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Wu R, Li H, Sun C, Liu J, Chen D, Yu H, Huang Z, Lin S, Chen Y, Zheng Q. Exosome-based strategy for degenerative disease in orthopedics: Recent progress and perspectives. J Orthop Translat 2022; 36:8-17. [PMID: 35891923 PMCID: PMC9283806 DOI: 10.1016/j.jot.2022.05.009] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Revised: 05/07/2022] [Accepted: 05/19/2022] [Indexed: 02/09/2023] Open
Abstract
BACKGROUND Degenerative diseases in orthopaedics have become a significant global public health issue with the aging of the population worldwide. The traditional medical interventions, including physical therapy, pharmacological therapy and even surgery, hardly work to modify degenerative progression. Stem cell-based therapy is widely accepted to treat degenerative orthopaedic disease effectively but possesses several limitations, such as the need for strict monitoring of production and storage and the potential risks of tumorigenicity and immune rejection in clinical translation. Furthermore, the ethical issues surrounding the acquisition of embryonic stem cells are also broadly concerned. Exosome-based therapy has rapidly grown in popularity in recent years and is regarded as an ideal alternative to stem cell-based therapy, offering a promise to achieve 'cell-free' tissue regeneration. METHODS Traditionally, the native exosomes extracted from stem cells are directly injected into the injured site to promote tissue regeneration. Recently, several modified exosome-based strategies were developed to overcome the limitations of native exosomes, which include mainly exogenous molecule loading and exosome delivery through scaffolds. In this paper, a systematic review of the exosome-based strategy for degenerative disease in orthopaedics is presented. RESULTS Treatment strategies based on the native exosomes are effective but with several disadvantages such as rapid diffusion and insufficient and fluctuating functional contents. The modified exosome-based strategies can better match the requirements of the regeneration in some complex healing processes. CONCLUSION Exosome-based strategies hold promise to manage degenerative disease in orthopaedics prior to patients reaching the advanced stage of disease in the future. The timely summary and highlights offered herein could provide a research perspective to promote the development of exosome-based therapy, facilitating the clinical translation of exosomes in orthopaedics. TRANSLATIONAL POTENTIAL OF THIS ARTICLE Exosome-based therapy is superior in anti-senescence and anti-inflammatory effects and possesses lower risks of tumorigenicity and immune rejection relative to stem cell-based therapy. Exosome-based therapy is regarded as an ideal alternative to stem cell-based therapy, offering a promise to achieve 'cell-free' tissue regeneration.
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Affiliation(s)
- Rongjie Wu
- Department of Orthopedics, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
- Shantou University Medical College, Shantou, China
| | - Haotao Li
- Department of Orthopedics, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
- Shantou University Medical College, Shantou, China
| | - Chuanwei Sun
- Department of Burn and Wound Repair Surgery and Research Department of Medical Science, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
| | - Jialin Liu
- Rehabilitation Center, Shengjing Hospital Affiliated to China Medical University, Shenyang, Liaoning, PR China
| | - Duanyong Chen
- Department of Orthopedics, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
| | - Haiyang Yu
- Department of Orthopedics, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
| | - Zena Huang
- Department of General Medicine, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China
| | - Sien Lin
- Department of Orthopaedics & Traumatology, Stem Cells and Regenerative Medicine Laboratory, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Special Administrative Region of China
| | - Yuanfeng Chen
- Department of Orthopedics, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
- Research Department of Medical Science, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
| | - Qiujian Zheng
- Department of Orthopedics, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, PR China
- Southern Medical University, Guangzhou, PR China
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Laagland LT, Bach FC, Creemers LB, Le Maitre CL, Poramba-Liyanage DW, Tryfonidou MA. Hyperosmolar expansion medium improves nucleus pulposus cell phenotype. JOR Spine 2022; 5:e1219. [PMID: 36203869 PMCID: PMC9520765 DOI: 10.1002/jsp2.1219] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2022] [Revised: 07/21/2022] [Accepted: 07/21/2022] [Indexed: 12/02/2022] Open
Abstract
Background Repopulating the degenerated intervertebral disc (IVD) with tissue-specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell-based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model. Methods Dog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read-out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production. Results Increasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion. Conclusions Altogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell-based therapies for IVD regeneration.
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Affiliation(s)
- Lisanne T Laagland
- Department of Clinical Sciences, Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands
| | - Frances C Bach
- Department of Clinical Sciences, Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands
| | - Laura B Creemers
- Department of Orthopedics University Medical Centre Utrecht Utrecht The Netherlands
| | | | - Deepani W Poramba-Liyanage
- Department of Clinical Sciences, Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands
| | - Marianna A Tryfonidou
- Department of Clinical Sciences, Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands
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Panebianco CJ, Rao S, Hom WW, Meyers JH, Lim TY, Laudier DM, Hecht AC, Weir MD, Weiser JR, Iatridis JC. Genipin-crosslinked fibrin seeded with oxidized alginate microbeads as a novel composite biomaterial strategy for intervertebral disc cell therapy. Biomaterials 2022; 287:121641. [PMID: 35759923 PMCID: PMC9758274 DOI: 10.1016/j.biomaterials.2022.121641] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Revised: 06/10/2022] [Accepted: 06/14/2022] [Indexed: 11/02/2022]
Abstract
Discectomy procedures alleviate disability caused by intervertebral disc (IVD) herniation, but do not repair herniation-induced annulus fibrosus (AF) defects. Cell therapy shows promise for IVD repair, yet cell delivery biomaterials capable of sealing AF defects and restoring biomechanical function have poor biological performance. To balance the biomechanical and biological demands of IVD cell delivery biomaterials, we engineered an injectable composite biomaterial using cell-laden, degradable oxidized alginate (OxAlg) microbeads (MBs) to deliver AF cells within high-modulus genipin-crosslinked fibrin (FibGen) hydrogels (FibGen + MB composites). Conceptually, the high-modulus FibGen would immediately stabilize injured IVDs, while OxAlg MBs would protect and release cells required for long-term healing. We first showed that AF cells microencapsulated in OxAlg MBs maintained high viability and, upon release, displayed phenotypic AF cell morphology and gene expression. Next, we created cell-laden FibGen + MB composites and demonstrated that OxAlg MBs functionalized with RGD peptides (MB-RGD) minimized AF cell apoptosis and retained phenotypic gene expression. Further, we showed that cell-laden FibGen + MB composites are biomechanically stable and promote extracellular matrix (ECM) synthesis in long-term in vitro culture. Lastly, we evaluated cell-laden FibGen + MB-RGD composites in a long-term bovine caudal IVD organ culture bioreactor and found that composites had low herniation risk, provided superior biomechanical and biological repair to discectomy controls, and retained anabolic cells within the IVD injury space. This novel injectable composite hydrogel strategy shows promise as an IVD cell delivery sealant with potentially broad applications for its capacity to balance biomechanical and biological performance.
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Affiliation(s)
- Christopher J Panebianco
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Sanjna Rao
- Department of Chemical Engineering, The Cooper Union for the Advancement of Science and Art, New York, NY, USA
| | - Warren W Hom
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - James H Meyers
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Tiffany Y Lim
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Damien M Laudier
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Andrew C Hecht
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Michael D Weir
- Department of Advanced Oral Sciences and Therapeutics, University of Maryland School of Dentistry, Baltimore, MD, USA
| | - Jennifer R Weiser
- Department of Chemical Engineering, The Cooper Union for the Advancement of Science and Art, New York, NY, USA
| | - James C Iatridis
- Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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Liang W, Han B, Hai Y, Sun D, Yin P. Mechanism of Action of Mesenchymal Stem Cell-Derived Exosomes in the Intervertebral Disc Degeneration Treatment and Bone Repair and Regeneration. Front Cell Dev Biol 2022; 9:833840. [PMID: 35096846 PMCID: PMC8795890 DOI: 10.3389/fcell.2021.833840] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2021] [Accepted: 12/27/2021] [Indexed: 12/19/2022] Open
Abstract
Exosomes are extracellular vesicles formed by various donor cells that regulate gene expression and cellular function in recipient cells. Exosomes derived from mesenchymal stem cells (MSC-Exos) perform the regulatory function of stem cells by transporting proteins, nucleic acids, and lipids. Intervertebral disc degeneration (IDD) is one of the main causes of low back pain, and it is characterized by a decreased number of nucleus pulposus cells, extracellular matrix decomposition, aging of the annulus fibrosus, and cartilage endplate calcification. Besides, nutrient transport and structural repair of intervertebral discs depend on bone and cartilage and are closely related to the state of the bone. Trauma, disease and aging can all cause bone injury. However, there is a lack of effective drugs against IDD and bone injury. Recent MSC-Exos fine tuning has led to significant progress in the IDD treatment and bone repair and regeneration. In this review, we looked at the uniqueness of MSC-Exos, and the potential treatment mechanisms of MSC-Exos with respect to IDD, bone defects and injuries.
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Affiliation(s)
- Weishi Liang
- Department of Orthopedic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China
| | - Bo Han
- Department of Orthopedic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China
| | - Yong Hai
- Department of Orthopedic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China
| | - Duan Sun
- Department of Orthopedic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China
| | - Peng Yin
- Department of Orthopedic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China
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Integrin and syndecan binding peptide-conjugated alginate hydrogel for modulation of nucleus pulposus cell phenotype. Biomaterials 2021; 277:121113. [PMID: 34492582 DOI: 10.1016/j.biomaterials.2021.121113] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 08/17/2021] [Accepted: 08/27/2021] [Indexed: 01/05/2023]
Abstract
Biomaterial based strategies have been widely explored to preserve and restore the juvenile phenotype of cells of the nucleus pulposus (NP) in degenerated intervertebral discs (IVD). With aging and maturation, NP cells lose their ability to produce necessary extracellular matrix and proteoglycans, accelerating disc degeneration. Previous studies have shown that integrin or syndecan binding peptide motifs from laminin can induce NP cells from degenerative human discs to re-express juvenile NP-specific cell phenotype and biosynthetic activity. Here, we engineered alginate hydrogels to present integrin- and syndecan-binding peptides alone or in combination (cyclic RGD and AG73, respectively) to introduce bioactive features into the alginate gels. We demonstrated human NP cells cultured upon and within alginate hydrogels presented with cRGD and AG73 peptides exhibited higher cell viability, biosynthetic activity, and NP-specific protein expression over alginate alone. Moreover, the combination of the two peptide motifs elicited markers of the NP-specific cell phenotype, including N-Cadherin, despite differences in cell morphology and multicellular cluster formation between 2D and 3D cultures. These results represent a promising step toward understanding how distinct adhesive peptides can be combined to guide NP cell fate. In the future, these insights may be useful to rationally design hydrogels for NP cell-transplantation based therapies for IVD degeneration.
