Download The Human Intervertebral Disc

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
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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:
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More condensed: Around notorchord to become annulus
Less condensed: Become vertebral bodies
From Orthop Clin N Am 42 (2011) 447–464
Disc Genetic Factors
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Key Developmental Genes:
• Sox (5,6,9):
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Chondrogenesis.
Critical to collagen II, inner annulus and matrix
formation.2,3
Reduced Sox9 expression correlated with degenerative
changes.
• TGFβ:
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Regulates cell proliferation and matrix production.
In murine model, remains active at maturity4
Cellular Biology
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Nucleus Pulposus
• Cells very similar to notochord cells at birth.1
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Large with vacuoles containing glycosaminoglycans.
By 10 years of age, notochordal cells disappear.
In other species, connote disc repair6
• NP cells:
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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
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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
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Vascularity
• Fetal/infant (up to 2 years old)5:
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Inner and outer annulus
Anterior, central, posterior endplates
• Juvenile/adolescent:
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Avascular except small capillaries in outermost annulus
• Adult ( > 21 years old):
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Avascular except small capillaries in outermost annulus
May have vascular ingrowth with annular tears or complete
disc destruction/scar
Vascular Supply
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1
2
3
4
=
=
=
=
segmental radicular artery
interosseous artery
capillary tuft
disc annulus
Disc Nutrition
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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
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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
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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
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Maintain immune privilege
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Restore matrix milieu

Reduce clinical symptoms!
Potential Targets
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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…

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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
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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.
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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.