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Intervertebral Discs and Their Interactions with Different Environments Jin Yoon Introduction Back pain and joint pain from cartilage degeneration are major problems in the world. More than 80 percent of adult population sufers from back pain at some time in their lives [1]. Majority of the pain is due to degeneration of the cartilage that forms intervertebral discs (IVD). IVD’s are located between vertebral bodies and serve three major functions: acting as a ligament to hold the vertebrae of the spine together, absorbing shock, and enabling the spine to rotate and bend [2]. hey can wear down from overuse, injury and aging. Because of the pain’s debilitating efects, there have been many attempts to ix the problem, without much success. One method to ix the problem is to replace the damaged IVD, but IVD’s have numerous functions and complex properties that are diicult to imitate. A replacement tissue must be able to distribute the load evenly, resist compression, have viscoelastic property, and provide smooth surface for pivoting. If the replacement tissue cannot fulill even one of these properties, the patient cannot function fully. Many have turned to fusing vertebrae together with prosthesis, while other patients, with smaller lesions, have attempted to regenerate the cartilage. here are a few ways people have employed to restore damaged cartilages. A popular restoration method is by implanting replacement tissue grafts. he patient can either replace the damaged cartilage with small sections from a less weight bearing joint or with a full allograft. his replacement treatment has shown to decrease pain in 70 percent of the patients for two to ive years [2]; however there are few problems associated with this treatment. he replaced cartilage does not last long, so the patient must replace it continuously to avoid pain. Furthermore, allografts often induce immune response, which can be dangerous. A big concern in the U.S. is that these tissue grafts can break down and cause osteolysis [2]. To eliminate the need for donor sites, many have tried to heal or regenerate existing cartilage through natural processes. hese processes focused on either enhancing the environment for regeneration or transplanting chondrocytes to form more tissue. hese techniques have not shown completely successful results, and more so in older populations [1]. he reason for such poor results in regeneration methods is because cartilage is an avascular tissue, which means that nutrients transport and waste removal are much more complicated processes that rely solely on difusion. Another common treatment is by exciting the cartilage
through physical, energy, or pharmacological stimulation. Physical stimulation involves penetrating the subchondral bone through abrasion or drilling. he stimulation creates a full-thickness defect [1] which causes a clot to form and provides a scafold that allows mesenchymal stem cells (MSC) to migrate. Even though this treatment is very common, the results have been mixed, due to random differentiation of MSC into diferent cartilage cells or, sometimes, not even a cartilage cell. Moreover, the new tissue has mechanical properties and durability that are less than that of the original tissue. Both energy stimulation and pharmacological stimulations have also shown ambiguous results, requiring further research. An important aspect of further research that is required to engineer a satisfactory functional scafold or tissue is to understand how a cell knows and interacts with matrix. We currently know that cells react to its environment through physical senses, or phenotypic responses. Knowing the speciic interactions of IVD cells with matrix components could yield more information on the degenerative process and provide novel methods for repair.
Structure of IVDs IVD is a cartilage and a joint that allows lexible motion and absorbs shock. It holds the vertebrae together and limits excessive motion. An IVD is composed of 3 main parts: nucleus pulposus (NP), annulus ibrosus (AF), and vertebral endplate (VEP). NP is a gelatinous structure that contains hydrophilic proteoglycan (PG) and glycosaminoglycan (GAG) chains. hese chains are negatively charged, and they maintain a large amount of water in the IVD. Main role of NP is to support the load and distribute the weight evenly. AF is a concentric, multilayered structure with a regular pattern of collagen type I ibers. AF surrounds NP and supports it by preventing NP from deforming when compressed. VEP is positioned on top and bottom of each IVD. It allows nutrients and waste to travel across it by difusion. Because lower IVDs are so big and the rate of difusion for nutrients is slow, lower IVDs are at much greater risk of degeneration.
Cell-substrate interaction Usually, tissue cells need to adhere to a solid to be viable; hence, they are called anchorage dependent. Yet, we do not know how tissue cells are able to distinguish the stifness of diferent matrices. It is hypothesized that cells anchor and pull on their surroundings to determine the stifness [3]. hese processes partly rely on myosin-based Volume 2 | 2012-2013 | 63
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Figure 1. MRI of IVD showing NP and AF in distinct regions (left). Schematic of spinal column (middle). Anatomy of normal disc with histological stain (right) [2]. contractility and transcellular adhesions to apply forces to substrates. However, tissue cells not only apply forces, but also respond to the resistance by the substrate through cytoskeleton organization [3]. It has been found that cellcell contact promotes the cells to have indistinguishable morphologies, while cells on stif surface difer in their spreading and cytoskeletal organization. his research is done in order to try to understand how cells know to exert greater contractile traction forces on stifer substrates.