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Zhang Q, Shen Y, Zhao S, Jiang Y, Zhou D, Zhang Y. Exosomes miR-15a promotes nucleus pulposus-mesenchymal stem cells chondrogenic differentiation by targeting MMP-3. Cell Signal 2021; 86:110083. [PMID: 34252537 DOI: 10.1016/j.cellsig.2021.110083] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 06/29/2021] [Accepted: 07/07/2021] [Indexed: 12/27/2022]
Abstract
The physiology of the nucleus pulposus (NP) in intervertebral disc degeneration (IVD) has been studied widely. However, interactions involving nucleus pulposus -mesenchymal stem cells (NP-MSCs) are less understood. MicroRNA 15a (miR-15a) is known to target and modulate genes involved in cellular proliferation and apoptosis. This study aimed to understand the interactions and impact of miR-15a and NP-MSCs on chondrogenic differentiation and IVD degeneration. Exosomes secreted by NP cells were purified by differential centrifugation and identified by transmission electron microscopy and exosomal markers. Further, by co-culture these exosomes were re-introduced into the NP-MSC cells, which were confirmed by fluorescence confocal microscopy. NP-MSCs treated with exo-miR-15a increases aggrecan and collagen II mRNA and protein levels while decreasing mRNA and protein levels of ADAMTS4/5 and MMP-3/-13. Toluidine blue staining confirmed that chondrogenic differentiation was increased in NP-MSCs treated with exo-miR-15a. NP-MSCs treated with exo-anti-miR-15a inhibit aggrecan and collagen II expression while increasing ADAMTS4/5 and MMP-3/-13 expression and decreasing chondrogenic differentiation. Dual-luciferase reporter assays revealed that miR-15a directly targets MMP-3 and downregulates its expression. Overexpression of miR-15a increased proliferation and colony formation, whereas combinatorial overexpression with MMP3, suppressed miR-15a's effects. This was also evident through the decreased phosphorylation of PI3K and Akt, upregulation of Wnt3a and β-catenin in the presence of miR-15a, but overexpression of MMP3 indicated an opposite effect. Overall, these data demonstrate that exo-miR-15a promotes NP-MSCs chondrogenic differentiation by downregulating MMP-3 through PI3K/Akt and Wnt3a/β-catenin axis.
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Affiliation(s)
- Qiang Zhang
- Department of Orthopedics, The Affiliated Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213003, China
| | - Yifei Shen
- Department of Orthopedics, The Affiliated Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213003, China
| | - Shujie Zhao
- Department of Orthopedics, The People's Hospital of Jiangsu Province, Nanjing, Jiangsu 210029, China
| | - Yuqing Jiang
- Department of Orthopedics, The Affiliated Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213003, China
| | - Dong Zhou
- Department of Orthopedics, The Affiliated Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213003, China.
| | - Yunkun Zhang
- Department of Orthopedics, The Affiliated Changzhou No. 2 People's Hospital, Nanjing Medical University, Changzhou, Jiangsu 213003, China.
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Guerrero J, Häckel S, Croft AS, Albers CE, Gantenbein B. The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency. JOR Spine 2021; 4:e1131. [PMID: 33778405 PMCID: PMC7984018 DOI: 10.1002/jsp2.1131] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 10/29/2020] [Accepted: 11/04/2020] [Indexed: 02/06/2023] Open
Abstract
INTRODUCTION Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. OBJECTIVE Here, we studied the influence of the culture method and of the microenvironment on the proliferation rate and the differentiation potential of human NP progenitors in vitro. METHOD Cells were obtained from human NP tissue from trauma patients. Briefly, the NP tissue cells were cultured in two-dimensional (2D) (monolayer) or three-dimensional (3D) (alginate beads) conditions. After 1 week, cells from 2D or 3D culture were expanded on fibronectin-coated flasks. Subsequently, expanded NP cells were then characterized by cytometry and tri-lineage differentiation, which was analyzed by qPCR and histology. Moreover, experiments using Tie2+ and Tie2- NP cells were also performed. RESULTS The present study aims to demonstrate that 3D expansion of NP cells better preserves the Tie2+ cell populations and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. Moreover, the cell sorting experiments reveal that only Tie2+ cells were able to maintain the pluripotent gene expression if cultured in 3D within alginate beads. Therefore, our results highly suggest that the maintenance of the cell's multipotency is mainly, but not exclusively, due to the higher presence of Tie2+ cells due to 3D culture. CONCLUSION This project not only might have a scientific impact by evaluating the influence of a two-step expansion protocol on the functionality of NP progenitors, but it could also lead to an innovative clinical approach.
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Affiliation(s)
- Julien Guerrero
- Tissue Engineering for Orthopaedics & Mechanobiology, Department for BioMedical Research (DBMR) of the Faculty of Medicine of the University of BernUniversity of BernSwitzerland
| | - Sonja Häckel
- Department of Orthopaedic Surgery & Traumatology, InselspitalBern University HospitalBernSwitzerland
| | - Andreas S. Croft
- Tissue Engineering for Orthopaedics & Mechanobiology, Department for BioMedical Research (DBMR) of the Faculty of Medicine of the University of BernUniversity of BernSwitzerland
| | - Christoph E. Albers
- Department of Orthopaedic Surgery & Traumatology, InselspitalBern University HospitalBernSwitzerland
| | - Benjamin Gantenbein
- Tissue Engineering for Orthopaedics & Mechanobiology, Department for BioMedical Research (DBMR) of the Faculty of Medicine of the University of BernUniversity of BernSwitzerland
- Department of Orthopaedic Surgery & Traumatology, InselspitalBern University HospitalBernSwitzerland
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Takeoka Y, Kang JD, Mizuno S. In vitro nucleus pulposus tissue model with physicochemical stresses. JOR Spine 2020; 3:e1105. [PMID: 33015578 PMCID: PMC7524234 DOI: 10.1002/jsp2.1105] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Revised: 05/12/2020] [Accepted: 06/04/2020] [Indexed: 12/21/2022] Open
Abstract
Intervertebral discs (IVDs) are exposed to changes in physicochemical stresses including hydrostatic and osmotic pressure via diurnal spinal motion. Homeostasis, degeneration, and regeneration in IVDs have been studied using in vitro, ex vivo, and animal models. However, incubation of nucleus pulposus (NP) cells in medium has limited capability to reproduce anabolic turnover and regeneration under physicochemical stresses. We developed a novel pressure/perfusion cell culture system and a semipermeable membrane pouch device for enclosing isolated NP cells for in vitro incubation under physicochemical stresses. We assessed the performance of this system to identify an appropriate stress loading regimen to promote gene expression and consistent accumulation of extracellular matrices by bovine caudal NP cells. Cyclic hydrostatic pressure (HP) for 4 days followed by constant HP for 3 days in high osmolality (HO; 450 mOsm/kg H2O) showed a trend towards upregulated aggrecan expression and dense accumulation of keratan sulfate without gaps by the NP cells. Furthermore, a repetitive regimen of cyclic HP for 2 days followed by constant HP for 1 day in HO (repeated twice) significantly upregulated gene expression of aggrecan (P < .05) compared to no pressure and suppressed matrix metalloproteinase-13 expression (P < .05) at 6 days. Our culture system and pouches will be useful to reproduce physicochemical stresses in NP cells for simulating anabolic, catabolic, and homeostatic turnover under diurnal spinal motion.
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Affiliation(s)
- Yoshiki Takeoka
- Department of Orthopaedic SurgeryBrigham and Women's Hospital and Harvard Medical SchoolBostonMassachusettsUSA
| | - James D. Kang
- Department of Orthopaedic SurgeryBrigham and Women's Hospital and Harvard Medical SchoolBostonMassachusettsUSA
| | - Shuichi Mizuno
- Department of Orthopaedic SurgeryBrigham and Women's Hospital and Harvard Medical SchoolBostonMassachusettsUSA
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Jaworski LM, Kleinhans KL, Jackson AR. Effects of Oxygen Concentration and Culture Time on Porcine Nucleus Pulposus Cell Metabolism: An in vitro Study. Front Bioeng Biotechnol 2019; 7:64. [PMID: 31001527 PMCID: PMC6454860 DOI: 10.3389/fbioe.2019.00064] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Accepted: 03/07/2019] [Indexed: 01/07/2023] Open
Abstract
Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.
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Affiliation(s)
- Lukas M Jaworski
- Orthopaedic Biomechanics Laboratory, Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
| | - Kelsey L Kleinhans
- Orthopaedic Biomechanics Laboratory, Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
| | - Alicia R Jackson
- Orthopaedic Biomechanics Laboratory, Department of Biomedical Engineering, University of Miami, Coral Gables, FL, United States
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Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro. J Clin Med 2019; 8:jcm8040433. [PMID: 30934902 PMCID: PMC6518239 DOI: 10.3390/jcm8040433] [Citation(s) in RCA: 75] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Accepted: 03/26/2019] [Indexed: 01/21/2023] Open
Abstract
Curcumin and o-Vanillin cleared senescent intervertebral disc (IVD) cells and reduced the senescence-associated secretory phenotype (SASP) associated with inflammation and back pain. Cells from degenerate and non-mildly-degenerate human IVD were obtained from organ donors and from patients undergoing surgery for low back pain. Gene expression of senescence and SASP markers was evaluated by RT-qPCR in isolated cells, and protein expression of senescence, proliferation, and apoptotic markers was evaluated by immunocytochemistry (ICC). The expression levels of SASP factors were evaluated by enzyme-linked immunosorbent assay (ELISA). Matrix synthesis was verified with safranin-O staining and the Dimethyl-Methylene Blue Assay for proteoglycan content. Western blotting and ICC were used to determine the molecular pathways targeted by the drugs. We found a 40% higher level of senescent cells in degenerate compared to non-mildly-degenerate discs from unrelated individuals and a 10% higher level in degenerate compared to non-mildly-degenerate discs from the same individual. Higher levels of senescence were associated with increased SASP. Both drugs cleared senescent cells, and treatment increased the number of proliferating as well as apoptotic cells in cultures from degenerate IVDs. The expression of SASP factors was decreased, and matrix synthesis increased following treatment. These effects were mediated through the Nrf2 and NFkB pathways.
