The Human The Human Intervertebral DiscIntervertebral Disc

Developmental, Anatomic and Developmental, Anatomic and Physiologic Considerations for Potential Physiologic Considerations for Potential

Regenerative TherapiesRegenerative Therapies

Benjamin D. Levy, MD, FAAPMRBenjamin D. Levy, MD, FAAPMR

Interventional Pain ManagementInterventional Pain ManagementAmbulatory Care ServiceAmbulatory Care Service

U.S. Department of Veterans AffairsU.S. Department of Veterans AffairsVA New Jersey Health Care SystemVA New Jersey Health Care System

Topics of DiscussionTopics of Discussion

AnatomyAnatomy Cellular and Molecular BiologyCellular and Molecular Biology PathophysiologyPathophysiology Implications for Regenerative Implications for Regenerative

TherapiesTherapies

Financial DisclosuresFinancial Disclosures

NoneNone Happily employed by the United Happily employed by the United

States Federal GovernmentStates Federal Government

Disc AnatomyDisc Anatomy

30° angle-ply architecture30° angle-ply architecture11

Disease Models & Mechanisms 4, 31-41 (2011)Disease Models & Mechanisms 4, 31-41 (2011)

Disc EmbryologyDisc Embryology Notochord: Notochord:

• Mesoderm-derived involved in cell signaling and Mesoderm-derived involved in cell signaling and differentiationdifferentiation22

• Becomes nucleus pulposusBecomes nucleus pulposus Somites: Somites:

• Blocks of mesoderm flanking the notochord paraxiallyBlocks of mesoderm flanking the notochord paraxially• Cells of somites become sclerotomeCells of somites become sclerotome• Sclerotomes become alternating more & less condensedSclerotomes become alternating more & less condensed22::

More condensed: Around notorchord to become annulusMore condensed: Around notorchord to become annulus Less condensed: Become vertebral bodiesLess condensed: Become vertebral bodies

From Orthop Clin N Am 42 (2011) 447–464

Disc Genetic FactorsDisc Genetic Factors Key Developmental Genes: Key Developmental Genes:

• Sox (5,6,9): Sox (5,6,9): Chondrogenesis. Chondrogenesis. Critical to collagen II, inner annulus and matrix Critical to collagen II, inner annulus and matrix

formation.formation.2,32,3

Reduced Sox9 expression correlated with degenerative Reduced Sox9 expression correlated with degenerative changes.changes.

• TGFTGFββ: : Regulates cell proliferation and matrix production. Regulates cell proliferation and matrix production. In murine model, remains active at maturityIn murine model, remains active at maturity44

Cellular BiologyCellular Biology Nucleus PulposusNucleus Pulposus

• Cells very similar to notochord cells at birth.Cells very similar to notochord cells at birth.11

Large with vacuoles containing glycosaminoglycans.Large with vacuoles containing glycosaminoglycans. By 10 years of age, notochordal cells disappear.By 10 years of age, notochordal cells disappear. In other species, connote disc repairIn other species, connote disc repair66

• NP cells: NP cells: Appear similar to chondrocytes.Appear similar to chondrocytes. Humans are termed “chondrodystrophoid”Humans are termed “chondrodystrophoid”66

Aggrecan and some Collagen Type II Aggrecan and some Collagen Type II productionproduction1,21,2

Express FasL, which induces apoptosis of any Express FasL, which induces apoptosis of any cell with Fas receptorcell with Fas receptor77::

• T-cellsT-cells77

• Nucleus pulposus cellsNucleus pulposus cells88

Cellular BiologyCellular Biology

Annulus FibrosusAnnulus Fibrosus• Outer annulus fibroblastic cellsOuter annulus fibroblastic cells1,21,2

Collagen type I (like tendon)Collagen type I (like tendon)

• Inner annulus chondrocyte-like cellsInner annulus chondrocyte-like cells1,21,2

Collagen type II (like hyaline cartilage, eye vitreus)Collagen type II (like hyaline cartilage, eye vitreus)

