Declaration of Conflict of Interest or Relationship Speaker Name: Richard G. Spencer I have no conflicts of interest to disclose with regard to the subject.

Declaration of Conflict of Interest or Relationship

Speaker Name: Richard G. SpencerSpeaker Name: Richard G. Spencer

I have no conflicts of interest to disclose with regard to the subject matter ofI have no conflicts of interest to disclose with regard to the subject matter ofthis presentation.this presentation.

Unmet Needs for MR in the UnsolvedUnmet Needs for MR in the UnsolvedProblem of Tissue Engineering:Problem of Tissue Engineering:

Richard G. SpencerRichard G. SpencerMagnetic Resonance Imaging and Spectroscopy SectionMagnetic Resonance Imaging and Spectroscopy Section

National Institute on Aging, NIHNational Institute on Aging, NIHBaltimore, MDBaltimore, MD

Not just one problem, not just one solutionNot just one problem, not just one solution

ISMRM Toronto, 2008ISMRM Toronto, 2008

Definition of Tissue Engineering:Definition of Tissue Engineering:

"…an interdisciplinary field that applies the"…an interdisciplinary field that applies theprinciples of engineering and life sciencesprinciples of engineering and life sciences

towards the development of biological substitutes that towards the development of biological substitutes that restore, maintain, or improve tissue function."restore, maintain, or improve tissue function."

Langer and Vacanti, 1993Langer and Vacanti, 1993

Includes:Includes: Tissue EngineeringTissue Engineering

Excludes: Excludes: Organ transplantsOrgan transplants Artificial mechanical organsArtificial mechanical organs Metal jointsMetal joints Hemodialysis, external blood oxygenators,…Hemodialysis, external blood oxygenators,…

Implanted cells which will develop function in vivoImplanted cells which will develop function in vivo--stem cells for MI or stroke repair --stem cells for MI or stroke repair --cartilage matrix from implanted cells--cartilage matrix from implanted cells

Implanted acellular scaffoldsImplanted acellular scaffolds--bioactive bone graft scaffold--bioactive bone graft scaffold

Implanted cellular constructs designed in the labImplanted cellular constructs designed in the lab----off-the-shelf cartilage, pancreas, liver,…off-the-shelf cartilage, pancreas, liver,…

Motivation for TEMotivation for TE

Limitations of non-biologic repair:Limitations of non-biologic repair:

Duration < longevity of patient Duration < longevity of patient (metal implants)(metal implants)

Highly invasive Highly invasive (hemodialysis)(hemodialysis)

Typically, no growth or adaptation Typically, no growth or adaptation (c.f. advantage of demand pacemakers)(c.f. advantage of demand pacemakers)

Funding OpportunitiesFunding Opportunities

NIH:NIH: regenerative medicine funding 2008-- regenerative medicine funding 2008-- US$575 M (approx. 2% of NIH budget)US$575 M (approx. 2% of NIH budget)

DoD:DoD: Armed Forces Institute of Regenerative Armed Forces Institute of Regenerative Medicine (AFIRM), April, 2008: Medicine (AFIRM), April, 2008: US$265 M over 5 yearsUS$265 M over 5 years

Other: Other: NIST, NSF, Arthritis Foundation, NIST, NSF, Arthritis Foundation, Musculoskeletal Transplant Foundation…Musculoskeletal Transplant Foundation…

Medtronic: INFUSE bone graftsMedtronic: INFUSE bone grafts

RESTORE small intestine submucosa (duPont)RESTORE small intestine submucosa (duPont)

Apligraf skin patch (Organogenesis)Apligraf skin patch (Organogenesis)

Cord blood stem cell bankingCord blood stem cell banking

The product list is limited but activeThe product list is limited but active

Lysaght, Tissue Engineering, 2008Lysaght, Tissue Engineering, 2008

(SIS)

# of firms or business units > 50# of firms or business units > 50

# of employees > 3000# of employees > 3000

Annual sales > US$1.3 billionAnnual sales > US$1.3 billion

# of patients treated > 1M# of patients treated > 1M

# products in preclinical stages > 50# products in preclinical stages > 50

Private sector activity, 2007 (worldwide)Private sector activity, 2007 (worldwide)

Lysaght, Tissue Engineering, 2008Lysaght, Tissue Engineering, 2008

"Structural imaging" may be less important "Structural imaging" may be less important than other capabilities of MR, such asthan other capabilities of MR, such as

more specific microstructural and molecular more specific microstructural and molecular analyses, and metabolic analysis analyses, and metabolic analysis

What Can MRI and MRS Contribute?What Can MRI and MRS Contribute?

