Scientific update on functional tissue

engineering of cartilage

Department of Biomedical Engineering

Columbia University

New York, NY 10027

Clark T. Hung, Ph.D.

Professor

2016 ICRS Sorrento Italy

Cartilage Repair: Basic Science Towards Clinical Translation

Approach 1

Cartilage

Allografts

Native Cartilage

Tissue

Engineered

Cartilage

Engineered

Cartilage

Approach 2

NIAMS 2002, Lawrence+ 1989

Focal Defect

Defect Repair

Cartilage Repair: Basic Science Towards Clinical Translation

Approach 1

Cartilage

Allografts

NIAMS 2002, Lawrence+ 1989

Focal Defect

Defect Repair

Native Cartilage

Cartilage allograft

Challenges:

• Declining cell viability

• Loss of matrix components

• Decreasing cell synthesis

Standard cartilage storage protocol • Fetal bovine serum (FBS)

• Hypothermal refrigeration (4˚C)

To develop a tissue culture protocol (37˚C) for

preserving cartilage allografts • Serum-free chondrogenic media (CM) (Lima+, OAC 2007)

• Dexamethasone

Brighton+ 1979; Dumont+ 1999; Bugbee+ 2002; Williams+ 2004; Pearsall+ 2004; Williams+2005

Cook+, Vet Surg, 2008

Compromised clinical outcome

FBS

0

0.5

1

1.5

2

2.5

day 14 day 28 day 42

Norm

aliz

ed V

olu

me

† † †

• Serum constructs swell

• Cells are alive in both media

Explants: Serum (FBS) vs. serum-free medium (CM)

FBS CM CM

Bian+ J Biomech 2008; Bian+ Am J Sports Med 2010

37˚C CO2 incubator

53 days preserved OCA

MOPS: serum-free, room temperature preservation media

Stoker+ Tran ORS, 2016

1/25/2016 M

izzo

u B

ioJ

oin

t C

en

ter

Cartilage Repair: Basic Science Towards Clinical Translation

Tissue

Engineered

Cartilage

Engineered

Cartilage

Approach 2

NIAMS 2002, Lawrence+ 1989

Focal Defect

Defect Repair

• Dynamic Loading

• Scale Up & Channels

• Chemical Protection

• Tissue Integration

Functional Tissue Engineering

Hypothesis:

Physiologic Loading enhances chondrocyte

biosynthetic activities and development of

functional tissue construct properties.

Basic Science Tissue

Engineering

Agarose Scaffold: (Benya & Shaffer, 1982; Buschmann et al. 1992, 1995; Sittinger et al., 1994; Rahfoth

et al., 1998; Mauck et al., 2000; Weisser et al., 2001; Cook et al., 1997, 2003, Mouw et al., 2005, Ng et

al. 2010; Selmi et al. 2007, 2008; Clave et al. 2016)

Mauck+ J Biomech Eng, 2000

Dynamic Loading

Axial Loading: unconfined compression

0

100

200

300

400

500

600

700

Day 0 Day 14 Day 28 Day 42 Day 56

EY(k

Pa

)

Free Swelling

• Expansion Media (DMEM, 10% FBS, TGF-1, FGF-2, PDGF-)

• Allogeneic, Passage 2 cells, Loading starting day 28 (10% deformation, 1 Hz, 3h/day)

• Continuous growth factor supplementation (TGF-3)

native knee cartilage

Bian+ Tissue Eng 2010

Dynamic Loading

*

* *

Ng+ Tissue Eng 2010

Our solution is effective

ECHON

Envisioned Product: The proposed

ECHON solution provides an off-the-shelf

consistent living cartilage graft that is

customizable and can be provided on

demand in one surgical procedure.

Engineered OCA

Scale Up Challenges

Digitized Topography

Data

CAD Rendering of

Mold

G-Code Automated MRI

Segmentation

CNC Milling

Machine

Hung+ J Biomech Eng 2002

Roach+ Methods 2014

Nutrient limitations Human Patella

Articular Surface-Sized Constructs

FEBio Modeling Glucose Consumption

Rate

3-piece mold to

create channels Rack and Shaking

Cadaver

hum

an k

nee

P2 cells, EY: 450 kPa; [GAG]: 8.1%

ww; [Collagen]: 2.6% ww

Bian+ OAC, 2009; Nims+ Tissue Eng C

Methods, 2015; Cigan+ Trans ORS 2016

Day 56

Initial diameter: 40 mm; 2.33 mm thick

Cells and Construct

Fabrication

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

14 28 42 56GA

G R

ete

nti

on

Fra

cti

on

Days in Culture

B §

19 15 29 35

0

50

100

150

200

250

300

14 28 42 56

Ey (

kP

a)

Days in Culture

40M

90M

†

*§

† †

*§

* C

4 4 5 3 3 3 2 0

2

4

6

8

10

12

14

0

0,5

1

1,5

2

2,5

28 42 56 14 28 42 56

%/D

0 w

w

GA

G (

µg

)

Days in Culture

Media

Construct

40M

90M D

4 4 5 3 2 3 3

R² = 0,7578

0

50

100

150

200

250

300

0 2 4 6 8

Ey (

kP

a)

[GAG] (%/ww)

Donor 1

Donor 2

Donor 3

Donor 4

Donor 5

A N = 92

Constructs: 3 × 2.3 mm

Translation to Human Constructs Cigan+ Tissue Eng 2016 in press

Recently expired

grafts

*Best to Date: Ey: 367.8±121 kPa, 8.91±0.46 %GAG/ww, 1.67±0.14 %COL/ww, 30M cells/ml (n=6)- AR Tan

Chemical Protection of Engineered Grafts

• Incorporation of dexamethasone delivery for

chondroprotection and pain/inflammation therapy.

Intra-articular steroid injections for

pain/inflammation, 3X per year maximum

due to concerns of local and systemic

side effects. Huebner+ J Orthop Res 2014

Heard+ J Orthop Res 2015

Roach+ Tissue Eng 2016

Dexamethasone Release from within Constructs

Repair with

engineered

cartilage

Large cartilage

focal knee

defect Chondrocytes with dex-PLGA

µspheres in hydrogel scaffold

Roach+ Tissue Eng 2016

Rubin+ J Drug Target 2009

Results: Dex Microsphere Study

Dex microspheres promote growth of

human cartilage constructs and protects

against proinflammatory cytokines

(treatment for 1 week, days 28-35).

Pellet GAG Content

inte

rleu

kin

inte

rleu

kin

inte

rleu

kin

Integrative Repair: Electric field induced migration

Synovial Cells- DiI membrane dye: yellow

Chondrocytes- DAPI nuclear dye: blue

CONTROL E: 25 V/cm

Synovial Explant Placed Adjacent to Cartilage Explant with Central Defect

Top

Side

Stefani+ 2017 ORS submitted

defect defect

Integrative Repair: High Intensity Focused Ultrasound

Nover+ Med Eng Phys 2016; Tan+ 2017 ORS submitted

Albumin solder

Push-out Test

Acknowledgements

NIH R01 AR068133

NIH R01 AR060361

NIH 5P41EB002520

NIH T32AR059038 (AMS, BLR)

NSF GRF (ART, BLR)

Gerard A. Ateshian

Elisa E. Konofagou

James L. Cook

Kacey G. Marra