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Inhibitory Effects of Human Primary Intervertebral Disc Cells on Human Primary Osteoblasts in a Co-Culture System. Int J Mol Sci 2018; 19:ijms19041195. [PMID: 29652862 PMCID: PMC5979604 DOI: 10.3390/ijms19041195] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2018] [Revised: 04/10/2018] [Accepted: 04/11/2018] [Indexed: 11/16/2022] Open
Abstract
Spinal fusion is a common surgical procedure to address a range of spinal pathologies, like damaged or degenerated discs. After the removal of the intervertebral disc (IVD), a structural spacer is positioned followed by internal fixation, and fusion of the degenerated segment by natural bone growth. Due to their osteoinductive properties, bone morphogenetic proteins (BMP) are applied to promote spinal fusion. Although spinal fusion is successful in most patients, the rates of non-unions after lumbar spine fusion range from 5% to 35%. Clinical observations and recent studies indicate, that the incomplete removal of disc tissue might lead to failure of spinal fusion. Yet, it is still unknown if a secretion of BMP antagonists in intervertebral disc (IVD) cells could be the reason of inhibition in bone formation. In this study, we co-cultured human primary osteoblasts (OB) and IVD cells i.e., nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC), to test the possible inhibitory effect from IVD cells on OB. Although we could see a trend in lower matrix mineralization in OB co-cultured with IVD cells, results of alkaline phosphatase (ALP) activity and gene expression of major bone genes were inconclusive. However, in NPC, AFC and CEPC beads, an up-regulation of several BMP antagonist genes could be detected. Despite being able to show several indicators for an inhibition of osteoinductive effects due to IVD cells, the reasons for pseudarthrosis after spinal fusion remain unclear.
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Palacio-Mancheno PE, Evashwick-Rogler TW, Laudier DM, Purmessur D, Iatridis JC. Hyperosmolarity induces notochordal cell differentiation with aquaporin3 upregulation and reduced N-cadherin expression. J Orthop Res 2018; 36:788-798. [PMID: 28853179 PMCID: PMC5832547 DOI: 10.1002/jor.23715] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2017] [Accepted: 08/19/2017] [Indexed: 02/04/2023]
Abstract
The nucleus pulposus (NP) of intervertebral discs (IVD) undergoes dramatic changes with aging including loss of its gelatinous structure and large, vacuolated notochordal cells (NCs) in favor of a matrix-rich structure populated by small NP cells (sNPCs). NP maturation also involves a loading-pattern shift from pressurization to matrix deformations, and these events are thought to predispose to degeneration. Little is known of the triggering events and cellular alterations involved with NP maturation, which remains a fundamental open spinal mechanobiology question. A mouse IVD organ culture model was used to test the hypotheses that hyperosmotic overloading will induce NP maturation with transition of NCs to sNPCs while also increasing matrix accumulation and altering osmoregulatory and mechanotransductive proteins. Results indicated that static hyperosmolarity, as might occur during growth, caused maturation of NCs to sNPCs and involved a cellular differentiation process since known NC markers (cytokeratin-8, -19, and sonic hedgehog) persisted without increased cell apoptosis. Osmosensitive channels Aquaporin 3 (Aqp3) and transient receptor potential vanilloid-4 (TRPV4) expression were both modified with altered osmolarity, but increased Aqp3 with hyperosmolarity was associated with NC to sNPC differentiation. NC to sNPC differentiation was accompanied by a shift in cellular mechanotransduction proteins with decreased N-cadherin adhesions and increased Connexin 43 connexons. We conclude that hyperosmotic overloading can promote NC differentiation into sNPCs. This study identified osmolarity as a triggering mechanism for notochordal cell differentiation with associated shifts in osmoregulatory and mechanotransductive proteins that are likely to play important roles in intervertebral disc aging. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:788-798, 2018.
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Affiliation(s)
| | | | - Damien M. Laudier
- Dept. of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY
| | - Devina Purmessur
- Dept. of Biomedical Engineering, Ohio State University, Columbus, OH
| | - James C. Iatridis
- Dept. of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY
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Bach FC, Laagland LT, Grant MP, Creemers LB, Ito K, Meij BP, Mwale F, Tryfonidou MA. Link-N: The missing link towards intervertebral disc repair is species-specific. PLoS One 2017; 12:e0187831. [PMID: 29117254 PMCID: PMC5679057 DOI: 10.1371/journal.pone.0187831] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2017] [Accepted: 10/26/2017] [Indexed: 01/07/2023] Open
Abstract
Introduction Degeneration of the intervertebral disc (IVD) is a frequent cause for back pain in humans and dogs. Link-N stabilizes proteoglycan aggregates in cartilaginous tissues and exerts growth factor-like effects. The human variant of Link-N facilitates IVD regeneration in several species in vitro by inducing Smad1 signaling, but it is not clear whether this is species specific. Dogs with IVD disease could possibly benefit from Link-N treatment, but Link-N has not been tested on canine IVD cells. If Link-N appears to be effective in canines, this would facilitate translation of Link-N into the clinic using the dog as an in vivo large animal model for human IVD degeneration. Materials and methods This study’s objective was to determine the effect of the human and canine variant of Link-N and short (s) Link-N on canine chondrocyte-like cells (CLCs) and compare this to those on already studied species, i.e. human and bovine CLCs. Extracellular matrix (ECM) production was determined by measuring glycosaminoglycan (GAG) content and histological evaluation. Additionally, the micro-aggregates’ DNA content was measured. Phosphorylated (p) Smad1 and -2 levels were determined using ELISA. Results Human (s)Link-N induced GAG deposition in human and bovine CLCs, as expected. In contrast, canine (s)Link-N did not affect ECM production in human CLCs, while it mainly induced collagen type I and II deposition in bovine CLCs. In canine CLCs, both canine and human (s)Link-N induced negligible GAG deposition. Surprisingly, human and canine (s)Link-N did not induce Smad signaling in human and bovine CLCs. Human and canine (s)Link-N only mildly increased pSmad1 and Smad2 levels in canine CLCs. Conclusions Human and canine (s)Link-N exerted species-specific effects on CLCs from early degenerated IVDs. Both variants, however, lacked the potency as canine IVD regeneration agent. While these studies demonstrate the challenges of translational studies in large animal models, (s)Link-N still holds a regenerative potential for humans.
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Affiliation(s)
- Frances C. Bach
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
| | - Lisanne T. Laagland
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
| | - Michael P. Grant
- Department of Surgery, McGill University, Montreal, Canada
- Orthopedic Research Laboratory, Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, Montreal, Canada
| | - Laura B. Creemers
- Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands
| | - Keita Ito
- Department of Orthopedics, University Medical Center Utrecht, Utrecht, the Netherlands
- Orthopedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands
| | - Björn P. Meij
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
| | - Fackson Mwale
- Department of Surgery, McGill University, Montreal, Canada
| | - Marianna A. Tryfonidou
- Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands
- * E-mail:
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Wan S, Borland S, Richardson SM, Merry CL, Saiani A, Gough JE. Self-assembling peptide hydrogel for intervertebral disc tissue engineering. Acta Biomater 2016; 46:29-40. [PMID: 27677593 DOI: 10.1016/j.actbio.2016.09.033] [Citation(s) in RCA: 94] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2016] [Revised: 09/12/2016] [Accepted: 09/23/2016] [Indexed: 12/22/2022]
Abstract
Cell-based therapies for regeneration of intervertebral discs are regarded to hold promise for degenerative disc disease treatment, a condition that is strongly linked to lower back pain. A de novo self-assembling peptide hydrogel (SAPH), chosen for its biocompatibility, tailorable properties and nanofibrous architecture, was investigated as a cell carrier and scaffold for nucleus pulposus (NP) tissue engineering. Oscillatory rheology determined that the system would likely be deliverable via minimally invasive procedure and mechanical properties could be optimised to match the stiffness of the native human NP. After three-dimensional culture of NP cells (NPCs) in the SAPH, upregulation of NP-specific genes (KRT8, KRT18, FOXF1) confirmed that the system could restore the NP phenotype following de-differentiation during monolayer culture. Cell viability was high throughout culture whilst, similarly to NPCs in vivo, the viable cell population remained stable. Finally, the SAPH stimulated time-dependent increases in aggrecan and type II collagen deposition, two important NP extracellular matrix components. Results supported the hypothesis that the SAPH could be used as a cell delivery system and scaffold for the treatment of degenerative disc disease. STATEMENT OF SIGNIFICANCE Lower back pain (LBP) prevalence is widespread due to an aging population and the limited efficacy of current treatments. As LBP is strongly associated with intervertebral disc (IVD) degeneration, it is thought that cell-based therapies could alleviate LBP by repairing IVD tissue. Various natural and synthetic biomaterials have been investigated as potential IVD tissue engineering scaffolds. Self-assembling peptide hydrogels (SAPHs) combine advantages of both natural and synthetic biomaterials; for example they are biocompatible and have easily modifiable properties. The present study demonstrated that a de novo SAPH had comparable strength to the native tissue, was injectable, restored the IVD cell phenotype and stimulated deposition of appropriate matrix components. Results illustrated the promise of SAPHs as scaffolds for IVD tissue engineering.
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Kang YM, Hong SH, Yang JH, Oh JC, Park JO, Lee BH, Lee SY, Kim HS, Lee HM, Moon SH. Pamidronate Down-regulates Tumor Necrosis Factor-alpha Induced Matrix Metalloproteinases Expression in Human Intervertebral Disc Cells. J Bone Metab 2016; 23:165-73. [PMID: 27622181 PMCID: PMC5018610 DOI: 10.11005/jbm.2016.23.3.165] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/24/2016] [Revised: 08/12/2016] [Accepted: 08/12/2016] [Indexed: 01/08/2023] Open
Abstract
Background N-containing bisphosphonates (BPs), such as pamidronate and risedronate, can inhibit osteoclastic function and reduce osteoclast number by inducing apoptotic cell death in osteoclasts. The aim of this study is to demonstrate the effect of pamidronate, second generation nitrogen-containing BPs and to elucidate matrix metallo-proteinases (MMPs) mRNA expression under serum starvation and/or tumor necrosis factor alpha (TNF-α) stimulation on metabolism of intervertebral disc (IVD) cells in vitro. Methods Firstly, to test the effect of pamidronate on IVD cells in vitro, various concentrations (10-12, 10-10, 10-8, and 10-6 M) of pamidronate were administered to IVD cells. Then DNA and proteoglycan synthesis were measured and messenger RNA (mRNA) expressions of type I collagen, type II collagen, and aggrecan were analyzed. Secondly, to elucidate the expression of MMPs mRNA in human IVD cells under the lower serum status, IVD cells were cultivated in full serum or 1% serum. Thirdly, to elucidate the expression of MMPs mRNA in IVD cells under the stimulation of 1% serum and TNF-α (10 ng/mL) In this study, IVD cells were cultivated in three dimensional alginate bead. Results Under the lower serum culture, IVD cells in alginate beads showed upregulation of MMP 2, 3, 9, 13 mRNA. The cells in lower serum and TNF-α also demonstrated upregulation of MMP-2, 3, 9, and 13 mRNA. The cells with various doses of pamidronate and lower serum and TNF-α were reveled partial down-regulation of MMPs. Conclusions Pamidronate, N-containing second generation BPs, was safe in metabolism of IVD in vitro maintaining chondrogenic phenotype and matrix synthesis, and down-regulated TNF-α induced MMPs expression.
