (MMPs) are known to be involved in the degradation of the nucleus pulposus (NP) during intervertebral disc (IVD) degeneration. This study investigated MMP-10 (stromelysin-2) expression in the NP during IVD degeneration and correlated its expression with pro-inflammatory cytokines and molecules involved in innervation and nociception during degeneration which results in low back pain (LBP). Methods Human NP tissue was obtained at postmortem (PM) from patients without a history of back pain and graded as histologically normal or degenerate. Symptomatic degenerate NP samples were also obtained at surgery for LBP. Expression of MMP-10 mRNA and protein was analysed using real-time polymerase chain reaction and immunohistochemistry. Gene expression for pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α), nerve growth factor (NGF) and the pain-associated neuropeptide substance P were also analysed. Correlations between MMP-10 and IL-1, TNF-α and NGF were assessed along with NGF with substance P. Results MMP-10 mRNA was significantly increased in surgical degenerate NP when compared to PM normal and PM degenerate samples. MMP-10 protein was also significantly higher in degenerate surgical NP samples compared to PM normal. IL-1 and MMP-10 mRNA demonstrated a significant correlation in surgical degenerate samples, while TNF-α was not correlated with MMP-10 mRNA. NGF was significantly correlated with both MMP-10 and substance P mRNA in surgical degenerate NP samples. Conclusions MMP-10 expression is increased in the symptomatic degenerate IVD, where it may contribute to matrix degradation and initiation of nociception. Importantly, this study suggests differences in the pathways involved in matrix degradation between painful and pain-free IVD degeneration.
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Affiliation(s)
- Stephen M Richardson
- Tissue Injury and Repair Group, School of Clinical and Laboratory Sciences, Stopford Building, The University of Manchester, Oxford Road, Manchester, UK.
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Topographical variation in the distributions of versican, aggrecan and perlecan in the foetal human spine reflects their diverse functional roles in spinal development. Histochem Cell Biol 2009; 132:491-503. [PMID: 19669783 DOI: 10.1007/s00418-009-0623-z] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/08/2009] [Indexed: 02/06/2023]
Abstract
We evaluated the immunohistochemical distribution of three major proteoglycans of cartilage, i.e., aggrecan, versican and perlecan vis-a-vis collagens I and II in the developing human spine of first-trimester foetuses. Aggrecan and perlecan were prominently immunolocalised in the cartilaginous vertebral body rudiments and to a lesser extent within the foetal intervertebral disc. In contrast, versican was only expressed in the developing intervertebral disc interspace. Using domain-specific monoclonal antibodies against the various modules of versican, we discovered the V0 isoform as the predominant form present. Versican immunolocalisations conducted with antibodies directed to epitopes in its N and C termini and GAG-alpha and GAG-beta core protein domains provided evidence that versican in the nucleus pulposus was either synthesised devoid of a G3 domain or this domain was proteolytically removed in situ. The V0 versican isoform was localised with prominent fibrillar components in the annular lamellae of the outer annulus fibrosus. Perlecan was a notable pericellular proteoglycan in the annulus fibrosus and nucleus pulposus but poorly immunolocalised in the marginal tissues of the developing intervertebral disc, apparently delineating the intervertebral disc-vertebral body interface region destined to become the cartilaginous endplate in the mature intervertebral disc. The distribution of collagens I and II in the foetal spine was mutually exclusive with type I present in the outer annulus fibrosus, marginal tissues around the vertebral body rudiment and throughout the developing intervertebral disc, and type II prominent in the vertebral rudiment, absent in the outer annulus fibrosus and diffusely distributed in the inner annulus fibrosus and nucleus pulposus. Collectively, our findings suggest the existence of an intricate and finely balanced interplay between various proteoglycans and collagens and the spinal cell populations which synthesise and assemble these components during spinal development.
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Primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 BMPs and Sox9 maintain appropriate phenotype. Am J Phys Med Rehabil 2009; 88:455-63. [PMID: 19454853 DOI: 10.1097/phm.0b013e3181a5f0aa] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
OBJECTIVE To confirm that primary intervertebral disc cells cultured in monolayer transduced with adenovirus maintained their phenotype, hence is an appropriate system to test gene therapy agents. DESIGN Adult bovine nucleus pulposus and anulus fibrosus cells cultured in monolayer were transduced with adenoviruses expressing human bone morphogenetic proteins (AdBMPs) or Sox9 (AdSox9), or green fluorescence protein (AdGFP, as control). Chondrocyte phenotypic markers (e.g., type II collagen and aggrecan) and the chondrocyte hypertrophy marker (type X collagen) were measured 6 days after viral transduction by reverse-transcription polymerase chain reaction. RESULTS Primary nucleus pulposus and anulus fibrosus cells transduced with AdBMPs, AdSox9, or adenovirus-expressing green fluorescence protein only (AdGFP, as control) continue to express healthy chondrocyte phenotypic markers and showed no evidence of the expression of the chondrocyte hypertrophy marker (type X collagen gene). Thus, we have shown that bovine nucleus pulposus and anulus fibrosus cells transduced with adenovirus overexpressing 12 different bone morphogenetic proteins or Sox9 maintain their phenotype in short-term culture. CONCLUSIONS In this study, primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 bone morphogenetic proteins or Sox9 preserved their phenotype in short-term culture. These cells did not express the type X collagen gene, an undesirable chondrocyte hypertrophic gene that could lead to ossification. Therefore, low-passage intervertebral disc cells cultured in monolayer is an appropriate culture system to test therapeutic genes. We further suggest that these cells may also be appropriate for engineering tissues or for cell therapy for degenerative disc diseases.
