Scaffolds, Growth Factors, and Tissue Constructs

Hasan Uludag

Department of Chemical & Materials Engineering,

Department of Biomedical Engineering, and

Faculty of Pharmacy & Pharmaceutical Sciences,

University of Alberta, Edmonton, AB

Delivery Systems

Bone Healing:an orchestrated process of molecular and cellular events

Hematoma

Angiogenesis

Influx of Stem Cells

Osteogenic Differentiation

Bone Deposition

Remodelling

• chemotaxis, proliferation, differentiation, osteogenesis, and apoptosis• all processes regulated by growth factors and the extracellular matrix

Shier D. et al., Hole's Human Anatomy & Physiology, 1996.

Approaches for Bone Regeneration:

• Extracellular Matrices: dynamic scaffolding capable of modulating cell activity: natural (collagen) or synthetic (apatite fillers)

• Growth Factors: endogenous regulators of cellular activity: chemotaxis, proliferation, and differentiation (BMPs)

• Osteogenic Cells: cells that can differentiate under the influence of local factors (bone marrow cells or engineered cells)

A Brief History of BMP Development

• 1965 ~ Description of bone morphogenetic activity from demineralized bone matrix(M. Urist, Science)

• 1988 ~ Cloning of recombinant human BMP-2 and reproduction of BMP activity by a single protein (Wozney et al., Science)

• 1988-2000 ~ Development of a delivery system

• >2000 ~ Regulatory approval, publications on specific clinical indications, success rates, and alternatives.

TORONTO

BOSTON

GENETICS INSTITUTE INC.

Osteoinductive Devices in Clinics

BMP-2 Soaked Collagen Sponge Wet Sponge in Fusion Cage

Demineralized Bone Powder to be Soaked with OP-1 (BMP-7)

Representation of an Osteoinductive Device

cell flux

cell flux

cell flux

BMP release

biomaterialscaffold

Effect of biomaterial scaffold on BMP release kinetics from implants

release kinetics ≈ 'retention'

Subsequent Pharmacokinetics

0 20 40 60 80

%Implanted Dose

Dexon

deLip-Bovine Bone

Synhetic HA

TCP

bovine Mineral

human Mineral

coral HA

rat DBM

human DBM

Gelatin Sponge

Collagen Sponge (2)

Collagen Sponge (1)

Initial (3 hr) Recovery of 125I-BMP-2 in Rat Ectopic Model

Retention of different BMPs in implants

0.01

0.1

1

10

100

0 5 10 15

Succ. rhBMP-2

Acet. rhBMP-2

rhBMP-2 (E.coli)

rhBMP-6

rhBMP-4

rhBMP-2

0.01

0.1

1

10

100

0 5 10 15

Collagen Sponge Poly(glycolic acid)

Time (days)

Per

cen

t R

eten

tio

n

Relationship between BMP retention and osteoinductive activity in implants

Strategy-1Biomaterial-1

Biomaterial-2BMP-X

Strategy-2 Biomaterial

BMP-Y

BMP-Z

BMP-2 vs. BMP-4: Comparison of protein retention and bone formation in the rat ectopic model

Pharmacokinetics Osteoinduction

O

NH

NiPAM(N-isopropylacrylamide)

O

OR

Alkyl-methacrylates

O

ON

O

O

NASI(N-acryloxysuccinimide)

Engineered biomaterials for increased retention of BMPs after injection

NiPAM/EMA-49 kD

NiPAM/EMA/NASI-50 kD

NiPAM/EMA-404 kD

NiPAM/EMA/NASI-422 kD

Temperature-Sensitive Polymers

BMP-biomaterial interactions

BMPs

BMP-2 Retention by NiPAM-Polymers (intramuscular injection model in rats)

Time (days)

% R

eten

tio

n

0.1

1

10

100

0 7 14

BMA/NASI (15.6 oC)

BMA (14.8 oC)

BMA/NASI (24.7 oC)

BMA (25.6 oC)

BMP-2 alone

An Osteoinductive Device by Gene Delivery

cell flux

cell flux

cell flux

release of osteoinductive (BMP-2) genes

biomaterialscaffold

... Lieberman et al. (UCLA) 1998.

... Huard et al. (U. Pittsburg) 1999.

... Helm et al. (U. Virginia) 1999.

MedicinalAgent

MedicinalAgent

Growth Factors for Systemic Bone Therapy

BP conjugation as a means to impart boneaffinity (Zhang et al., Chem. Soc. Rev. 2006)

BisphosphonatesQuickTime™ and a

TIFF (LZW) decompressorare needed to see this picture.

Protein

Protein chemistry to couple bisphosphonates therapeutic proteins

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

sulfoSMCC

aminoBP ( )

O

O

N

O

ONaO3S

NO

O

sulfoSMCC

Protein

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

MedicinalAgent

New bisphosphonates engineered specifically for targeting proteins to bone

MedicinalAgent

(Bansal et al., J. Pharm. Sci. 2004; Bansal et al., Angew Chem. 2005)

Di-Bisphosphonate Tetra-Bisphosphonate

Effectiveness of BP-coupled proteins to seek bone after systemic administration ?

Targeting Efficiency:Bone Delivery of BP-Conjugate

Bone Delivery of Native Protein

MedicinalAgent

(Zhang et al., Chem. Soc. Rev. 2006)

3-6 Hours 1 Day 2-4 Days

Protein Linker Substitution Route Tibia Femur Tibia Femur Tibia Femur

BSA-aBP SMCC 11.0 IV 2.2 2.0 3.7 3.7

BSA-aBP* SMCC 11.0 IV 2.2 3.0 7.5 5.9

BSA-aBP SMCC 11.0 SC 2.5 1.9

LYZ-aBP SMCC 3.9 IV 4.1 5.3 5.6 3.7

LYZ-aBP SMCC 3.9 SC 3.1 4.5

Fetuin-aBP SMCC 6.1 IV 0.9 1.0 0.9 0.6 0.6 0.8

Fetuin-aBP SMCC 6.1 IP 0.7 0.6

Fetuin-aBP MMCCH 7.1 IV 1.0 1.0

Fetuin-diBP EDC/NHS 1.2 IV 1.8 2.1

BSA-diBP EDC/NHS 2.6 IV 3.7 2.9

BSA-tetraBP EDC/NHS 3.6 IV 4.1 4.7

BSA-tetraBP EDC/NHS 3.6 IP 3.4 3.7

IgG-diBP EDC/NHS 6.3 IV 2.6 1.5

BMP-2-aBP SMCC ~3 IV 2.8 4.3

ACKNOWLEDGEMENTS

Co-workers and Collaborators

S. Gittens G. Bansal

T.J. Gao G. Wohl

S. Zhang T. Haque

A. Jalil J. Yang

C. Kucharski J.E.I. Wright

J. Wozney (Genetics Institute)

W. Sebald (U. of Würzburg)

R.F. Zernicke, J.R. Matyas (UC)A. Lavasanifar (UA)

Financial SupportCanadian Institutes of Health Research, AHFMR/CFI,Genetics Institute Inc., Millenium Biologics, Whitaker Foundation, NSERC