The Human Intervertebral Disc Developmental, Anatomic and Physiologic Considerations for Potential Regenerative Therapies Benjamin D. Levy, MD, FAAPMR Interventional Pain Management Ambulatory Care Service U.S. Department of Veterans Affairs VA New Jersey Health Care System Topics of Discussion Anatomy Cellular and Molecular Biology Pathophysiology Implications for Regenerative Therapies Financial Disclosures None Happily employed by the United States Federal Government Disc Anatomy 30° angle-ply architecture1 Disease Models & Mechanisms 4, 31-41 (2011) Disc Embryology Notochord: • Mesoderm-derived involved in cell signaling and differentiation2 • Becomes nucleus pulposus Somites: • Blocks of mesoderm flanking the notochord paraxially • Cells of somites become sclerotome • Sclerotomes become alternating more & less condensed2: More condensed: Around notorchord to become annulus Less condensed: Become vertebral bodies From Orthop Clin N Am 42 (2011) 447–464 Disc Genetic Factors Key Developmental Genes: • Sox (5,6,9): Chondrogenesis. Critical to collagen II, inner annulus and matrix formation.2,3 Reduced Sox9 expression correlated with degenerative changes. • TGFβ: Regulates cell proliferation and matrix production. In murine model, remains active at maturity4 Cellular Biology Nucleus Pulposus • Cells very similar to notochord cells at birth.1 Large with vacuoles containing glycosaminoglycans. By 10 years of age, notochordal cells disappear. In other species, connote disc repair6 • NP cells: Appear similar to chondrocytes. Humans are termed “chondrodystrophoid”6 Aggrecan and some Collagen Type II production1,2 Express FasL, which induces apoptosis of any cell with Fas receptor7: • T-cells7 • Nucleus pulposus cells8 Cellular Biology Annulus Fibrosus • Outer annulus fibroblastic cells1,2 Collagen type I (like tendon) • Inner annulus chondrocyte-like cells1,2 Collagen type II (like hyaline cartilage, eye vitreus) Molecular Biology Main molecules in nucleus: • Aggrecan: Large proteoglycan for water retention (220 kDa). Anionic chondroitin sulfate GAG chains • Biglycan: Small proteoglycan with chondroitin / dermatan sulfate GAG chans. (38 kDa). • Collagen type II, elastin Disc homeostasis1: • Balance of proteoglycan synthesis and degradation (ADAMTS, MMP) • Ratio of small to large proteoglycans Vascular Supply Vascularity • Fetal/infant (up to 2 years old)5: Inner and outer annulus Anterior, central, posterior endplates • Juvenile/adolescent: Avascular except small capillaries in outermost annulus • Adult ( > 21 years old): Avascular except small capillaries in outermost annulus May have vascular ingrowth with annular tears or complete disc destruction/scar Vascular Supply 1 2 3 4 = = = = segmental radicular artery interosseous artery capillary tuft disc annulus Disc Nutrition Diffusion from limited blood vessels9: • Glucose and oxygen most important. • Endplates (vertebral) vessels only. Terminate in loops. • Any vascular portion of annulus only supplies the annulus. • Endplate vessels have muscarinic receptors: will constrict in response to cigarette smoke.9 Convection: • Movement of solutes from periphery to center of disc from changes in mechanical load. • MINIMAL contribution compared to diffusion down concentration gradient. • Zero-gravity state can cause hyperhydration10 Disc Nutrition Endplate selective permeability9: • Small solutes (oxygen, glucose) easy. • Growth factors and matrix macromolecules cannot pass. • Prevention of lactic acid build-up; pH > 6.7 Proteoglycan role: • Impedes movement of larger proteins. • Higher proteoglycan concentration = smaller diffusion pore size. Effect of diurnal cycle: • Fluid loss decreases disc height by 20% = higher proteoglycan concentration. • Smaller disc height decreases distance for diffusion. Innervation Pathophysiology Classically begun with tear of annulus. Endplate microfractures now felt to be sentinel event (~65% of time). Subclinical avulsion + time = disc herniation Acute annular tear with disc herniation also common Neovascularization / innervation Pathophysiology Endplate compromise = loss of nutrition From Spine (Phila Pa 1976). 2005 Jan 15;30(2):167-73. Calcification of endplate = decreased pore size. Pathophysiology Change in pore size = disruption of diffusion From Spine (Phila Pa 1976). 