Cell-matrix interaction It is crucial for proper interaction to exist between a cell and its extracellular matrix (ECM) because the interaction is a key factor in regulating cell survival, diferentiation, and response to environmental stimuli [4]. Integrin receptors on cell surface link cells to their ECM and are responsible for aforementioned functions. It was found that stained NP cells tested highly positive for laminin, while AF cells had minimal attachment to laminin. his signiies that NP cells readily attach to laminin substrates. In mesenchymal stem cells (MSC), it has been found that the elasticity of the matrix can specify lineage of the cells [7]. Using crosslinking of collagen-I, tissues with a range of matrix elasticity values were created. Elasticity is measured by elastic constant, E, which is the resistance that a cell feels when it deforms the ECM. It was found that MSCs on soft substrates (E of 0.1 -1 kPa) branched and spread, and their branching density approached those of primary neurons under similar conditions. MSCs on stifer substrates (E of 8-17 kPa) became spindle-shaped similar to myoblasts. Finally, on very stif substrates (E of 25-40 kPa), which mimic the crosslinked collagen of osteoids, MSCs’ morphology was similar to that of osteoblasts. Besides the elasticity of the matrix, it was found that non64 | 2012-2013 | Volume 2
muscle myosin II (NMM II)—which is predicted to exert force on the substrate through focal adhesions to sense the matrix elasticity—plays a role in lineage speciication. he researchers have found that when MSCs are treated with blebbistatin, they are prevented from diferentiating. Blebbistatin is a selective and potent myosin inhibitor that inhibits actin activation of NMM II ATPase activity and blocks migration and cytokinesis in vertebrate cells [7]. Adding blebbistatin during plating MSCs can prevent the cells from branching or spreading; however, if bleb is added 24 hours after plating of the MSCs, no signiicant changes are observed. From this research, it is clear that matrix elasticity needs to be optimized for regeneration.
Cellular responses to load IVD cells are made to withstand pressure. However, it has been found that these cells respond diferently based on the type, duration and magnitude of the load [6]. Diferent loads can cause IVD cells to exhibit either anabolic or catabolic responses. Usually, low to moderate magnitudes of compression or pressure causes an increase in anabolic cell responses; contrarily, high magnitude increases catabolic cell responses. It has been observed that a range of magnitudes or frequency exists for each condition in a cell type that promotes biosynthesis. his means that there is a physiological range of stimuli that can promote maximum biosynthesis and cell-mediated repair. Furthermore, inner AF and NP showed similar responses to diferent loads, while outer AF showed a diferent response; having similar responses suggest that they experience similar stimuli and may respond similarly than cells of the outer AF.
Mechanosensing A study aligning with our goal has been previously
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Conclusion here have been many attempts to correct damaged IVDs, but they have all had drawbacks. If we understand how and why IVD’s become degenerated, we can come up with a solution. So to understand the process behind regeneration, researchers have made investigations as to how cells behave and react given an outside force, but many more interactions to be researched. Furthermore, we are looking into ways in which cadherins can change
Figure 2. Neonatal ventricular rat myocytes plated on gels of varying stifness (A-F). G (top of page) is a comparative bar graph of cell-spreading area on extracellular matrices of varying stifness [8]. Volume 2 | 2012-2013 | 65
Street Broad Scientific downstream signaling cascades. Cadherins are important because these protein molecules help cells adhere to one another. Without cadherins, cells would not be able to function together and accomplish their goals. At this point, we need to understand how the changes in the substrate can afect cell responses. Elucidating this process can help us ind the mechanical signaling targets and perhaps reverse the degeneration of IVD cells.
References [1] Johnna S Temenof, Antonios G Mikos, Review: tissue engineering for regeneration of articular cartilage, Biomaterials, Volume 21, Issue 5, March 2000, Pages 431-440, ISSN 0142-9612, 10.1016/S0142-9612(99)00213-6. [2] Benjamin R. Whatley, Xuejun Wen, Intervertebral disc (IVD): Structure, degeneration, repair and regeneration, Materials Science and Engineering: C, Volume 32, Issue 2, 1 March 2012, Pages 61-77, ISSN 0928-4931, 10.1016/j.msec.2011.10.011. [3] Discher, Dennis E, Paul Janmey, and Yu-Li Wang. “Tissue cells feel and respond to the stifness of their substrate.” Science 310.5751 (2005) : 1139-1143. [4] Gilchrist, C. L., et al. “Functional Integrin Subunits Regulating Cell-Matrix Interactions in the Intervertebral Disc.” J Orthop Res 25.6 (2007): 829-40. NLM. [5] C.L. Gilchrist, A.T. Francisco, G.E. Plopper, J. Chen, L.A. Setton. Eur Cell Mater. Author manuscript; available in PMC 2012 April 22. Published in inal edited form as: Eur Cell Mater. 2011 June 20; 21: 523–532. [6] Setton LA, Chen J, 2004, Intervertebral disc cell mechanics and biological responses to load. Current Opinion in Orthopedics. 15(5):331-340, October 2004. [7] Adam J. Engler, Shamik Sen, H. Lee Sweeney, Dennis E. Discher. Matrix elasticity directs stem cell lineage speciication. Cell. 2006 August 25; 126(4): 677–689. doi: 10.1016/j.cell.2006.06.044. [8] Chopra A, Tabdanov E, Patel H, Janmey PA, Kresh JY. Cardiac myocyte remodeling mediated by N-cadherindependent mechanosensing. Am J Physiol Heart Circ Physiol. 2011 April. 200(4):H1252-66.
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