Molecular BiologyMolecular Biology Main molecules in nucleus:Main molecules in nucleus:

• Aggrecan: Large proteoglycan for water retention Aggrecan: Large proteoglycan for water retention (220 kDa). Anionic chondroitin sulfate GAG (220 kDa). Anionic chondroitin sulfate GAG chainschains

• Biglycan: Small proteoglycan with chondroitin / Biglycan: Small proteoglycan with chondroitin / dermatan sulfate GAG chans. (38 kDa).dermatan sulfate GAG chans. (38 kDa).

• Collagen type II, elastinCollagen type II, elastin

Disc homeostasisDisc homeostasis11::• Balance of proteoglycan synthesis and degradation Balance of proteoglycan synthesis and degradation

(ADAMTS, MMP)(ADAMTS, MMP)• Ratio of small to large proteoglycans Ratio of small to large proteoglycans

Vascular SupplyVascular Supply

VascularityVascularity• Fetal/infant (up to 2 years oldFetal/infant (up to 2 years old)5)5::

Inner and outer annulusInner and outer annulus Anterior, central, posterior endplatesAnterior, central, posterior endplates

• Juvenile/adolescent:Juvenile/adolescent: Avascular except small capillaries in outermost annulusAvascular except small capillaries in outermost annulus

• Adult ( > 21 years old):Adult ( > 21 years old): Avascular except small capillaries in outermost annulusAvascular except small capillaries in outermost annulus May have vascular ingrowth with annular tears or complete May have vascular ingrowth with annular tears or complete

disc destruction/scardisc destruction/scar

Vascular SupplyVascular Supply

1 = segmental radicular artery1 = segmental radicular artery 2 = interosseous artery2 = interosseous artery 3 = capillary tuft3 = capillary tuft 4 = disc annulus4 = disc annulus

Disc NutritionDisc Nutrition Diffusion from limited blood vesselsDiffusion from limited blood vessels99::

• Glucose and oxygen most important.Glucose and oxygen most important.• Endplates (vertebral) vessels only. Terminate in loops.Endplates (vertebral) vessels only. Terminate in loops.• Any vascular portion of annulus only supplies the Any vascular portion of annulus only supplies the

annulus.annulus.• Endplate vessels have muscarinic receptors: will Endplate vessels have muscarinic receptors: will

constrict in response to cigarette smoke.constrict in response to cigarette smoke.99

Convection:Convection:• Movement of solutes from periphery to center of disc Movement of solutes from periphery to center of disc

from changes in mechanical load.from changes in mechanical load.• MINIMAL contribution compared to diffusion down MINIMAL contribution compared to diffusion down

concentration gradient.concentration gradient.• Zero-gravity state can cause hyperhydrationZero-gravity state can cause hyperhydration1010

Disc NutritionDisc Nutrition Endplate selective permeabilityEndplate selective permeability99: :

• Small solutes (oxygen, glucose) easy.Small solutes (oxygen, glucose) easy.• Growth factors and matrix macromolecules cannot pass.Growth factors and matrix macromolecules cannot pass.• Prevention of lactic acid build-up; pH > 6.7Prevention of lactic acid build-up; pH > 6.7

Proteoglycan role: Proteoglycan role: • Impedes movement of larger proteins.Impedes movement of larger proteins.• Higher proteoglycan concentration = smaller diffusion Higher proteoglycan concentration = smaller diffusion

pore size.pore size.

Effect of diurnal cycle:Effect of diurnal cycle:• Fluid loss decreases disc height by 20% = higher Fluid loss decreases disc height by 20% = higher

proteoglycan concentration.proteoglycan concentration.• Smaller disc height decreases distance for diffusion.Smaller disc height decreases distance for diffusion.

InnervationInnervation

PathophysiologyPathophysiology

Classically begun with tear of Classically begun with tear of annulus.annulus.