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Affiliation(s)
- Young-Mi Kang
- The Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Seong-Hwan Hong
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Jae-Ho Yang
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Jin-Cheol Oh
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Jin-Oh Park
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Byung Ho Lee
- Department of Orthopedic Surgery, Catholic Kwandong University College of Medicine, Incheon, Korea
| | - Sang-Yoon Lee
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Hak-Sun Kim
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Hwan-Mo Lee
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Seong-Hwan Moon
- The Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.; Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
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16
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Melrose J. The knee joint loose body as a source of viable autologous human chondrocytes. Eur J Histochem 2016; 60:2645. [PMID: 27349321 PMCID: PMC4933832 DOI: 10.4081/ejh.2016.2645] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2016] [Revised: 05/05/2016] [Accepted: 05/05/2016] [Indexed: 12/15/2022] Open
Abstract
Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present study we assessed if loose bodies could be used as a source of autologous human chondrocytes for experimental purposes. Histochemical examination of loose bodies and differential enzymatic digestions were undertaken, the isolated cells were cultured in alginate bead microspheres and immunolocalisations were undertaken for chondrogenic markers such as aggrecan, and type II collagen. Isolated loose body cells had high viability (≥90% viable), expressed chondrogenic markers (aggrecan, type II collagen) but no type I collagen. Loose bodies may be a useful source of autologous chondrocytes of high viability.
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Affiliation(s)
- J Melrose
- Royal North Shore Hospital University of Sydney University of NSW.
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17
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Vadalà G, Russo F, Ambrosio L, Loppini M, Denaro V. Stem cells sources for intervertebral disc regeneration. World J Stem Cells 2016; 8:185-201. [PMID: 27247704 PMCID: PMC4877563 DOI: 10.4252/wjsc.v8.i5.185] [Citation(s) in RCA: 73] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/30/2015] [Revised: 12/18/2015] [Accepted: 02/16/2016] [Indexed: 02/06/2023] Open
Abstract
Intervertebral disc regeneration field is rapidly growing since disc disorders represent a major health problem in industrialized countries with very few possible treatments. Indeed, current available therapies are symptomatic, and surgical procedures consist in disc removal and spinal fusion, which is not immune to regardable concerns about possible comorbidities, cost-effectiveness, secondary risks and long-lasting outcomes. This review paper aims to share recent advances in stem cell therapy for the treatment of intervertebral disc degeneration. In literature the potential use of different adult stem cells for intervertebral disc regeneration has already been reported. Bone marrow mesenchymal stromal/stem cells, adipose tissue derived stem cells, synovial stem cells, muscle-derived stem cells, olfactory neural stem cells, induced pluripotent stem cells, hematopoietic stem cells, disc stem cells, and embryonic stem cells have been studied for this purpose either in vitro or in vivo. Moreover, several engineered carriers (e.g., hydrogels), characterized by full biocompatibility and prompt biodegradation, have been designed and combined with different stem cell types in order to optimize the local and controlled delivery of cellular substrates in situ. The paper overviews the literature discussing the current status of our knowledge of the different stem cells types used as a cell-based therapy for disc regeneration.
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18
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Byvaltsev VA, Stepanov IA, Bardonova LA, Belykh EG. [The Use of Stem Cells in the Treatment of Intervertebral Disc Degeneration]. ACTA ACUST UNITED AC 2016; 71:359-66. [PMID: 29297665 DOI: 10.15690/vramn729] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
The paper presents a review of current data on the use of stem cells in the treatment of intervertebral disc degeneration. Acute spinal pain is often a consequence of the pathology affecting the intervertebral disc. Many applied therapeutic techniques do not provide effective results as expected because most of them address symptoms, but do not treat the underlying disease. We have outlined current findings on the molecular mechanisms of intervertebral disc degeneration, analyzed international experimental studies demonstrating the feasibility of a stem cell therapy for intervertebral disc degeneration. The conducted studies reported on the clinical application of mesenchymal stem cells or stem cells derived from adipose, synovium, and bone marrow tissue. The most pressing and undetermined issues that require further experimental and clinical studies are indicated and defined in the article.
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19
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Chan SCW, Tekari A, Benneker LM, Heini PF, Gantenbein B. Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells. Arthritis Res Ther 2015; 18:29. [PMID: 26809343 PMCID: PMC4727301 DOI: 10.1186/s13075-015-0900-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Accepted: 12/14/2015] [Indexed: 02/04/2023] Open
Abstract
Background Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family. Methods Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry. Results Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs. Conclusions We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.
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Affiliation(s)
- Samantha C W Chan
- Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, Bern, CH-3014, Switzerland. .,Biointerfaces, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, St Gallen, CH-9014, Switzerland.
| | - Adel Tekari
- Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, Bern, CH-3014, Switzerland.
| | - Lorin M Benneker
- Department for Orthopedic Surgery and Traumatology, Inselspital, University of Bern, Freiburgstrasse 4, Bern, CH-3010, Switzerland. .,AOSpine Research Network, Stettbachstrasse 6, Dübendorf, CH-8600, Switzerland.
| | - Paul F Heini
- Orthopedic Department, Sonnenhof Clinic, Buchserstrasse 30, Bern, CH-3006, Switzerland.
| | - Benjamin Gantenbein
- Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, Bern, CH-3014, Switzerland. .,AOSpine Research Network, Stettbachstrasse 6, Dübendorf, CH-8600, Switzerland.
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Mavrogonatou E, Papadimitriou K, Urban JP, Papadopoulos V, Kletsas D. Deficiency in the α1 subunit of Na+/K+-ATPase enhances the anti-proliferative effect of high osmolality in nucleus pulposus intervertebral disc cells. J Cell Physiol 2015; 230:3037-48. [PMID: 25967398 DOI: 10.1002/jcp.25040] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2015] [Accepted: 05/05/2015] [Indexed: 12/31/2022]
Abstract
Intervertebral disc cells are constantly exposed to a hyperosmotic environment. Among cellular responses towards this stress is the inhibition of proliferation through the activation of p38 MAPK and p53. In an effort to further elucidate the biochemical pathways triggered by hyperosmotic stress, we assessed the high osmolality-induced transcriptional changes of bovine nucleus pulposus cells using whole-genome arrays. A 5- and a 24-h hyperosmotic treatment led to the differential expression of >100 and >200 genes, respectively, including nine genes encoding transporters (SLC4A11, SLC5A3, ATP1A1, SLC38A2, KCNK17, KCTD20, KCTD11, SLC7A5, and CLCA2). Differences in the transcriptional profile of these selected genes, as indicated by the microarrays experiments, were validated by qRT-PCR in 2D and 3D cell cultures, under hyperosmolar salt and sorbitol conditions, revealing the presence of a common triggering signal for osmotic adaptation. The key signaling molecules p38 MAPK and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters. Finally, siRNA-mediated knocking-down of each one of the three transporters with the highest and sustained over-expression (i.e., SLC4A11, SLC5A3, and ATP1A1) had a distinct outcome on the transcriptional profile of the other transporters, on p38 MAPK and p53 phosphorylation and consequently on cell cycle progression. The inhibition of ATP1A1 had the most prominent effect on the transcription of the rest of the transporters and was found to enhance the anti-proliferative effect of hyperosmotic conditions through an increased G2/M cell cycle block, ascribing to this pump a central role in the osmoregulatory response of nucleus pulposus cells.
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Affiliation(s)
- Eleni Mavrogonatou
- Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", Athens, Greece
| | - Konstantinos Papadimitriou
- Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, Athens, Greece
| | - Jill P Urban
- Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, United Kingdom
| | - Vassilios Papadopoulos
- The Research Institute of the McGill University Health Centre, Department of Medicine, McGill University, Montreal, Quebec, Canada
| | - Dimitris Kletsas
- Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", Athens, Greece
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Sun Y, Lv M, Zhou L, Tam V, Lv F, Chan D, Wang H, Zheng Z, Cheung KMC, Leung VYL. Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation. Osteoarthritis Cartilage 2015; 23:1194-203. [PMID: 25749011 DOI: 10.1016/j.joca.2015.02.166] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2014] [Revised: 01/30/2015] [Accepted: 02/24/2015] [Indexed: 02/02/2023]
Abstract
OBJECTIVE Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation. METHOD Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis. RESULTS Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18, KRT19, and CDH2, as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1, COL3A1, and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture. CONCLUSION We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function.
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Affiliation(s)
- Y Sun
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region
| | - M Lv
- Advanced Technology Research Institution of China Science Institution, Shenzhen, China
| | - L Zhou
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region
| | - V Tam
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region; Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region
| | - F Lv
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region; Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region
| | - D Chan
- Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region
| | - H Wang
- Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China
| | - Z Zheng
- Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangdong, China
| | - K M C Cheung
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region.
| | - V Y L Leung
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong Special Administrative Region.
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Chuah YJ, Lee WC, Wong HK, Kang Y, Hee HT. Three-dimensional development of tensile pre-strained annulus fibrosus cells for tissue regeneration: An in-vitro study. Exp Cell Res 2015; 331:176-182. [DOI: 10.1016/j.yexcr.2014.09.022] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2014] [Revised: 09/16/2014] [Accepted: 09/17/2014] [Indexed: 01/13/2023]
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Gantenbein B, Calandriello E, Wuertz-Kozak K, Benneker LM, Keel MJB, Chan SCW. Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia. BMC Musculoskelet Disord 2014; 15:422. [PMID: 25496082 PMCID: PMC4295479 DOI: 10.1186/1471-2474-15-422] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2014] [Accepted: 12/01/2014] [Indexed: 12/14/2022] Open
Abstract
Background Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). Methods Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. Results We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. Conclusions Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified. Electronic supplementary material The online version of this article (doi:10.1186/1471-2474-15-422) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Benjamin Gantenbein
- Tissue & Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland.
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24
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Omlor GW, Nerlich AG, Tirlapur UK, Urban JP, Guehring T. Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair. Arch Orthop Trauma Surg 2014; 134:1673-81. [PMID: 25348151 DOI: 10.1007/s00402-014-2097-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Indexed: 02/05/2023]
Abstract
INTRODUCTION Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. MATERIALS AND METHODS NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. RESULTS Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. CONCLUSIONS 3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.
