Differentiation of Human Marrow-Differentiation of Human Marrow-Derived Stromal Cells in Derived Stromal Cells in

Carboxymethylcellulose (CMC) Carboxymethylcellulose (CMC) HydrogelsHydrogels

Presented by Estefany Condo

Mentor Professor Steven B. Nicoll

(Raj PP. Pain Practice. 8(1):18-44. 2008.) (Raj PP. Pain Practice. 8(1):18-44. 2008.)

(Courtesy of Dawn Elliot PhD) (Courtesy of Dawn Elliot PhD)

NP OAIA

Intervertebral Disc BiologyIntervertebral Disc Biology

Intervertebral Disc (IVD)Intervertebral Disc (IVD)

Balances flexibility and stability of Balances flexibility and stability of spine under physiologic loadingspine under physiologic loading

IVD AnatomyIVD Anatomy

Nucleus Pulposus: Coll II and PG Nucleus Pulposus: Coll II and PG unorganized gelatinous matrixunorganized gelatinous matrix

Inner Annulus Fibrosus: Coll I & II Inner Annulus Fibrosus: Coll I & II and PGsand PGs

Outer Annulus Fibrosus: Coll I Outer Annulus Fibrosus: Coll I fibrous lamellar ring, minimal PGsfibrous lamellar ring, minimal PGs

Cartilaginous End Plates: Hyaline Cartilaginous End Plates: Hyaline cartilagecartilage

(Adams MA. Acupunct Med. (Adams MA. Acupunct Med. 22(4):178-88. 2004.) 22(4):178-88. 2004.)

DegenerationDegeneration- Imbalance between anabolic and catabolic Imbalance between anabolic and catabolic

metabolism of matrix moleculesmetabolism of matrix molecules

- Enzymatic: cathepsins, MMPs, aggrecanaseEnzymatic: cathepsins, MMPs, aggrecanase

- Nonenzymatic GlycationNonenzymatic Glycation

Contributing FactorsContributing Factors- Abnormal mechanical loadingAbnormal mechanical loading

- GeneticsGenetics

- Lack of nutrient supplyLack of nutrient supply

Herniated DiscsHerniated Discs- Disc bulge or rupture due to NP displacement down Disc bulge or rupture due to NP displacement down

a radial fissurea radial fissure

- Common site is at insertion of posterior OA into the Common site is at insertion of posterior OA into the vertebral bodyvertebral body

Intervertebral Disc DegenerationIntervertebral Disc Degeneration

Intervertebral Discogenic PainIntervertebral Discogenic Pain

IVD degeneration associated with lower back painIVD degeneration associated with lower back pain- Afflicts 80% of Americans during their lifetime (Frymoyer JW, et al. 1991)Afflicts 80% of Americans during their lifetime (Frymoyer JW, et al. 1991)

- Costs over $80 billion dollars in annual-medical related expenses (Martin Costs over $80 billion dollars in annual-medical related expenses (Martin BI, et al. 2008)BI, et al. 2008)

Mechanisms contributing to back painMechanisms contributing to back pain- Loss of disc structure alters load and vertebral body alignmentLoss of disc structure alters load and vertebral body alignment

- Increased nerve and blood vessel penetration in IA and NP with Increased nerve and blood vessel penetration in IA and NP with degenerationdegeneration

- Increased sensitivity of nerve endings through release of inflammatory Increased sensitivity of nerve endings through release of inflammatory moleculesmolecules

IVD Tissue Engineering StrategyIVD Tissue Engineering Strategy

(O’Halloran DM. Tissue Engineering. 13(8):1927-454. 2007.) (O’Halloran DM. Tissue Engineering. 13(8):1927-454. 2007.)