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Le Maitre CL, Baird P, Freemont AJ, Hoyland JA. An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue. Arthritis Res Ther 2009; 11:R20. [PMID: 19210770 PMCID: PMC2688252 DOI: 10.1186/ar2611] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2008] [Revised: 01/14/2009] [Accepted: 02/11/2009] [Indexed: 12/24/2022] Open
Abstract
Introduction The decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures. Methods Human MSCs extracted from bone marrow were expanded in monolayer culture and, after labelling with adenoviral vectors carrying the green fluorescent protein transcript, were injected into NP tissue explants (sourced from bovine caudal discs) and maintained in culture for 2, 7, 14 and 28 days post injection. Following fixation and paraffin embedding, cell viability was assessed using in situ hybridisation for polyA-mRNA and using immunohistochemistry for caspase 3. Immunohistochemistry/fluorescence for aggrecan, Sox-9 and types I, II and X collagen together with Alizarin red staining was employed to investigate the MSC phenotype and matrix formation. Results MSCs were identified in all injected tissue samples and cell viability was maintained for the 4 weeks investigated. MSCs displayed cellular staining for Sox-9, and displayed cellular and matrix staining for aggrecan and type II collagen that increased during culture. No type I collagen, type X collagen or Alizarin red staining was observed at any time point. Conclusions MSCs from older individuals differentiate spontaneously into chondrocyte-like NP cells upon insertion into NP tissue in vitro, and thus may not require additional stimulation or carrier to induce differentiation. This is a key finding, as such a strategy would minimise the level of external manipulation required prior to insertion into the patient, thus simplifying the treatment strategy and reducing costs.
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Affiliation(s)
- Christine L Le Maitre
- Biomedical Research Centre, Biosciences, Faculty of Health and Wellbeing, Sheffield Hallam University, City Campus, Owen Building, Howard Street, Sheffield S11WB, UK.
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ISSLS prize winner: integrating theoretical and experimental methods for functional tissue engineering of the annulus fibrosus. Spine (Phila Pa 1976) 2008; 33:2691-701. [PMID: 19018251 PMCID: PMC3424511 DOI: 10.1097/brs.0b013e31818e61f7] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
STUDY DESIGN Integrating theoretical and experimental approaches for annulus fibrosus (AF) functional tissue engineering. OBJECTIVE Apply a hyperelastic constitutive model to characterize the evolution of engineered AF via scalar model parameters. Validate the model and predict the response of engineered constructs to physiologic loading scenarios. SUMMARY OF BACKGROUND DATA There is need for a tissue engineered replacement for degenerate AF. When evaluating engineered replacements for load-bearing tissues, it is necessary to evaluate mechanical function with respect to the native tissue, including nonlinearity and anisotropy. METHODS Aligned nanofibrous poly-epsilon-caprolactone scaffolds with prescribed fiber angles were seeded with bovine AF cells and analyzed over 8 weeks, using experimental (mechanical testing, biochemistry, histology) and theoretical methods (a hyperelastic fiber-reinforced constitutive model). RESULTS The linear region modulus for phi = 0 degrees constructs increased by approximately 25 MPa, and for phi = 90 degrees by approximately 2 MPa from 1 day to 8 weeks in culture. Infiltration and proliferation of AF cells into the scaffold and abundant deposition of s-GAG and aligned collagen was observed. The constitutive model had excellent fits to experimental data to yield matrix and fiber parameters that increased with time in culture. Correlations were observed between biochemical measures and model parameters. The model was successfully validated and used to simulate time-varying responses of engineered AF under shear and biaxial loading. CONCLUSION AF cells seeded on nanofibrous scaffolds elaborated an organized, anisotropic AF-like extracellular matrix, resulting in improved mechanical properties. A hyperelastic fiber-reinforced constitutive model characterized the functional evolution of engineered AF constructs, and was used to simulate physiologically relevant loading configurations. Model predictions demonstrated that fibers resist shear even when the shearing direction does not coincide with the fiber direction. Further, the model suggested that the native AF fiber architecture is uniquely designed to support shear stresses encountered under multiple loading configurations.