Endplate microfractures now felt to Endplate microfractures now felt to be sentinel event (~65% of time).be sentinel event (~65% of time).

Subclinical avulsion + time = disc Subclinical avulsion + time = disc herniationherniation

Acute annular tear with disc Acute annular tear with disc herniation also commonherniation also common

Neovascularization / innervationNeovascularization / innervation

PathophysiologyPathophysiology

Endplate compromise = loss of nutritionEndplate compromise = loss of nutrition

From From Spine (Phila Pa 1976). 2005 Jan 15;30(2):167-73.

Calcification of endplate = decreased pore Calcification of endplate = decreased pore size.size.

PathophysiologyPathophysiology Change in pore size = disruption of diffusionChange in pore size = disruption of diffusion

From From Spine (Phila Pa 1976). 2005 Jan 15;30(2):167-73.

Normal animal modelNormal animal model Human disc herniationHuman disc herniation

PathophysiologyPathophysiology

Decreased oxygen tension, glucose and Decreased oxygen tension, glucose and pH = cell deathpH = cell death

Reduced proteoglycan concentrationReduced proteoglycan concentration Loss of selective permeabilityLoss of selective permeability Inflammatory cytokines (TNF, IL-1, IL-6, Inflammatory cytokines (TNF, IL-1, IL-6,

etc) can enter nucleusetc) can enter nucleus Cytokines upregulate MMP expression; Cytokines upregulate MMP expression;

TIMP cannot keep up.TIMP cannot keep up. Additional proteoglycan destructionAdditional proteoglycan destruction Loss of water content and disc morphologyLoss of water content and disc morphology

Goals for New TherapiesGoals for New Therapies

Efficacy/survival in hostile Efficacy/survival in hostile environmentenvironment

Maintain immune privilegeMaintain immune privilege

Restore matrix milieuRestore matrix milieu

Reduce clinical symptoms!Reduce clinical symptoms!

Potential TargetsPotential Targets

Chemodenervation of annular nerve Chemodenervation of annular nerve ingrowth: methylene blueingrowth: methylene blue

Recruitment of remaining NP cells: Recruitment of remaining NP cells: platelet rich plasma (via TGFplatelet rich plasma (via TGFββ, IGF1), IGF1)

Replacement of NP cellsReplacement of NP cells

Careful ConsiderationsCareful Considerations Stem cell implantation:Stem cell implantation:

• Embryonic stem cells controversial and may retain Embryonic stem cells controversial and may retain tumorigenic potential.tumorigenic potential.66

• Cell type needs to be similar to NP cells.Cell type needs to be similar to NP cells. Mesenchymal is derived from mesoderm embryologically.Mesenchymal is derived from mesoderm embryologically.

• Need cells to survive in low oxygen tension / low pH.Need cells to survive in low oxygen tension / low pH. Bone marrow derived mesenchymal stem cells may survive better than Bone marrow derived mesenchymal stem cells may survive better than

adipose (in rat model).adipose (in rat model).1212

• Should NOT provoke immune responseShould NOT provoke immune response66

• Need to keep cells within nucleus.Need to keep cells within nucleus.1313

• Identify ideal cell amount: prevent oxygen deprivation and Identify ideal cell amount: prevent oxygen deprivation and over-pressurizationover-pressurization6,146,14

Careful ConsiderationsCareful Considerations Platelet rich plasma:Platelet rich plasma:

• Inject to coax remaining cells to produce Inject to coax remaining cells to produce proteoglycans / collagen type IIproteoglycans / collagen type II

• Possible transient efficacyPossible transient efficacy• Incomplete knowledge of effects…Incomplete knowledge of effects…

Ex. PRP contains VEGF,Ex. PRP contains VEGF,1515 but disc milieu is avascular but disc milieu is avascular Some preparations contain white blood cellsSome preparations contain white blood cells1616, but NP cells , but NP cells

express FasL. May induce IL-1 and TNF-express FasL. May induce IL-1 and TNF-αα1717