Early StageEarly Stage

Growth factor supplementation to Growth factor supplementation to stimulate cell synthesis of ECMstimulate cell synthesis of ECM

Middle StageMiddle Stage

Cell-seeded NP scaffold replacement Cell-seeded NP scaffold replacement following nucleotomyfollowing nucleotomy

Late StageLate Stage

Full IVD replacement with composite Full IVD replacement with composite cell-seeded scaffoldcell-seeded scaffold

- Crosslinked, atellocolagen type II scaffolds Crosslinked, atellocolagen type II scaffolds (Halloran (Halloran DO, et al. 2008)DO, et al. 2008)

- MSC encapsulated uncrosslinked hyaluronic acid MSC encapsulated uncrosslinked hyaluronic acid hydrogels hydrogels (Crevensten G, et al. 2004)(Crevensten G, et al. 2004)

- Porcine disc cells encapsulated in ionically Porcine disc cells encapsulated in ionically crosslinked alginate hydrogel beads crosslinked alginate hydrogel beads (Baer AE, et al. (Baer AE, et al. 2001)2001)

Biomaterials for Engineering Nucleus Pulposus Tissue Biomaterials for Engineering Nucleus Pulposus Tissue

SyntheticSynthetic- Canine NP seeded PLGA porous scaffold (Ruan DK, et al. 2010)Canine NP seeded PLGA porous scaffold (Ruan DK, et al. 2010)- Acellular PNIPAAm – PEG injectable copolymer (Vernengo J, et al. 2008)Acellular PNIPAAm – PEG injectable copolymer (Vernengo J, et al. 2008)

NaturalNatural- MSC seeded agarose hydrogels (Nerurkar NL, et al. 2010)MSC seeded agarose hydrogels (Nerurkar NL, et al. 2010)- NP encapsulated chitosan-glycerophosphate hydrogels (Richardson SM, et al. 2008)NP encapsulated chitosan-glycerophosphate hydrogels (Richardson SM, et al. 2008)

Photocrosslinked HydrogelsPhotocrosslinked Hydrogels

(Elisseeff J. Plast Reconstr Surg. 104(4):1014-(Elisseeff J. Plast Reconstr Surg. 104(4):1014-22. 1999.) 22. 1999.)

PhotopolymerizationPhotopolymerization- Biocompatible light-sensitive photoinitiators create covalently crosslinked 3D Biocompatible light-sensitive photoinitiators create covalently crosslinked 3D

networks via free radical polymerization with UV exposurenetworks via free radical polymerization with UV exposure

- Modify polymer backbone with functional methacrylate groups to create stable, Modify polymer backbone with functional methacrylate groups to create stable, crosslinked hydrogel networks crosslinked hydrogel networks

Articular Cartilage Photopolymerized Articular Cartilage Photopolymerized ScaffoldsScaffolds

- PEO gels encapsulating chondrocytes PEO gels encapsulating chondrocytes (Elisseeff J, et al. 1999)(Elisseeff J, et al. 1999)

- PEG gels encapsulating chondrocytes PEG gels encapsulating chondrocytes (Bryant SJ, et al. 2002)(Bryant SJ, et al. 2002)

- Hyaluronic acid gels encapsulating Hyaluronic acid gels encapsulating chondrocytes (Burdick JA, et al. 2005)chondrocytes (Burdick JA, et al. 2005)

CelluloseCellulose

Primary component of plant cell walls Primary component of plant cell walls

Comprised D-glucose subunits with Comprised D-glucose subunits with ββ(1-4)glycosidic bonds (1-4)glycosidic bonds

H-bonding of hydroxyl groups forms H-bonding of hydroxyl groups forms non-soluble crystalline structurenon-soluble crystalline structure

CarboxymethylcelluloseCarboxymethylcellulose

Water-soluble derivative of celluloseWater-soluble derivative of cellulose

Non-toxic, FDA-approvedNon-toxic, FDA-approved

Biocompatible, available in high purity Biocompatible, available in high purity formsforms

DegradabilityDegradability

InexpensiveInexpensive

Cellulose-Based HydrogelsCellulose-Based Hydrogels

(Reza and Nicoll, (Reza and Nicoll, Acta Biomater.Acta Biomater., 6:179-186, 2010), 6:179-186, 2010)

Photocrosslinked CarboxymethylcellulosePhotocrosslinked Carboxymethylcellulose

(Reza and Nicoll, (Reza and Nicoll, Acta Biomater.Acta Biomater., 6:179-186, 2010), 6:179-186, 2010)

CMC successfully modified with functional CMC successfully modified with functional methacrylate groups to create methacrylate groups to create photocrosslinked CMC hydrogelsphotocrosslinked CMC hydrogels

