Engineering Tissue-To-Tissue Interfaces and the Formation of Complex Tissues

Engineering Tissue-to-Tissue Interfaces and the Formation of Complex Tissues

Helen H. Lu, Ph.D.

US Frontier of Engineering (USFOE) September 15, 2012

Biomaterials and Interface Tissue Engineering Laboratory Department of Biomedical Engineering Columbia University in the City of New York Tissue Engineering

Skalak 1988, Langer and Vacanti 1993

Business Week, July, 1998 Tissue Engineering: the Next Generation Engineering Complex Tissues

• Assemble or connect more than one type of tissue • Interfaces between these different tissue types are critical for engineering collective functionality

Tissue Engineering, 2006 Challenges in Orthopedic Tissue Engineering

• Soft tissues - Articular cartilage - Ligaments - Tendons

• Lack of graft-bone integration –Compromises long term functionality

• Challenge – How to achieve BIOLOGICAL FIXATION of soft tissue to bone? Interface Tissue Engineering

TissueSignificance-to-Tissue Clinical Challenge Interfaces• Soft tissues have limited • Ligament-to- regeneration Interface Cell-Cell bonepotential interface Characterization Interactions

– Anterior cruciate • Lackligament of tissue (ACL) graft-to-bone Scaffold integration Design • Tendon -to-Bone • interfaceBridging tissues to form organ In Vitro In Vivo • Osteochondralsystems Testing Testing interface How to Connect a Rope to the Wall?

• Multiple Tissue and Cell Types (Cooper and Miscel, 1970, Arnoczky et al, 1993, Niyibizi et al., 1996, Visconti et al., 1996, Thomapoulus et al, 1999, Benjamin et al, 2002, Wang et al., 2006) – Ligament (L) – Fibroblasts L FC B – Fibrocartilage – Fibrochondrocytes • Non-Mineralized Fibrocartilage (FC) • Mineralized Fibrocartilage (MFC) L – Bone (B) – Osteoblasts Neonatal Bovine ACL FemurInsertion – TrichromeFC Stain • A gradient of cellular, chemical NFC MFC and mechanical properties B ACL – Minimize the formation L of stress concentrations (Butler et al., 1978, Woo et al., 1988, FC Matyas et al., 1995, Gao et al., 1996) Tibia – Load transfer between 200 μm soft and hard tissues (Woo SL, et al. 1988) Neonatal BovineB ACL Insertion – von Kossa Stain Age-Related Changes

Picro Sirius Stain under polarized light, 50x. Wang et al., JOR 24:1745-1755, 2006 Interface Characterization Mechanical Properties

• Mechanical properties of the interface is not known • Ultrasound Elastography (Konofagou et al, Ultrasound in Medicine and Biology, 1998) – Provides information on tissue mechanical properties – Permits the imaging of strain distribution within tissues – RF data acquired at 5 MHz, 54 frames/s during loading – Speckle tracking analysis Spalazzi et al, J. Orthop. Res., 2006 Gradient of Mechanical Response

100 FI • Displacement Map 200 300 ACL 400 FI 500 TI – Variations in displacement 600 700 ACL from ligament to bone 800 900 TI 1000

1100 • Strain Map 5 15 25 35 Spalazzi et al., JOR, 2006 – Yellow – Red  Tensile Strain (+εyy)

– Light Blue – Dark Blue  Compressive Strain (-εyy) 0.1 0.16 0.080.09 20 0.14 0.060.08 40 0.12 0.07 0.04 60 0.06 0.1 0.02 0.05 80 0 0.08

0.04 Depth (mm) Depth Depth (mm) Depth 100 -0.02

0.03 0.06 Displacement Displacement (mm) -0.04 0.02 120 0.04 -0.060.01 140 0.02 -0.080

160 -0.1-0.01 0 5 15 25 35 5 15 25 35 Length (mm) Length (mm) Interface Characterization Mechanical Properties

• Unconfined compression and image analyses L FC B (Schinagl et al., 1999, Wang et al., 2002)

• Region-dependent 50 NFC MFC 45 Incremental variations in 40 Strain 0-5% 35 Incremental displacement and 30 Strain 5-10% 25 Incremental incremental strain, Strain 10-15% 20 Incremental

across insertion 15 Strain 15-20% Displacement(μm) 10 – Strain: FC > B 5 0 0 200 400 600 800 1000 1200 1400 Sample Depth (μm) (Moffat et al., ASME 2005 Masters Research Award) Region-Dependent Mechanical Inhomogeneity

• Axial stress and Young’s modulus exhibit depth- dependent variations for tibial and femoral samples

– σMFC > σNFC and EMFC > ENFC

0.14 Femoral NFC Femoral MFC 1.00 0.12 Femoral NFC Femoral MFC 0.10 0.80 0.08 0.60 0.06 0.40 0.04

Axial Stress (MPa) Stress Axial 0.02 0.20

0.00 Young's(MPa) Modulus 0.00 0.00 0.05 0.10 0.10 Resulting Strain Resulting Strain (n=4) (n=4) Moffat et al., PNAS, 2008 Structure-Function: Mineral

NFC

MFC

Bone How to Regenerate the Interface?