Non-invasive Non-invasive Monitoring after Implantation Monitoring after Implantation Positional stabilityPositional stability Integration with surroundingsIntegration with surroundings Metabolic activityMetabolic activity Need for and response to ongoing interventionNeed for and response to ongoing intervention

Examples of Current MR/TE StudiesExamples of Current MR/TE Studies

TE Cartilage Development in Hollow-fiber BioreactorTE Cartilage Development in Hollow-fiber Bioreactor

● from bovine cartilage

● 150 million cells/mL● Cultured for 4 weeks ● Sectioned 5mm each

A B C D E F G H I J

OutflowInflow BR3

● from bovine cartilage

● 150 million cells/mL● Cultured for 4 weeks ● Sectioned 5mm each

A B C D E F G H I J

OutflowInflow BR3

Bovine neocartilageBovine neocartilagedeveloping from developing from

chondrocyteschondrocytes

ATP

PCr

MDP

TR (s)

2.5

Pi

3131P spectrum of avian neocartilageP spectrum of avian neocartilage

30

20

10

1 / T

2, s

-1

0.150.100.050.00Tissue GAG Concentration, w/w

R = 0.81, p < 0.0011 week 2 weeks

3 weeks 4 weeks

Blue: MT< 0.4 Red: MT> 0.8

MT of TE CartilageMT of TE Cartilage

P = 0.04

0.0

1.0

2.0

Control Ibuprofen

MT

C k

m, s

-1

Effect of ibuprofen on Effect of ibuprofen on developing cartilagedeveloping cartilage

Day 14Day 14Matrix containing Matrix containing iron-labeled cellsiron-labeled cells Non-labeled matrixNon-labeled matrix

Prussian blue Prussian blue stained sectionstained sectionMR ImageMR Image

FCDFCD S-GAG/S-GAG/Dry Wt.Dry Wt.

Dynamic Dynamic ModulusModulus

Equilibrium Equilibrium ModulusModulus

Collagen/Collagen/Dry Wt.Dry Wt.

Effect of chondroitinase on developing cartilageEffect of chondroitinase on developing cartilage

Neocartilate in BioreactorNeocartilate in Bioreactor

culturemedium

collagen I gel

5x5x1 mm voxel

time

internal chemical shift standard TSP 10mM

glucose diffusion front

Metabolic Mapping in TE cartilage Non MRI: Model Based Estimates

(Sengers B. G. et al. JBME 2005)

Fick’s law

Michaelis-Menten

Computed spatial glucose concentration

Reference geometryof collagen I gel

Time course of glucose diffusion into ROI_1

Direct measurement of spatial glucose concentration

1H Localized Spectroscopy in Collagen I Constructs

Courtesy David ReiterCourtesy David Reiter

100µm

defect regionrepair tissue

T2 = 49.5 ms

repair tissuedefect region

T2 = 83.4 ms

repair tissue

native cartilage

defect

Courtesy S. Ramaswamy, Bioengineering, Univ of Pittsburgh

Hydrogel defect repair in rabbitHistology vs MRI

Cartilage in hydrogelBiochem vs MRI

9 days 21 days 28 days 43 days

100µm

T2 = -1.0144 X (% tissue fill) + 121.12

R2 = 0.6715

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100

% Tissue fill

Tra

nsv

erse

rel

ax

ati

on

tim

e,

T2

(ms)

10 mM CuCl2 10 mM CuCl2 landmark bulblandmark bulb

Defect 1Defect 1

Subchondral boneSubchondral bone

Defect 2Defect 2CartilageCartilage

TidemarkTidemark

Cartilage/Hydrogel StudiesCartilage/Hydrogel Studies

Asssessment of Cartilage formed inSelf-Aggregating Suspension Culture

MR-based methods offer advantages of providing noninvasive,

nondestructive, and quantitative surrogate measures of material

properties.

Characteristics of Culture Model Grows in suspension at high

density (1-2 X107 cells/ml) Forms a mass quickly (110 weeks mass >6 fold) Maintains cartilage phenotype(cartilage specific ECM, no Coll I )

Courtesy of George DodgeBone & Cartilage Research Lab, Nemours Biomedical Research

T1ρ = 105 ms T1ρ = 107 ms

Bovine patellar cartilage Engineered cartilage Previous studies: Direct correlation of T1ρ MR measurements with FCD and PG in cartilage

Source: Trattnig et al., 2007

MRI Following Matrix-assisted Autologous Chondrocyte TransplantationMRI Following Matrix-assisted Autologous Chondrocyte Transplantation

4 Weeks Post-Surgery:4 Weeks Post-Surgery:

Fluid-like contrast in repair zoneFluid-like contrast in repair zone

24 Weeks Post-Surgery:24 Weeks Post-Surgery:

Heterogeneous contrastHeterogeneous contrast

52 Weeks Post-Surgery:52 Weeks Post-Surgery:

Repair zone isointense Repair zone isointense with native cartilagewith native cartilage

TT22-weighted fast spin echo images reveal longitudinal changes in -weighted fast spin echo images reveal longitudinal changes in

repair zone containing implanted chondrocytesrepair zone containing implanted chondrocytes