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Affiliation(s)
- G W Omlor
- Department of Orthopaedic Surgery and Trauma Surgery, Heidelberg University Hospital, 69118, Heidelberg, Germany
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Wang SZ, Rui YF, Lu J, Wang C. Cell and molecular biology of intervertebral disc degeneration: current understanding and implications for potential therapeutic strategies. Cell Prolif 2014; 47:381-390. [PMID: 25112472 PMCID: PMC6495969 DOI: 10.1111/cpr.12121] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Accepted: 05/09/2014] [Indexed: 12/25/2022] Open
Abstract
Intervertebral disc degeneration (IDD) is a chronic, complex process associated with low back pain; mechanisms of its occurrence have not yet been fully elucidated. Its process is not only accompanied by morphological changes, but also by systematic changes in its histological and biochemical properties. Many cellular and molecular mechanisms have been reported to be related with IDD and to reverse degenerative trends, abnormal conditions of the living cells and altered cell phenotypes would need to be restored. Promising biological therapeutic strategies still rely on injection of active substances, gene therapy and cell transplantation. With advanced study of tissue engineering protocols based on cell therapy, combined use of seeding cells, bio-active substances and bio-compatible materials, are promising for IDD regeneration. Recently reported progenitor cells within discs themselves also hold prospects for future IDD studies. This article describes the background of IDD, current understanding and implications of potential therapeutic strategies.
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Affiliation(s)
- SZ. Wang
- Department of OrthopaedicsZhongda HospitalMedical School of Southeast UniversityNanjingJiangsu210009China
| | - YF. Rui
- Department of OrthopaedicsZhongda HospitalMedical School of Southeast UniversityNanjingJiangsu210009China
- Surgical Research CenterMedical School of Southeast UniversityNanjingJiangsu210009China
| | - J. Lu
- Department of OrthopaedicsZhongda HospitalMedical School of Southeast UniversityNanjingJiangsu210009China
| | - C. Wang
- Department of OrthopaedicsZhongda HospitalMedical School of Southeast UniversityNanjingJiangsu210009China
- Surgical Research CenterMedical School of Southeast UniversityNanjingJiangsu210009China
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Gawri R, Ouellet J, Önnerfjord P, Alkhatib B, Steffen T, Heinegård D, Roughley P, Antoniou J, Mwale F, Haglund L. Link N is cleaved by human annulus fibrosus cells generating a fragment with retained biological activity. J Orthop Res 2014; 32:1189-97. [PMID: 24861010 DOI: 10.1002/jor.22653] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 05/02/2014] [Indexed: 02/04/2023]
Abstract
Presently, there are no established treatments to prevent, stop or even retard back pain arising from disc degeneration. Previous studies have shown that Link N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens, in IVD. However, the sequences in Link N involved in modulating cellular activity are not well understood. To determine if disc cells can proteolytically process Link N, human disc cells were exposed to native Link N over a 48 h period and mass spectrometric analysis revealed that a peptide spanning residues 1-8 was generated in the presence of AF cells but not NP cells. Link N 1-8 significantly induced proteoglycan production in the presence of IL-1β NP and AF cells, confirming that the biological effect is maintained in the first 8 amino acids of the peptide and indicating that the effect is sustained in an inflammatory environment. Thus Link-N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration.
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Affiliation(s)
- Rahul Gawri
- Orthopaedic Research Laboratory, Royal Victoria Hospital, McGill University, Montréal, Canada; Orthopaedic Research Laboratory, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Canada; McGill Scoliosis and Spine Center, McGill University, Montréal, Canada; Department of Surgery, McGill University, Montréal, Canada
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Disc cell therapies: critical issues. 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 2014; 23 Suppl 3:S375-84. [PMID: 24509721 DOI: 10.1007/s00586-014-3177-2] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/31/2012] [Revised: 12/02/2013] [Accepted: 01/08/2014] [Indexed: 12/13/2022]
Abstract
BACKGROUND Disc cell therapies, in which cells are injected into the degenerate disc in order to regenerate the matrix and restore function, appear to be an attractive, minimally invasive method of treatment. Interest in this area has stimulated research into disc cell biology in particular. However, other important issues, some of which are discussed here, need to be considered if cell-based therapies are to be brought to the clinic. PURPOSE Firstly, a question which is barely addressed in the literature, is how to identify patients with 'degenerative disc disease' who would benefit from cell therapy. Pain not disc degeneration is the symptom which drives patients to the clinic. Even though there are associations between back pain and disc degeneration, many people with even severely degenerate discs, with herniated discs or with spinal stenosis, are pain-free. It is not possible using currently available techniques to identify whether disc repair or regeneration would remove symptoms or prevent symptoms from occurring in future. Moreover, the repair process in human discs is very slow (years) because of the low cell density which can be supported nutritionally even in healthy human discs. If repair is necessary for relief of symptoms, questions regarding quality of life and rehabilitation during this long process need consideration. Also, some serious technical issues remain. Finding appropriate cell sources and scaffolds have received most attention, but these are not the only issues determining the feasibility of the procedure. There are questions regarding the safety of implanting cells by injection through the annulus whether the nutrient supply to the disc is sufficient to support implanted cells and whether, if cells are able to survive, conditions in a degenerate human disc will allow them to repair the damaged tissue. CONCLUSIONS If cell therapy for treatment of disc-related disorders is to enter the clinic as a routine treatment, investigations must examine the questions related to patient selection and the feasibility of achieving the desired repair in an acceptable time frame. Few diagnostic tests that examine whether cell therapies are likely to succeed are available at present, but definite exclusion criteria would be evidence of major disc fissures, or disturbance of nutrient pathways as measured by post-contrast MRI.
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Cho H, Park SH, Park K, Shim JW, Huang J, Smith R, Elder S, Min BH, Hasty KA. Construction of a tissue-engineered annulus fibrosus. Artif Organs 2013; 37:E131-8. [PMID: 23621741 DOI: 10.1111/aor.12066] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The intervertebral disc is composed of load-bearing fibrocartilage that may be subjected to compressive forces up to 10 times the body weight. The multilaminated outer layer, the annulus fibrosus (AF), is vulnerable to damage and its regenerative potential is limited, sometimes leading to nuclear herniation. Scaffold-based tissue engineering of AF using stem cell technology has enabled the development of bi-laminate constructs after 10 weeks of culture. It is difficult to know if these constructs are limited by the differentiation state of the stem cells or the culture system. In this study, we have characterized an expandable scaffold-free neoconstruct using autologous AF cells. The construct was prepared from pellet cultures derived from monolayer cultures of AF cells from mature pigs that became embedded in their own extracellular matrix. The pellet cultures were incubated for 24 h in a standardized conical tube and then carefully transferred intact to a culture flask and incubated for 21 days to allow continued matrix synthesis. Cell viability was maintained above 90% throughout the culture period. The engineered scaffold-free construct was compared with the native AF tissue by characterization of gene expression of representative markers, histological architecture, and biochemical composition. The morphological and biochemical characteristics of the cultured disc construct are very similar to that of native AF. The cell number per gram of construct was equal to that of native AF. Expression of aggrecan was elevated in the engineered construct compared with RNA extracted from the AF. The glycosaminoglycan content in the engineered construct showed no significant difference to that from native construct. These data indicate that scaffold-free tissue constructs prepared from AF cells using a pellet-culture format may be useful for in vitro expansion for transplantation into damaged discs.
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Affiliation(s)
- Hongsik Cho
- University of Tennessee Health Science Center, 1030 Jefferson Avenue, Memphis, TN 38138, USA
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Iatridis JC, Nicoll SB, Michalek AJ, Walter BA, Gupta MS. Role of biomechanics in intervertebral disc degeneration and regenerative therapies: what needs repairing in the disc and what are promising biomaterials for its repair? Spine J 2013; 13:243-62. [PMID: 23369494 PMCID: PMC3612376 DOI: 10.1016/j.spinee.2012.12.002] [Citation(s) in RCA: 229] [Impact Index Per Article: 19.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2011] [Revised: 07/26/2012] [Accepted: 12/09/2012] [Indexed: 02/03/2023]
Abstract
BACKGROUND CONTEXT Degeneration and injuries of the intervertebral disc (IVD) result in large alterations in biomechanical behaviors. Repair strategies using biomaterials can be optimized based on the biomechanical and biological requirements of the IVD. PURPOSE To review the present literature on the effects of degeneration, simulated degeneration, and injury on biomechanics of the IVD, with special attention paid to needle puncture injuries, which are a pathway for diagnostics and regenerative therapies and the promising biomaterials for disc repair with a focus on how those biomaterials may promote biomechanical repair. STUDY DESIGN A narrative review to evaluate the role of biomechanics on disc degeneration and regenerative therapies with a focus on what biomechanical properties need to be repaired and how to evaluate and accomplish such repairs using biomaterials. Model systems for the screening of such repair strategies are also briefly described. METHODS Articles were selected from two main PubMed searches using keywords: intervertebral AND biomechanics (1,823 articles) and intervertebral AND biomaterials (361 articles). Additional keywords (injury, needle puncture, nucleus pressurization, biomaterials, hydrogel, sealant, tissue engineering) were used to narrow the articles down to the topics most relevant to this review. RESULTS Degeneration and acute disc injuries have the capacity to influence nucleus pulposus (NP) pressurization and annulus fibrosus (AF) integrity, which are necessary for an effective disc function and, therefore, require repair. Needle injection injuries are of particular clinical relevance with the potential to influence disc biomechanics, cellularity, and metabolism, yet these effects are localized or small and more research is required to evaluate and reduce the potential clinical morbidity using such techniques. NP replacement strategies, such as hydrogels, are required to restore the NP pressurization or the lost volume. AF repair strategies including cross-linked hydrogels, fibrous composites, and sealants offer promise for regenerative therapies to restore AF integrity. Tissue engineered IVD structures, as a single implantable construct, may promote greater tissue integration due to the improved repair capacity of the vertebral bone. CONCLUSIONS IVD height, neutral zone characteristics, and torsional biomechanics are sensitive to specific alterations in the NP pressurization and AF integrity and must be addressed for an effective functional repair. Synthetic and natural biomaterials offer promise for NP replacement, AF repair, as an AF sealant, or whole disc replacement. Meeting mechanical and biological compatibilities are necessary for the efficacy and longevity of the repair.