• Mechanism of interface Bone regeneration is not known

• Neo-fibrocartilage formation was FC observed where soft tissue and bone are in direct contact (Rodeo et al., 1993; Weiler et al., 2002; Liu et al., 1997; Yoshiya et al., 2000; Anderson et al., 2001; Panni et al., 1997; Chen et al., 2003; Grana et al., 1994) Tendon – Non-anatomical – Fibrocartilage can be regenerated Rodeo et al, 2005

• Examine the role of fibroblast- osteoblast interactions in initiating fibrochondrogenic differentiation • Stem cell niche at various tissue-to-tissue junctions Fibroblasts Osteoblasts BMSC Cellular Interactions

• Osteoblast-Fibroblast Co-Culture – Homotypic and heterotypic interactions – Paracrine/Autocrine interactions

Microchannel design

(Wang et al., JOR, 2007) Cellular Interactions

• Fibroblast Osteoblast interactions during co-culture

Day 0, 5x Day 11, 5x DayDay 22, 5x Cell-Cell Osteoblast-Fibroblast Interactions Interactions

• Co-culturing osteoblasts and fibroblasts results in changes in respective phenotypes (Wang et al., JOR, 2007) – Suppression of cell proliferation – Suppression of osteoblast ALP activity – Suppression of mineralization A co-culture well – Increased ectopic fibroblast after cell seeding mineralization and ALP activity (Wang et al. 2007) – Expression of fibrocartilage interface- related markers MSC • Triculture studies revealed that osteoblast-fibroblast interaction Ob Fb promoted the fibrochondrogenic differentiation of mesenchymal Tri-Culture Model stem cells (Wang et al. IEEE, 2007) (Wang et al., 2007)

Cellular Interaction Mechanism

• Mode of cellular interactions – Secreted soluble factors and cytokines • Chemical messengers directing both local and systemic cellular communications (Canalis et al., 1988; Bhatia et al., 1999; Lu et al., 2007) Fibroblasts Osteoblasts – Direct physical contact between cells • Important in the dynamic regulation of cell-cell adhesion, communication and tissue development (Gumbiner, 2005; Kii et al., 2004 ; Leckband et al., 2006)

Soluble Factor • Maintenance and repair of tissue Communication could be tightly controlled by these cellular interactions (Waldman et al., 2003; Alsberg et al., 2002; Lu&Jiang, 2005)

Physical Contact Scaffold Bioinspired Design Criteria Design

0.09 20 Collagen Mineral 0.08 • Three phases to support ligament-, 40 ACL 55 ACL 9 Interface 0.07 250 25050 8 fibrocartilage- and bone-like tissues 60 0.06 45 ACL 7 500 500 0.05 – Controlled matrix heterogeneity 80 40 6 35 0.04

Depth (mm) Depth FC FC 100750 750 30 5 0.03

• Continuous & interconnected phases Bone micrometers micrometers 25 4 0.02 1201000 1000 to support heterotypic interactions of 20 3 0.01 140 15 interface-relevant cell populations 1250 1250 2 0 Bone 10 Bone 160 1 -0.01 1500 5 1515005 25 35 • Gradient of mechanical properties ACLLength (mm) 125 250 375 500 125 250 375 500 comparable to those of the ligament micrometers micrometers insertion site

• Biodegradable for host-mediated FC interface regeneration

Spalazzi et al, 2006, 2008; Moffat et al., 2008 Bone Wang et al, 2006, 2007, Tsai et al., 2005, Lu and Jiang, 2005 Scaffold for Interface Regeneration • Biomimetic Triphasic Scaffold A – Phase A: Polylactide-co-glycolide (PLGA) B – Phase B: PLGA Microspheres C – Phase C: PLGA–Bioactive Glass Composite (Lu et al, JBMR 2003; Lu et al., Biomaterials, 2005) 2 mm

• Co-culture of human osteoblasts and fibroblasts on 3-D scaffolds Seeding Density: 5x104 cells/cm2 Phase A Phase B Phase C

hFB hOB

Spalazzi et al, Tissue Engineering, 2006 In Vitro Testing Cell Migration and Growth

Phase A, Day 0, x10 Phase B, Day 0, x10 Phase C, Day 0, x10

Phase A, Day 28, x10 Phase B, Day 28, x10 Phase C, Day 28, x10 In Vivo Cell Tracking

Phase A Phase B Phase C

Week 2 Week

200 μm

fibroblasts chondrocytes osteoblasts

Week 4 Week

200 μm In Vivo Mechanical Properties

140 Acellular *p<0.05 120 Co-Cultured n=6

100

40 Compressive Modulus Compressive (MPa) 20

0 Week 0 Week 4 Week 8 Phase-specific Mineral Deposition

• Mineralized matrix formation Phase A observed in all groups

• Zonal distribution of mineral confined to Phase C

TriAcellular-Cultured Co-Cultured Tri-Culture Tri-Cultured Phase B Week 4, 5x Von Kossa Stain