TT11 weighted image weighted image

of of osteochondral plugosteochondral plug

Courtesy Richard Magin, UICCourtesy Richard Magin, UIC

TT11 and T and T22 values for values for

developing TE bonedeveloping TE bone

Conclusion: TConclusion: T22 is an is an

effective marker for effective marker for ossificationossification

11.7 T SE; TE/TR=1000/3011.7 T SE; TE/TR=1000/30

Ostogenesis in a Tissue Engineered ConstructOstogenesis in a Tissue Engineered Construct

MSC’s from adult bone marrow gelatin sponge osteogenic cx medium

constructfat

Courtesy of Hai-Ling Margaret Cheng, PhDThe Hospital for Sick Children and University of Toronto

Non-invasive MRI of tissue-engineered bladder construct

Major challenge to successful regeneration: creating immediate angiogenesisRole for imaging: non-invasive quantitative assessment of angiogenesis

Exposed bladder Angiogenesis at different VEGF levelsCD31-immunostaining

VEGF microvessel density

0

10

20

30

5 7 9 11 13

AU

C1m

in r = 0.784, P = 0.003

MVD (%)

r = 0.437, P = 0.1790

10

20

30

40

50

5 7 9 11 13

Gadomer

Gd-DTPA

MVD (%)

v p (

%)

r = 0.696, P = 0.012

r = 0.228, P = 0.50

Gadomer

Gd-DTPA

Tissue-engineered bladder construct

Gadomer (blood pool cx agent): correlation with microvessel density (MVD) through either fractional plasma volume (vp) or area under uptake curve (AUC); superior to Gd-DTPA

Gd-DTPA (extracellular cx agent): correlation with MVD is best obtained with AUC

SIGNIFICANCE: MRI measurement of absolute blood volume is feasible in therapeutic angiogenesis paradigms

Constantinidis et al. et al.

MR Monitors Viable Cell Number, Bioenergetics and Oxygen Distribution MR Monitors Viable Cell Number, Bioenergetics and Oxygen Distribution for Mouse Insulinoma for Mouse Insulinoma TC Cells in Tissue-Engineered PancreasTC Cells in Tissue-Engineered Pancreas

31P 1D CSI yields bioenergetic data with both spatial and temporal

resolution.

T1 of 19F PFTBA shows changes in dissolved oxygen content during hypoxic intervention.

Stem Cell Therapeutics—Cell PersistenceStem Cell Therapeutics—Cell Persistence

D. Kraitchman--JHUD. Kraitchman--JHU

Hypointense regions showing sites ofHypointense regions showing sites ofmesenchymal stem cell injectionmesenchymal stem cell injection

Loss of cells Loss of cells from from

noninfarctednoninfarctedtissuetissue

Tissue properties can often be evaluated Tissue properties can often be evaluated by highly by highly sensitivesensitive

but but invasiveinvasive techniques techniques

Gene expression analysisGene expression analysis for genotype and tissue quality for genotype and tissue quality

Electron microscopyElectron microscopy for microstructure for microstructure

HistologyHistology for exquisite structural detail for exquisite structural detail

ImmunohistochemistryImmunohistochemistry for protein expression for protein expression

Optical spectroscopyOptical spectroscopy for more specific matrix components for more specific matrix components

Microelectrodes / biochemical analysisMicroelectrodes / biochemical analysis for metabolic analysis for metabolic analysis

Mechanical testing Mechanical testing for functional support propertiesfor functional support properties

Unmet NeedsUnmet NeedsReplacement of gold-standard techniques with non-invasive versionsReplacement of gold-standard techniques with non-invasive versions

Molecule-specific MR:Molecule-specific MR: example: molecular imaging for collagen I vs collagen IIexample: molecular imaging for collagen I vs collagen II

Mechanical measurements: Mechanical measurements: example: bone/cartilage elastography; strain measurementsexample: bone/cartilage elastography; strain measurements

Sequences and analyses more specific to tissue componentsSequences and analyses more specific to tissue components example: water compartment analysis; multiparametric approachesexample: water compartment analysis; multiparametric approaches

Functional analysis (esp. metabolism)Functional analysis (esp. metabolism) example: localized spectroscopy for cell number and viabilityexample: localized spectroscopy for cell number and viability

Histologic-like resolutionHistologic-like resolution example: microscopyexample: microscopy

Opportunities Abound!Opportunities Abound!

Caveat:Caveat:

Capital value of publicly traded TE companies:Capital value of publicly traded TE companies:

2000: $2.5 billion US2000: $2.5 billion US

2003: $300 million US2003: $300 million US

2007: $4.7 billion US2007: $4.7 billion US

Lysaght, Tissue Engineering, 2008Lysaght, Tissue Engineering, 2008

Tissue engineering is currently in a growth phaseTissue engineering is currently in a growth phase

ConclusionsConclusions

MR can actually contribute to further progressMR can actually contribute to further progress

Fundable through several sourcesFundable through several sources

There is a substantial market presence There is a substantial market presence (contrast gene therapy)(contrast gene therapy)

Come to the educational session in Hawaii!Come to the educational session in Hawaii!