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Affiliation(s)
- James C. Iatridis
- Leni and Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, New York, NY
| | - Steven B. Nicoll
- Department of Biomedical Engineering, The City College of New York, New York, NY
| | - Arthur J. Michalek
- Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT
| | - Benjamin A. Walter
- Leni and Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, New York, NY,Department of Biomedical Engineering, The City College of New York, New York, NY
| | - Michelle S. Gupta
- Department of Biomedical Engineering, The City College of New York, New York, NY
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Fang Z, Yang Q, Luo W, Li GH, Xiao J, Li F, Xiong W. Differentiation of GFP-Bcl-2-engineered mesenchymal stem cells towards a nucleus pulposus-like phenotype under hypoxia in vitro. Biochem Biophys Res Commun 2013; 432:444-50. [PMID: 23416353 DOI: 10.1016/j.bbrc.2013.01.127] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Accepted: 01/29/2013] [Indexed: 01/08/2023]
Abstract
Differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into a nucleus pulposus-like phenotype under hypoxia has been proposed as a potential therapeutic approach for intervertebral disc degeneration. However, limited cell viability under hypoxic conditions has restricted MSC differentiation capacity and thus restricted its clinical application. In this study, we genetically modified MSCs with an anti-apoptotic GFP-Bcl-2 gene and evaluated cell survival and functional improvement under hypoxia in vitro. Rat bone marrow MSCs were transfected by lentiviral vectors with the GFP-Bcl-2 gene (GFP-Bcl-2-MSCs). Cell proliferation and apoptosis were assessed, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate phenotypic and biosynthetic activities. In addition, Alcian blue staining was used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells. We found that the Bcl-2 gene protected MSCs against apoptosis. We also observed that Bcl-2 over-expression reduced apoptosis by 40.61% in non-transfected MSCs and 38.43% in vector-MSCs to 18.33% in Bcl-2-MSCs. At 3days, the number of viable Bcl-2-MSCs was approximately two times higher than the number of MSCs or vector-MSCs under hypoxic conditions. RT-PCR showed higher expression of chondrocyte-related genes (Sox-9, aggrecan and type II collagen) in GFP-Bcl-2-MSCs cultured under hypoxia. The accumulation of proteoglycans in the pellet was 86% higher in GFP-Bcl-2-MSCs than in the control groups. Furthermore, the ratio of proteoglycans/collagen II in GFP-Bcl-2-MSCs was 6.2-fold higher compared to the MSC and vector-MSC groups, which denoted a nucleus pulposus-like differentiation phenotype. Our findings support the hypothesis that anti-apoptotic gene-modified MSCs can differentiate into cells with a nucleus pulposus-like phenotype in vitro, which may have value for the regeneration of intervertebral discs using cell transplantation therapy.
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Affiliation(s)
- Zhong Fang
- Department of Orthopaedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China
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Ma K, Wu Y, Wang B, Yang S, Wei Y, Shao Z. Effect of a synthetic link N peptide nanofiber scaffold on the matrix deposition of aggrecan and type II collagen in rabbit notochordal cells. JOURNAL OF MATERIALS SCIENCE. MATERIALS IN MEDICINE 2013; 24:405-415. [PMID: 23154913 DOI: 10.1007/s10856-012-4811-3] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2012] [Accepted: 11/01/2012] [Indexed: 06/01/2023]
Abstract
Self-assembling peptide nanofiber scaffolds have been studied extensively as biological materials for 3-dimensional cell culture and repairing tissue defects in animals. However, few studies have applied peptide nanofiber scaffolds in the tissue engineering of intervertebral discs (IVDs). In this study, a novel functionalized peptide scaffold was specifically designed for IVD tissue engineering, and notochordal cells (NCs) as an alternative cell source for IVD degeneration were selected to investigate the bioactive scaffold material. The novel RADA16-Link N self-assembling peptide scaffold material was designed by direct coupling to a bioactive motif link N. The link N nanofiber scaffold (LN-NS) material was obtained by mixing pure RADA16-I and RADA16-Link N (1:1) designer peptide solutions. Although live/dead cell assays showed that LN-NS and RADA16-I scaffold materials were both biocompatible with NCs, the LN-NS material significantly promoted NC adhesion compared with that of the pure RADA16-I SAP scaffold material. The depositions of aggrecan and type II collagen, which are significant markers for IVD cells, were remarkably increased. Furthermore, the results indicated that the link N motif, the matrix analog of the nucleus pulposus, significantly promoted the accumulation of other extracellular matrices in vitro. We conclude that the novel LN-NS material is a promising biological scaffold material, and may have a broad range of applications in IVD tissue engineering.
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Affiliation(s)
- Kaige Ma
- Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
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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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The challenge and advancement of annulus fibrosus tissue engineering. 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 2013; 22:1090-100. [PMID: 23361531 DOI: 10.1007/s00586-013-2663-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/07/2012] [Revised: 12/26/2012] [Accepted: 01/07/2013] [Indexed: 02/07/2023]
Abstract
BACKGROUND Intervertebral disc degeneration, a main cause of back pain, is an endemic problem and a big economic burden for the health care system. Current treatments are symptom relieving but do not address underlying problems-biological and structural deterioration of the disc. Tissue engineering is an emerging approach for the treatment of intervertebral disc degeneration since it restores the functionality of native tissues. Although numerous studies have focused on the nucleus pulposus tissue engineering and achieved successes in laboratory settings, disc tissue engineering without annulus fibrosus for the end stage of disc degeneration is deemed to fail. The purpose of this article is to review the advancement of annulus fibrosus tissue engineering. MATERIAL AND METHODS Relevant articles regarding annulus fibrosus tissue engineering were identified in PubMed and Medline databases. RESULTS The ideal strategy for disc regeneration is to restore the function and integrity of the disc by using biomaterials, native matrices, growth factors, and cells that producing matrices. In the past decades there are tremendous advancement in annulus fibrosus tissue engineering including cell biology, biomaterials, and whole disc replacement. The recent promising results on whole disc tissue engineering-a composite of annulus fibrosus and nucleus pulposus-make the tissue engineering approach more appealing. CONCLUSION Despite the promising results in disc tissue engineering, there is still much work to be done regarding the clinical application.
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Nilsson E, Larsson K, Rydevik B, Brisby H, Hammar I. Evoked thalamic neuronal activity following DRG application of two nucleus pulposus derived cell populations: an experimental study in rats. 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 2013; 22:1113-8. [PMID: 23341046 DOI: 10.1007/s00586-013-2669-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/24/2012] [Revised: 10/18/2012] [Accepted: 01/09/2013] [Indexed: 10/27/2022]
Abstract
PURPOSE To investigate the effects on evoked thalamic neuronal activity of application of notochordal cells and chondrocyte-like cells derived from nucleus pulposus (NP) onto a dorsal root ganglion (DRG) and to compare these effects with a previously reported increased thalamic activity induced by NP. METHODS Nucleus pulposus was harvested from tail discs of adult rats and the disc cells were separated into two cell populations, notochordal cells and chondrocyte-like cells. The two cell populations were applied separately, or in combination, to the L4 DRG of anaesthetised female Sprague-Dawley rats during acute electrophysiological experiments. In control experiments, cell suspension medium was applied on the DRG. Recordings from the contralateral thalamus were sampled for 40 min while electrically stimulating the ipsilateral sciatic nerve at above Aδ-fibre thresholds. RESULTS Application of notochordal cells resulted in a decrease in evoked thalamic activity within 10 min while chondrocyte-like cells did not induce any changes during the 40 min of recording. The difference in evoked thalamic activity 40 min after notochordal and chondrocyte-like cell application, respectively, was statistically significant. Neither an increased concentration of chondrocyte-like cells alone nor a combination of the two cell populations induced any changes in thalamic activity. CONCLUSIONS Separate exposure of the DRG to the two NP-derived cell populations induced different effects on evoked thalamic activity, but none of the tested cell samples induced an increase in neuronal activity similar to that previously observed with NP. This indicates a high complexity of the interaction between NP and nervous tissue.
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Affiliation(s)
- E Nilsson
- Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, PO Box 432, 405 30, Gothenburg, Sweden.
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Guterl CC, See EY, Blanquer SB, Pandit A, Ferguson SJ, Benneker LM, Grijpma DW, Sakai D, Eglin D, Alini M, Iatridis JC, Grad S. Challenges and strategies in the repair of ruptured annulus fibrosus. Eur Cell Mater 2013; 25:1-21. [PMID: 23283636 PMCID: PMC3655691 DOI: 10.22203/ecm.v025a01] [Citation(s) in RCA: 163] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Lumbar discectomy is the surgical procedure most frequently performed for patients suffering from low back pain and sciatica. Disc herniation as a consequence of degenerative or traumatic processes is commonly encountered as the underlying cause for the painful condition. While discectomy provides favourable outcome in a majority of cases, there are conditions where unmet requirements exist in terms of treatment, such as large disc protrusions with minimal disc degeneration; in these cases, the high rate of recurrent disc herniation after discectomy is a prevalent problem. An effective biological annular repair could improve the surgical outcome in patients with contained disc herniations but otherwise minor degenerative changes. An attractive approach is a tissue-engineered implant that will enable/stimulate the repair of the ruptured annulus. The strategy is to develop three-dimensional scaffolds and activate them by seeding cells or by incorporating molecular signals that enable new matrix synthesis at the defect site, while the biomaterial provides immediate closure of the defect and maintains the mechanical properties of the disc. This review is structured into (1) introduction, (2) clinical problems, current treatment options and needs, (3) biomechanical demands, (4) cellular and extracellular components, (5) biomaterials for delivery, scaffolding and support, (6) pre-clinical models for evaluation of newly developed cell- and material-based therapies, and (7) conclusions. This article highlights that an interdisciplinary approach is necessary for successful development of new clinical methods for annulus fibrosus repair. This will benefit from a close collaboration between research groups with expertise in all areas addressed in this review.
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Affiliation(s)
- Clare C. Guterl
- Department of Orthopaedics, Mount Sinai Medical Centre, New York, NY, USA,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Eugene Y. See
- Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Sebastien B.G. Blanquer
- Department of Biomaterials Science and Technology, University of Twente, Enschede, The Netherlands,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Abhay Pandit
- Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Stephen J. Ferguson
- Institute for Biomechanics, ETH Zurich, Zurich, Switzerland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Lorin M. Benneker
- Department of Orthopaedic Surgery, University of Bern, Bern, Switzerland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Dirk W. Grijpma
- Department of Biomaterials Science and Technology, University of Twente, Enschede, The Netherlands,Department of Biomedical Engineering, University Medical Centre Groningen and University of Groningen, Groningen, The Netherlands,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Daisuke Sakai
- Department of Orthopaedic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - David Eglin
- AO Research Institute Davos, Davos, Switzerland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Mauro Alini
- AO Research Institute Davos, Davos, Switzerland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - James C. Iatridis
- Department of Orthopaedics, Mount Sinai Medical Centre, New York, NY, USA,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland
| | - Sibylle Grad
- AO Research Institute Davos, Davos, Switzerland,Collaborative Research Partner Annulus Fibrosus Rupture Program of AO Foundation, Davos, Switzerland,Address for correspondence: Sibylle Grad, PhD, AO Research Institute Davos, Clavadelerstrasse 8, 7270 Davos, Switzerland, Telephone Number: +41 81 414 2480, FAX Number: +41 81 414 2288,
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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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Hiyama A, Arai F, Sakai D, Yokoyama K, Mochida J. The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells. Arthritis Res Ther 2012; 14:R105. [PMID: 22551380 PMCID: PMC3446482 DOI: 10.1186/ar3830] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2011] [Accepted: 05/02/2012] [Indexed: 01/10/2023] Open
Abstract
Introduction The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling. Methods Rat nucleus pulposus cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated β-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells. Results Nucleus pulposus cells exhibited increased β-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells. Conclusions These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.