Week 4 MicroCTWeek 4 MicroCT 3D ReconstructionsPhase C Multi-Tissue Formation

Compositionally Distinct & Structurally Contiguous

Week 4 5x Clinical Application • Incorporation of multi-phasic scaffold and onto ACL reconstruction grafts – Synthetic tissue engineered grafts – Soft tissue autografts or allografts

• Induction of Femur fibrocartilage formation on the tendon graft

Graft – Scaffold • Biological Fixation Complex • Multi-tissue formation Tibia

*modified from www.nucleusinc.com What is Next: Stem Cells

Phase A Phase B Phase C

fibroblasts chondrocytes osteoblasts

Week 4 Week

200 μm

• Feasibility – multiple cell sources required

• Stem Cell-Mediated Interface Regeneration - Fibroblasts - Osteoblasts - Chondrocytes

Growth Factor Gradient

Peret and Murphy, Adv. Funct. Mat., 2008 Gradient Scaffolds: Growth Factors

Polymer + Solvent + Growth Factor (TGF- or BMP-2) + Nanophase Material Aqueous non-solvent Sinter Microspheres To frequency generator

Piezoelectric transducer

Custom nozzle Scaffoldless Approach

Medial Isolate BMSCs In vitro 1-Month Engineered BLB

In vivo 6-Month Engineered BLB Tibia 1000 Femur In vivo 9-Month EngineeredLigament BLB Bone Contralateral ACL Pathway Pathway Lateral 100

GM GM + dex

BLB construct 10

Ligament(N/mm) monolayer

1 Geometric Stiffness Stiffness Geometric

SYLGARD 3-D bone construct

Ma, et. al. Tissue Engineering. Part A: 2011 What is NEXT?

• Higher mineral to matrix 7 Femoral Insertion 6 * ratio (mineral content) in Tibial Insertion bone and mineralized 5 fibrocartilage 4 • Mineral content higher in tibial 3 2

and femoral bone compared Ratio Mineral:Matrix to femoral insertion 1 0 Light Image Collagen Content Mineral:Matrix Ligament NFC MFC Bone

ACL 5555 99 250250 5050250 250

4545 77 500500 500 4040500 66 3535 750750 750 750 FC 3030 55

25 micrometers

micrometers 25 44 micrometers 10001000 micrometers 1000 1000 2020 33 15 1250 15 1250 1250 1250 22

Femoral Insertion Femoral 1010 5 11 Bone 15001500 15005 1500

125125250250375375500500 125 250125375250500375 500 micrometersmicrometers micrometersmicrometers Engineering Gradient Scaffolds

Electrospun Gas- Salt- Freeform Topography Hydrogels Nanofibers Foamed Leached Fabrication Library

Combine different fabrication approaches

• Chatterjee et al., • Ramalingam et • Chatterjee et al., J • Simon et al., Rev • Pirano et al, • Kumar et al., Biomaterials, 2010 al., J Biomat Funct. Biomat., in Sci Instrum, 2007 Lab Chip, 2012 Biomaterials, • Kloxin et al., 2011 Appl, in press press • Yang et al., Adv Biomaterials, 2010 • Li et al., Nano • Harris, et al. Mater, 2008 Reviews: Letters, 2009 Langmuir, 2006 • Simon et al., Comb Chem H T Scr, 2009 • Simon & Lin-Gibson, Adv Mater, 2011 • Seidi et al., Acta Biomateria, 2011 Gradations in Mineral

Ca/(Ca+C) (At%) Ca/(Ca+C) 15

0 0 2 4 6 8 10 12 Position (mm)

Li et al, Nano Letters, 2009 Cell-Mediated Gradient • Runx-2 transfection gradient to control fibroblast mineralization

Phillips et al, PNAS, 2008 Bioinspired Scaffold: Complex Tissues

Multi-Tissue Regional Biomimetic Multi-Tissue Multi-Tissue Native Collagen Multi-phased Formation Formation Interface Distribution Scaffold In Vitro In Vivo

Biological Graft Fixation and Integrative Soft Tissue Repair

Moffat et al, CSM, 2009 Challenges in Interface Tissue Engineering • Mechanism of Interface Regeneration – Biological: cell to cell interactions – Chemical: growth factors or proteins – Physical: mechanical, electrical

• Structure-Function Relationships at the Soft Tissue-Bone Interface

• Biomimetic and Bioactive Scaffold Design - Strategic Biomimecry

• Controlled Heterogeneity on Tissue Engineered Scaffolds In Vivo

ACKNOWLEDGEMENTS

• Presidential Early Career Award (PECASE) • Whitaker Foundation BME Research Award • Wallace H. Coulter Foundation Early Career Faculty Award (Phases I and II) • Musculoskeletal Transplant Foundation • National Football League (NFL) Charities • National Science Foundation (GK-12) • NIH-NIAMS - R01 AR055280 – R21 AR052402, R21 AR056459