Thrombin can be used to activate PRP, but may induce Thrombin can be used to activate PRP, but may induce antibodies against itantibodies against it1818

• May interfere with clotting cascade (post-op bleeding)May interfere with clotting cascade (post-op bleeding)• Animal studies implicate anti-thrombin antibodies in lupus-type Animal studies implicate anti-thrombin antibodies in lupus-type

syndromesyndrome1818

Combination therapy:Combination therapy: Pig model of PRP and MSC showed osteogenic differentiation Pig model of PRP and MSC showed osteogenic differentiation

instead of Collagen II / Aggrecan productioninstead of Collagen II / Aggrecan production1919

ReferencesReferences1.1. Chan WC, Sze KL, Samartzis D, Leung VY, Chan D. Structure and biology of the intervertebral disk in health and disease. 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. Orthop Clin North Am. 2011 Oct;42(4):447-64.

2.2. Smith LJ, Nerurkar NL, Choi KS, Harfe BD, Elliott DM. Degeneration and regeneration of the intervertebral disc: lessons 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.from development. Dis Model Mech. 2011 Jan;4(1):31-41.

3.3. Smits P1, Lefebvre V. Sox5 and Sox6 are required for notochord extracellular matrix sheath formation, notochord cell 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.survival and development of the nucleus pulposus of intervertebral discs. Development. 2003 Mar;130(6):1135-48.

4.4. Dahia CL, Mahoney EJ, Durrani AA, Wylie C. Intercellular signaling pathways active during intervertebral disc growth, 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.differentiation, and aging. Spine (Phila Pa 1976). 2009 Mar 1;34(5):456-62.

5.5. Nerlich AG, Schaaf R, Wälchli B, Boos N. Temporo-spatial distribution of blood vessels in human lumbar intervertebral 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.discs. Eur Spine J. 2007 Apr;16(4):547-55.

6.6. Oehme D, Goldschlager T, Ghosh P, Rosenfeld JV, Jenkin G. Cell-based therapies used to treat lumbar degenerative disc 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.disease: a systematic review of animal studies and human clinical trials. Stem Cells Int. 2015;2015:946031.

7.7. Sun Z, Wan ZY, Guo YS, Wang HQ, Luo ZJ. FasL on human nucleus pulposus cells prevents angiogenesis in the disc by 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.inducing Fas-mediated apoptosis of vascular endothelial cells. Int J Clin Exp Pathol. 2013 Oct 15;6(11):2376-85.

8.8. Park JB, Chang H, Kim KW. Expression of Fas ligand and apoptosis of disc cells in herniated lumbar disc tissue. Spine 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.(Phila Pa 1976). 2001 Mar 15;26(6):618-21.

9.9. Grunhagen T, Shirazi-Adl A, Fairbank JC, Urban JP. Intervertebral disk nutrition: a review of factors influencing 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.concentrations of nutrients and metabolites. Orthop Clin North Am. 2011 Oct;42(4):465-77.

10.10. Belavy DL, Adams M, Brisby H,5, Cagnie B, Danneels L, Fairbank J, Hargens AR, Judex S, Scheuring RA, Sovelius R, Urban 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 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.earth? Eur Spine J. 2015 Apr 18.

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13.13. Bertram H, Kroeber M, Wang H, Unglaub F, Guehring T, Carstens C, Richter W. Matrix-assisted cell transfer for 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):1185-92intervertebral disc cell therapy. Biochem Biophys Res Commun. 2005 Jun 17;331(4):1185-92

14.14. Ghosh P, Moore R, Vernon-Roberts B, Goldschlager T, Pascoe D, Zannettino A, Gronthos S, Itescu S. Immunoselected 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 STRO-3+ mesenchymal precursor cells and restoration of the extracellular matrix of degenerate intervertebral discs. J Neurosurg Spine. 2012 May;16(5):479-88.Neurosurg Spine. 2012 May;16(5):479-88.

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