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Affiliation(s)
- Akihiko Hiyama
- Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
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Vadalà G, Mozetic P, Rainer A, Centola M, Loppini M, Trombetta M, Denaro V. Bioactive electrospun scaffold for annulus fibrosus repair and regeneration. 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:S20-6. [PMID: 22411039 DOI: 10.1007/s00586-012-2235-x] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 02/13/2012] [Accepted: 02/19/2012] [Indexed: 01/07/2023]
Abstract
PURPOSE Annulus fibrosus (AF) tissue engineering is gathering increasing interest for the development of strategies to reduce recurrent disc herniation (DH) rate and to increase the effectiveness of intervertebral disc regeneration strategies. This study evaluates the use of a bioactive microfibrous poly(L-lactide) scaffold releasing Transforming Growth Factor (TGF)-β1 (PLLA/TGF) for the repair and regeneration of damaged AF. METHODS The scaffold was synthesized by electrospinning, with a direct incorporation of TGF-β1 into the polymeric solution, and characterized in terms of morphology and drug release profile. Biological evaluation was performed with bovine AF cells (AFCs) that were cultured on the scaffold up to 3 weeks to quantitatively assess glycosaminoglycans and total collagen production, using bare electrospun PLLA as a control. Histological evaluation was performed to determine the thickness of the deposited neo-ECM. RESULTS Results demonstrated that AFCs cultured on PLLA/TGF deposited a significantly greater amount of glycosaminoglycans and total collagen than the control, with higher neo-ECM thickness. CONCLUSIONS PLLA/TGF scaffold induced an anabolic stimulus on AFCs, mimicking the ECM three-dimensional environment of AF tissue. This bioactive scaffold showed encouraging results that allow envisaging an application for AF tissue engineering strategies and AF repair after discectomy for the prevention of recurrent DH.
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Affiliation(s)
- Gianluca Vadalà
- Department of Orthopaedics and Trauma Surgery, CIR-Center for Integrated Research, Università Campus Bio-Medico di Roma, via Álvaro del Portillo 200, 00128 Rome, Italy.
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Tilwani RK, Bader DL, Chowdhury TT. Biomechanical Conditioning Enhanced Matrix Synthesis in Nucleus Pulposus Cells Cultured in Agarose Constructs with TGFβ. J Funct Biomater 2012; 3:23-36. [PMID: 24956513 PMCID: PMC4031018 DOI: 10.3390/jfb3010023] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2011] [Revised: 12/23/2011] [Accepted: 12/28/2011] [Indexed: 11/24/2022] Open
Abstract
Biomechanical signals play an important role in normal disc metabolism and pathology. For instance, nucleus pulposus (NP) cells will regulate metabolic activities and maintain a balance between the anabolic and catabolic cascades. The former involves factors such as transforming growth factor-β (TGFβ) and mechanical stimuli, both of which are known to regulate matrix production through autocrine and paracrine mechanisms. The present study examined the combined effect of TGFβ and mechanical loading on anabolic activities in NP cells cultured in agarose constructs. Stimulation with TGFβ and dynamic compression reduced nitrite release and increased matrix synthesis and gene expression of aggrecan and collagen type II. The findings from this work has the potential for developing regenerative treatment strategies which could either slow down or stop the degenerative process and/or promote healing mechanisms in the intervertebral disc.
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Affiliation(s)
- Reshma K Tilwani
- School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
| | - Dan L Bader
- School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
| | - Tina T Chowdhury
- School of Engineering and Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
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Leung VYL, Tam V, Chan D, Chan BP, Cheung KMC. Tissue engineering for intervertebral disk degeneration. Orthop Clin North Am 2011; 42:575-83, ix. [PMID: 21944593 DOI: 10.1016/j.ocl.2011.07.003] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Many challenges confront intervertebral disk engineering owing to complexity and the presence of extraordinary stresses. Rebuilding a disk of native function could be useful for removal of the symptoms and correction of altered spine kinematics. Improvement in understanding of disk properties and techniques for disk engineering brings promise to the fabrication of a functional motion segment for the treatment of disk degeneration. Increasing sophistication of techniques available in biomedical sciences will bring its application into clinics. This review provides an account of current progress and challenges of intervertebral disk bioengineering and discusses means to move forward and toward bedside translation.
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Affiliation(s)
- Victor Y L Leung
- Department of Orthopaedics & Traumatology, The University of Hong Kong, Hong Kong SAR, China
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Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011; 11:947-60. [PMID: 21843975 DOI: 10.1016/j.spinee.2011.07.004] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/24/2010] [Revised: 05/17/2011] [Accepted: 07/05/2011] [Indexed: 02/03/2023]
Abstract
BACKGROUND Degenerative disc disease poses an increasing threat to our quality of life as we age. Existing treatments have limitations. New treatment modalities focusing on biologic rather than surgical approach would be appealing. PURPOSE Culturing intervertebral disc cells in a three-dimensional (3D) model that can retain cellular characteristics and phenotype is a critical step toward understanding how the disc cells respond to and interact with extrinsic signals before better therapeutics can be derived. STUDY DESIGN In this work, we studied the culture of rabbit nucleus pulposus (NP) cells in a collagen microsphere system and compared their cell morphology and expression of a few potential phenotypic markers with that in monolayer culture. METHODS Specifically, rabbit NP cells isolated from both young and old animals were encapsulated and cultured in collagen microspheres with different monomeric concentrations and with different cell encapsulation density for different period of time. Evaluation on the growth kinetics, the viability, the cell morphology, the expression of Types I and II collagen, glycosaminoglycans (GAGs), and Keratin 19, and the ultrastructure of the fiber meshwork were conducted to compare the microsphere 3D culture system and the traditional monolayer cultures. RESULTS Nucleus pulposus cells in two-dimensional culture lost the phenotypic expression of Type II collagen and keratin 19 and expressed Type I collagen. In contrast, the 3D collagen microsphere culture system consistently outperformed the traditional monolayer culture in maintaining a round morphology and preserving the phenotypes of NP cells with persistent expression of Type II collagen and Keratin 19. These cells also remodeled the template collagen matrix in the microspheres by depositing new matrices, including collagen Type II and GAGs in a cell seeding density and collagen concentration dependent manner. CONCLUSIONS This study demonstrates the appeal of the 3D collagen microsphere system for NP cell culture over traditional monolayer culture because it preserves the phenotypic characteristics of NP cells. This system also enables the NP cells to remodel the template collagen matrix by depositing new matrices, suggesting an innovative way to reconstitute cell-specific and native tissue-like environment in vitro for future studies on stem cell matrix niche and interactions of NP cell with extrinsic factors.
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Affiliation(s)
- Minting Yuan
- Tissue Engineering Laboratory, Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Rd, Hong Kong Special Administrative Region, China
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The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study. 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:S819-25. [PMID: 21953383 DOI: 10.1007/s00586-011-2026-9] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/07/2011] [Accepted: 09/08/2011] [Indexed: 10/17/2022]
Abstract
INTRODUCTION Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime. MATERIALS AND METHODS Here, we addressed the importance of cell ratio using alginate bead 3-D co-culture of bovine nucleus pulposus cells (bNPC) and porcine notochordal cells (pNCs) for 14 days using culture inserts. RESULT We found a significant stimulation of bNPC in the presence of pNC in terms of cell activity and glycosaminoglycan production, but not for proliferation (DNA content). Relative gene expression was significantly stimulated for collagen type 2 and aggrecan. CONCLUSION The stimulating effect of NC was confirmed and the ideal ratio of NPC: NC was found to be ~50:50. This has direct implications for tissue-engineering approaches, which aim to repopulate discs with NP-like precursor cells.
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Luo W, Xiong W, Qiu M, Lv Y, Li Y, Li F. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro. ACTA ACUST UNITED AC 2011; 31:199. [DOI: 10.1007/s11596-011-0252-3] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2010] [Indexed: 01/15/2023]
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Effects of intervertebral disc cells on neurite outgrowth from dorsal root ganglion explants in culture. Spine (Phila Pa 1976) 2011; 36:600-6. [PMID: 21124261 DOI: 10.1097/brs.0b013e3181d8bca7] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
STUDY DESIGN An experimental study investigating the effect of disc cells on neurite outgrowth in a rat dorsal root ganglion (DRG) culture system. OBJECTIVE To examine the effects of the 2 nucleus pulposus (NP) cell populations, notochordal cells (NC) and chondrocyte-like cells (CC) on neurite outgrowth from DRGs. SUMMARY OF BACKGROUND DATA NP consists of at least 2 cell populations, NC and CC. The cells in NP have been shown to be responsible for negative effects on neurite outgrowth in vitro and on nerve tissue in vivo. It is unknown whether 1 cell type or combinations of the 2 cell types are responsible for the reported effects. METHODS A total of 939 DRGs from newborn Sprague Dawley rats were harvested and placed in culture dishes. After 24 hours, the neurite outgrowth was measured. NP was harvested from tail discs of adult rats and the NP cells were separated into 2 populations, NC and CC. The cell populations were applied to the DRG culture in different cell concentrations and combinations, and compared to medium. After 24 hours of exposure, the neurite outgrowth was reassessed and expressed as the ratio between the outgrowth at 48 and 24 hours culture. RESULTS NC in intermediate and high concentration and CC in high concentration induced a significant inhibition of the neurite outgrowth compared to culture medium. Further, one of the combinations (low NC and high CC concentration) resulted in a significant inhibition of the neurite outgrowth. CONCLUSION The present study demonstrated negative effects of NP cells on nerve tissue culture explants. The combination of low NC and high CC concentrations may mimic the situation in humans, where we have an increased proportion of chondrocyte-like cells with age. The results from this study may provide a biologic explanation for the large variation of symptoms in disc herniation patients despite similar mechanical influence on nerve tissue.