2005 Jan 15;30(2):167-73. Normal animal model Human disc herniation Pathophysiology Decreased oxygen tension, glucose and pH = cell death Reduced proteoglycan concentration Loss of selective permeability Inflammatory cytokines (TNF, IL-1, IL-6, etc) can enter nucleus Cytokines upregulate MMP expression; TIMP cannot keep up. Additional proteoglycan destruction Loss of water content and disc morphology Goals for New Therapies Efficacy/survival in hostile environment Maintain immune privilege Restore matrix milieu Reduce clinical symptoms! Potential Targets Chemodenervation of annular nerve ingrowth: methylene blue Recruitment of remaining NP cells: platelet rich plasma (via TGFβ, IGF1) Replacement of NP cells Careful Considerations Stem cell implantation: • Embryonic stem cells controversial and may retain tumorigenic potential.6 • Cell type needs to be similar to NP cells. Mesenchymal is derived from mesoderm embryologically. • Need cells to survive in low oxygen tension / low pH. Bone marrow derived mesenchymal stem cells may survive better than adipose (in rat model).12 • Should NOT provoke immune response6 • Need to keep cells within nucleus.13 • Identify ideal cell amount: prevent oxygen deprivation and over-pressurization6,14 Careful Considerations Platelet rich plasma: • Inject to coax remaining cells to produce proteoglycans / collagen type II • Possible transient efficacy • Incomplete knowledge of effects… Ex. PRP contains VEGF,15 but disc milieu is avascular Some preparations contain white blood cells16, but NP cells express FasL. May induce IL-1 and TNF-α17 Thrombin can be used to activate PRP, but may induce antibodies against it18 • May interfere with clotting cascade (post-op bleeding) • Animal studies implicate anti-thrombin antibodies in lupus-type syndrome18 Combination therapy: Pig model of PRP and MSC showed osteogenic differentiation instead of Collagen II / Aggrecan production19 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Chan WC, Sze KL, Samartzis D, Leung VY, Chan D. Structure and biology of the intervertebral disk in health and disease. Orthop Clin North Am. 2011 Oct;42(4):447-64. Smith LJ, Nerurkar NL, Choi KS, Harfe BD, Elliott DM. Degeneration and regeneration of the intervertebral disc: lessons from development. Dis Model Mech. 2011 Jan;4(1):31-41. Smits P1, Lefebvre V. Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell survival and development of the nucleus pulposus of intervertebral discs. Development. 2003 Mar;130(6):1135-48. Dahia CL, Mahoney EJ, Durrani AA, Wylie C. Intercellular signaling pathways active during intervertebral disc growth, differentiation, and aging. Spine (Phila Pa 1976). 2009 Mar 1;34(5):456-62. Nerlich AG, Schaaf R, Wälchli B, Boos N. Temporo-spatial distribution of blood vessels in human lumbar intervertebral discs. Eur Spine J. 2007 Apr;16(4):547-55. Oehme D, Goldschlager T, Ghosh P, Rosenfeld JV, Jenkin G. Cell-based therapies used to treat lumbar degenerative disc disease: a systematic review of animal studies and human clinical trials. Stem Cells Int. 2015;2015:946031. Sun Z, Wan ZY, Guo YS, Wang HQ, Luo ZJ. FasL on human nucleus pulposus cells prevents angiogenesis in the disc by inducing Fas-mediated apoptosis of vascular endothelial cells. Int J Clin Exp Pathol. 2013 Oct 15;6(11):2376-85. Park JB, Chang H, Kim KW. Expression of Fas ligand and apoptosis of disc cells in herniated lumbar disc tissue. Spine (Phila Pa 1976). 2001 Mar 15;26(6):618-21. Grunhagen T, Shirazi-Adl A, Fairbank JC, Urban JP. Intervertebral disk nutrition: a review of factors influencing concentrations of nutrients and metabolites. Orthop Clin North Am. 2011 Oct;42(4):465-77. Belavy DL, Adams M, Brisby H,5, Cagnie B, Danneels L, Fairbank J, Hargens AR, Judex S, Scheuring RA, Sovelius R, Urban J, van Dieën JH, Wilke HJ. Disc herniations in astronauts: What causes them, and what does it tell us about herniation on earth? Eur Spine J. 2015 Apr 18. References 11. 12. 13. 14. 15. 16. 17. 18. 19. Benneker LM, Heini PF, Alini M, Anderson SE, Ito K. 2004 Young Investigator Award Winner: vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration. Spine (Phila Pa 1976). 2005 Jan 15;30(2):167-73. Han B, Wang HC, Li H, Tao YQ, Liang CZ, Li FC, Chen G, Chen QX. Nucleus pulposus mesenchymal stem cells in acidic conditions mimicking degenerative intervertebral discs give better performance than adipose tissue-derived mesenchymal stem cells. Cells Tissues Organs. 2014;199(5-6):342-52. Bertram H, Kroeber M, Wang H, Unglaub F, Guehring T, Carstens C, Richter W. Matrix-assisted cell transfer for intervertebral disc cell therapy. Biochem Biophys Res Commun. 2005 Jun 17;331(4):1185-92 Ghosh P, Moore R, Vernon-Roberts B, Goldschlager T, Pascoe D, Zannettino A, Gronthos S, Itescu S. Immunoselected STRO-3+ mesenchymal precursor cells and restoration of the extracellular matrix of degenerate intervertebral discs. J Neurosurg Spine. 2012 May;16(5):479-88. Pirvu TN, Schroeder JE, Peroglio M, Verrier S, Kaplan L, Richards RG, Alini M, Grad S. Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production. Eur Spine J. 2014 Apr;23(4):745-53. Arnoczky SP, Sheibani-Rad S. The basic science of platelet-rich plasma (PRP): what clinicians need to know. Sports Med Arthrosc. 2013 Dec;21(4):180-5. Riboh JC, Saltzman BM, Yanke AB, Fortier L, Cole BJ. Effect of Leukocyte Concentration on the Efficacy of Platelet-Rich Plasma in the Treatment of Knee Osteoarthritis. Am J Sports Med. 2015 Apr 29. Fufa D, Shealy B, Jacobson M, Kevy S, Murray MM. Activation of platelet-rich plasma using soluble type I collagen. J Oral Maxillofac Surg. 2008 Apr;66(4):684-90. Chen WH, Liu HY, Lo WC, Wu SC, Chi CH, Chang HY, Hsiao SH, Wu CH, Chiu WT, Chen BJ, Deng WP. Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials. 2009 Oct;30(29):5523-33.