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Kandel R, Roberts S, Urban JPG. Tissue engineering and the intervertebral disc: the challenges. EUROPEAN SPINE JOURNAL : OFFICIAL PUBLICATION OF THE EUROPEAN SPINE SOCIETY, THE EUROPEAN SPINAL DEFORMITY SOCIETY, AND THE EUROPEAN SECTION OF THE CERVICAL SPINE RESEARCH SOCIETY 2008; 17 Suppl 4:480-91. [PMID: 19005701 DOI: 10.1007/s00586-008-0746-2] [Citation(s) in RCA: 138] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/21/2008] [Revised: 07/13/2008] [Accepted: 07/13/2008] [Indexed: 12/25/2022]
Abstract
Disc degeneration is a common disorder. Although the back pain that can develop in association with this is rarely life-threatening, the annual cost in terms of morbidity, lost productivity, medical expenses and workers' compensation benefits is significant. Surgical intervention as practised currently is directed towards removing the damaged or altered tissue. Development of new treatment modalities is critical as there is a growing consensus that the strategies used currently for symptomatic degenerative disc disease may not be effective. Accordingly, there is a need to develop an entirely new way to treat this disorder; regenerative medicine and tissue engineering approaches appear particularly promising in this regard. This paper reviews some of the challenges that currently are limiting the clinical application of this approach to the treatment of disc degeneration.
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Affiliation(s)
- Rita Kandel
- CIHR-Bioengineering of Skeletal Tissues Team, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
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Recent advances in annular pathobiology provide insights into rim-lesion mediated intervertebral disc degeneration and potential new approaches to annular repair strategies. EUROPEAN SPINE JOURNAL : OFFICIAL PUBLICATION OF THE EUROPEAN SPINE SOCIETY, THE EUROPEAN SPINAL DEFORMITY SOCIETY, AND THE EUROPEAN SECTION OF THE CERVICAL SPINE RESEARCH SOCIETY 2008; 17:1131-48. [PMID: 18584218 DOI: 10.1007/s00586-008-0712-z] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/09/2007] [Revised: 06/04/2008] [Accepted: 06/18/2008] [Indexed: 12/14/2022]
Abstract
The objective of this study was to assess the impact of a landmark annular lesion model on our understanding of the etiopathogenesis of IVD degeneration and to appraise current IVD repairative strategies. A number of studies have utilised the Osti sheep model since its development in 1990. The experimental questions posed at that time are covered in this review, as are significant recent advances in annular repair strategies. The ovine model has provided important spatial and temporal insights into the longitudinal development of annular lesions and how they impact on other discal and paradiscal components such as the NP, cartilaginous end plates, zygapophyseal joints and vertebral bone and blood vessels. Important recent advances have been made in biomatrix design for IVD repair and in the oriented and dynamic culture of annular fibrochondrocytes into planar, spatially relevant, annular type structures. The development of hyaluronan hydrogels capable of rapid in situ gelation offer the possibility of supplementation of matrices with cells and other biomimetics and represent a significant advance in biopolymer design. New generation biological glues and self-curing acrylic formulations which may be augmented with slow delivery biomimetics in microcarriers may also find application in the non-surgical repair of annular defects. Despite major advances, significant technical challenges still have to be overcome before the biological repair of this intractable connective tissue becomes a realistic alternative to conventional surgical intervention for the treatment of chronic degenerate IVDs.
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Expression of glucose transporters GLUT-1, GLUT-3, GLUT-9 and HIF-1alpha in normal and degenerate human intervertebral disc. Histochem Cell Biol 2008; 129:503-11. [PMID: 18172662 DOI: 10.1007/s00418-007-0372-9] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/09/2007] [Indexed: 10/22/2022]
Abstract
The glucose transporters GLUT-1 and GLUT-3 are targets of the hypoxia-inducible transcription factor HIF-1alpha and it has been shown that nucleus pulposus (NP) cells in rat intervertebral discs (IVD) express both HIF-1alpha and GLUT-1. However, there is limited data on the expression of HIF-1alpha and GLUTs in human IVD. The aim here was to (1) determine whether, like articular chondrocytes, human IVD cells express GLUT-1, 3 and 9 and whether there was any co-expression with HIF-1alpha; and (2) to localise expression of the GLUT isoforms in the disc and identify any changes during degeneration. Real-time PCR was used to identify expression of GLUT1, 3, 9 and HIF-1alpha mRNAs and immunohistochemistry was used to analyse protein expression and localisation of GLUTs in normal and degenerate IVD biopsies. Results confirmed HIF-1alpha, GLUT1, 3 and 9 mRNA expression in NP and AF and co-expression of each GLUT isoform with HIF-1alpha in the NP, but not the AF. Immunohistochemistry demonstrated regional differences in GLUT expression, with the highest expression being in the NP. GLUT expression also changed as degeneration progressed. This study demonstrates that NP and AF cells have different GLUT expression profiles that suggest regional differences in the metabolic nature of the human IVD and that this environment changes during degeneration.
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