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Abstract
STUDY DESIGN the response of cells from the annulus fibrosus (AF) and nucleus pulposus (NP) to varying oxygen (O2) concentrations was examined when cultured in alginate. OBJECTIVE to study the effect of O2 concentration on AF and NP cells. SUMMARY OF BACKGROUND DATA AF and NP cells possess different metabolic profiles in situ. However, it is not clear whether this difference is maintained in in vitro culture conditions. AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply and O2 levels. In vivo, O2 levels vary from 1% to 5% within the intervertebral disc, and there is evidence that disc cell metabolism can vary with O2 concentrations. METHODS an alginate scaffold was seeded with bovine AF or NP cells and maintained in culture for up to 18 days under different O2 concentrations. The sulfated glycosaminoglycan (GAG) content in the culture medium and the expression of aggrecan, type I (COL1A2) and II (COL2A1) collagen genes were analyzed at day 9 and day 18. RESULTS in both NP and AF cells cultured either in normoxia (21% O2) or in hypoxia (5% and 1% O2), the GAG content of the culture medium increased with time, though the rate of increase was diminished in 5% O2. With a decrease in O2 levels, the expression of aggrecan mRNA increased in NP cells. There was little effect of O2 on aggrecan mRNA level in AF cells. However, there was a slight decrease with time. Interestingly, aggrecan mRNA levels did not reflect GAG release for either NP or AF cells. There was no effect with time or O2 levels on COL2A1 message in NP cells. The highest Aggrecan/COL2 message ratio for NP cells was with 1% O2, suggesting this to be the best condition for maintaining the NP phenotype. COL1A2 gene expression in NP and AF cells increased with time, but showed little change with O2 levels in NP cells. The highest COL2/COL1 ratio in NP cells was also observed with 1% O2. Finally, NP cells tended to remain localized in the alginate beads, whereas AF cells tended to migrate from the beads. CONCLUSION both NP and AF cells showed little change in GAG production with O2 levels ranging from 1% to 21%. Disc cell metabolism is not impaired at low O2 concentrations, which appear beneficial to matrix composition. Furthermore, low oxygen may promote a gelatinous NP matrix, whereas increased oxygen levels may promote a fibrous matrix.
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Lee HM, Kwon UH, Kim H, Kim HJ, Kim B, Park JO, Moon ES, Moon SH. Pulsed electromagnetic field stimulates cellular proliferation in human intervertebral disc cells. Yonsei Med J 2010; 51:954-9. [PMID: 20879066 PMCID: PMC2995961 DOI: 10.3349/ymj.2010.51.6.954] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
PURPOSE The purpose of this study is to investigate the mechanism of cellular proliferation of electromagnetic field (EMF) on human intervertebral disc (IVD) cells. MATERIALS AND METHODS Human IVD cells were cultured three-dimensionally in alginate beads. EMF was exposed to IVD cells with 650 Ω, 1.8 millitesla magnetic flux density, 60 Hz sinusoidal wave. Cultures were divided into a control and EMF group. Cytotoxicity, DNA synthesis and proteoglycan synthesis were measured by MTT assay, [(3)H]-thymidine, and [(35)S]-sulfate incorporation. To detect phenotypical expression, reverse transcription-polymerase chain reactions (RT-PCR) were performed for aggrecan, collagen type I, and type II mRNA expression. To assess action mechanism of EMF, IVD cells were exposed to EMF with N(G)-Monomethyl-L-arginine (NMMA) and acetylsalicylic acid (ASA). RESULTS There was no cytotoxicity in IVD cells with the EMF group in MTT assay. Cellular proliferation was observed in the EMF group (p < 0.05). There was no difference in newly synthesized proteoglycan normalized by DNA synthesis between the EMF group and the control. Cultures with EMF showed no significant change in the expression of aggrecan, type I, and type II collagen mRNA compared to the control group. Cultures with NMMA (blocker of nitric oxide) or ASA (blocker of prostaglandin E2) exposed to EMF demonstrated decreased DNA synthesis compared to control cultures without NMMA or ASA (p < 0.05). CONCLUSION EMF stimulated DNA synthesis in human IVD cells while no significant effect on proteoglycan synthesis and chondrogenic phenotype expressions. DNA synthesis was partially mediated by nitric oxide and prostaglandin E2. EMF can be utilized to stimulate proliferation of IVD cells, which may provide efficient cell amplification in cell therapy to degenerative disc disease.
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Affiliation(s)
- Hwan-Mo Lee
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Un-Hye Kwon
- Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Hyang Kim
- Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Ho-Joong Kim
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Boram Kim
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Jin-Oh Park
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Eun-Soo Moon
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
| | - Seong-Hwan Moon
- Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea
- Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
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Johnson J, Da Costa R, Allen M. Micromorphometry and Cellular Characteristics of the Canine Cervical Intervertebral Discs. J Vet Intern Med 2010; 24:1343-9. [DOI: 10.1111/j.1939-1676.2010.0613.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
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Bowles RD, Williams RM, Zipfel WR, Bonassar LJ. Self-assembly of aligned tissue-engineered annulus fibrosus and intervertebral disc composite via collagen gel contraction. Tissue Eng Part A 2010; 16:1339-48. [PMID: 19905878 DOI: 10.1089/ten.tea.2009.0442] [Citation(s) in RCA: 118] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Many cartilaginous tissues such as intervertebral disc (IVD) display a heterogeneous collagen microstructure that results in mechanical anisotropy. These structures are responsible for mechanical function of the tissue and regulate cellular interactions and metabolic responses of cells embedded within these tissues. Using collagen gels seeded with ovine annulus fibrosus cells, constructs of varying structure and heterogeneity were created to mimic the circumferential alignment of the IVD. Alignment was induced within gels by contracting annular gels around an inner boundary using both a polyethylene center and alginate center to create a composite engineered IVD. Collagen alignment and heterogeneity were measured using second harmonic generation microscopy. Decreasing initial collagen density from 2.5 mg/mL to 1 mg/mL produced greater contraction of constructs, resulting in gels that were 55% and 6.2% of the original area after culture, respectively. As a result, more alignment occurred in annular-shaped 1 mg/mL gels compared with 2.5 mg/mL gels (p < 0.05). This alignment was also produced in a composite-engineered IVD with alginate nucleus pulposus. The resulting collagen alignment could promote further aligned collagen development necessary for the creation of a mechanically functional tissue-engineered IVD.
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Affiliation(s)
- Robby D Bowles
- Department of Biomedical Engineering, Cornell University , Ithaca, NY, USA
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Differential gene expression profiling of metalloproteinases and their inhibitors: a comparison between bovine intervertebral disc nucleus pulposus cells and articular chondrocytes. Spine (Phila Pa 1976) 2010; 35:1101-8. [PMID: 20473119 DOI: 10.1097/brs.0b013e3181c0c727] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
STUDY DESIGN A comparative in vitro metalloproteinases and their inhibitors gene expression profile. OBJECTIVE To obtain a complete expression profile of matrix metalloproteinases (MMPs), family of proteases with a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS), and tissue inhibitors of metalloproteinases (TIMPs) in bovine adult nucleus pulposus (NP) cells and to compare this profile with the expression profile obtained from bovine adult articular chondrocytes cultured under identical conditions. SUMMARY OF BACKGROUND DATA The cells of the NP resemble articular chondrocytes morphologically but produce a matrix which, though consisting of similar components, has very different biomechanical properties. No specific markers for NP cells have yet been identified; they can be distinguished from chondrocytes only by differences in gene expression. Here we compare profiles of gene expression of metalloproteinases and their inhibitors between NP cells and chondrocytes to improve understanding of the differences between these cell types. METHODS NP cells and articular chondrocytes were harvested respectively from bovine caudal discs and the articular cartilage of metacarpal-phalangeal joints of 18- to 24-month-old steers. These cells were cultured under identical conditions for 96 hours in alginate beads. Expression levels of MMPs, ADAMTSs, and TIMPs were detected by real-time RT-PCR. RESULTS Gene profiling demonstrated distinct differences between levels of MMPs, ADAMTSs, and TIMPs produced by chondrocytes and NP cells. In particular, NP cells expressed considerably more MMP-2 and MMP-14 than chondrocytes, and expression of ADAMTS-1,-2,-17 and TIMP-1 was also higher. However, expression of MMP-1,-3,-7,-8,-10,-11,-13,-16,-19,-20,-21,-23,-24,-28, ADAMTS-4,-5,-6,-14,-18,-19, and TIMP-3 was lower in NP cells than in chondrocytes. Chondrocytes but not NP cells expressed MMP12 and MMP27; this difference is a potential marker for distinguishing between NP cells and chondrocytes. CONCLUSION Because culture conditions and animal age were identical, differences in metalloproteinase and inhibitor expression between NP cells and chondrocytes were intrinsic to cell phenotype and not induced by differences in the in situ extracellular environment.
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Abstract
STUDY DESIGN Micromass culture was assessed as a cell culture microenvironment for anulus cells from the human intervertebral disc. OBJECTIVE To determine whether the micromass culture technique might be useful for the culture of human anulus cells. SUMMARY OF BACKGROUND DATA Culture of cells in micromass has been traditionally used as a method to culture chondrocytes in a three-dimensional (3D) microenvironment with specialized chondrocyte media which allows expression of the chondrocytic phenotype. Recently it has also been used for disc cell 3D culture. METHODS Following approval of our human subjects Institutional Review Board, cells isolated from human anulus intervertebral disc tissue was cultured in micromass culture under control conditions or with addition of 5 ng/mL transforming growth factor-beta (TGF-beta). Cultures were grown for 7 days, and then analyzed for morphology with light microscopy, for extracellular matrix (ECM) production with transmission electron microscopy and quantitative measurement of total sulfated proteoglycan production. Immunohistochemistry was also performed to assess types I and II collagen, decorin, keratan sulfate, and chondroitin sulfate content of ECM. RESULTS Human anulus cells form multilayered colonies when cultured with minimal media and 20% fetal bovine serum in the micromass methodology. Stimulation of ECM production occurs when 5 ng/mL TGF-beta was added to the micromass media. TGF-beta also significantly increased the production of sulfated proteoglycans (P = 0.026). Under both control and TGF-beta-supplementation, the resulting micromass formed by anulus cells is not as compact as the micromass which results when stem cells cultured in chondrogenic media. Ultrastructural studies showed the presence of apoptotic cells and the presence of peroxisomes within cells. Immunohistochemical studies on production of type I collagen, decorin and keratan sulfate showed that there was localized production of these ECM components in focal regions; chondroitin sulfate and type II collagen, however, showed a more uniform overall production by cells within the micromass. CONCLUSION Human anulus cells were successfully cultured under micromass conditions in nonchondrogenic media and with TGF-beta supplementation which increased ECM production. The resulting anulus cell micromass, however, was not as rounded or compact as that which occurs with routine chondrocyte micromass or stem cells induced into chondrocyte differentiation. The presence of peroxisomes noted on ultrastructural studies may reflect cell stress or uneven distribution of nutrition within the micromass during the 7-day micromass culture period. Immunohistochemical studies showed nonuniform ECM gene expression and production within the micromass, suggesting variable gene expression patterns with this culture method.
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