Encapsulated bovine NP cells and Encapsulated bovine NP cells and promoted accumulation of ECMpromoted accumulation of ECM

Mechanical properties maintained in cell-Mechanical properties maintained in cell-laden gels compared to cell-freeladen gels compared to cell-free

1 2

3 4

(Reza and Nicoll, (Reza and Nicoll, Acta Biomater.Acta Biomater., 6:179-186, 2010), 6:179-186, 2010)

(Reza and Nicoll, (Reza and Nicoll, Acta Biomater.Acta Biomater., 6:179-186, 2010), 6:179-186, 2010)

Low Viscosity vs Medium ViscosityLow Viscosity vs Medium ViscosityBovine NP Encapsulated CMC HydrogelsBovine NP Encapsulated CMC Hydrogels

Cell Sources for NP Tissue ReplacementsCell Sources for NP Tissue Replacements

Autologous or Allograft Disc CellsAutologous or Allograft Disc Cells- Low yields during isolation due to acellularity of native tissueLow yields during isolation due to acellularity of native tissue

- Isolation from healthy vs. degenerated tissueIsolation from healthy vs. degenerated tissue

- Immortal human NP cell line (Iwashina T, et al. 2006) Immortal human NP cell line (Iwashina T, et al. 2006)

Notochordal CellsNotochordal Cells- Major role in biosynthetic activity of NP, but no human source availableMajor role in biosynthetic activity of NP, but no human source available

Mesenchymal Stem CellsMesenchymal Stem Cells- Nonhematopoietic, pluripotent progenitor cellsNonhematopoietic, pluripotent progenitor cells

- Extensive reserve of autologous cells with great ease of removalExtensive reserve of autologous cells with great ease of removal

- Successful chondrogenic differentiation capability of bone marrow vs Successful chondrogenic differentiation capability of bone marrow vs adispose MSCs (Afizah H, et al. 2007)adispose MSCs (Afizah H, et al. 2007)

- Gene markers to distinguish NP-like cells vs. chondrocytes (Minogue BM, et Gene markers to distinguish NP-like cells vs. chondrocytes (Minogue BM, et al. 2010)al. 2010)

Carboxymethylcellulose Tissue Scaffolds

(Gupta et al. Tissue Eng. 17:2903-10. 2011)

Photocrosslinked CMC hydrogels - Promote differentiation of encapsulated human mesenchymal stem cells (hMSCs) to form

a functional NP-like construct when supplemented with TGF-β3 - Medium Viscosity CMC ONLY!

Peak Stress

Day 21 (Scale bar = 50 μm)

Alcian Blue

Coll II

CSPG

CDM + TGF-β3CDM

CDM CDM + TGF-β3

Day 21

Objective

Evaluate how human MSCs respond to changes in polymer molecular weight when encapsulated in a three-dimensional CMC hydrogel scaffolds.

Materials & Methods

Macromer Synthesis:- Two molecular weights: 90 kDa and 250 kDa CMC

modified with methacrylic anhydride (9% and 10% methacrylation) (Gupta et al., 2011)

Hydrogel Preparation:

- 2.0% and 3.0% (w/v) for acellular and cellular hydrogels- Cellular constructs: hMSCs encapsulated in

photocrosslinked CMC at 20 x 106 cells/ml in the presence of UV light and photoinitiator (Gupta et al., 2011)

Cell Culture:

- In vitro culture in serum-free chemically defined medium supplemented with TGF-β3 (10ng/ml) for 28 days

Materials & Methods: Hydrogel Properties

Mechanical Properties: (Cellular & Acellular)- Unconfined compression to obtain Equilibrium modulus (Ey), Peak stress

(σpk), and % relaxation at day 1, 14, and 28

Swelling Ratio: (Cellular & Acellular)- Equilibrium weight swelling ratio (Qw) calculated as wet weight divided by dry

weight at day 1, 14, 28

ECM Accumulation and Deposition: (Cellular)- Histology for cellular and GAG distribution at day 28- Immunohistochemistry for Collagen II at day 28- Biochemical content of Collagen II, GAGs and DNA at day 1, 14 and 28

Statistics:- Two-way ANOVA with Tukey’s post hoc test (p < 0.05)