Prevascularized Acellular Lung Construct for Tissue Engineering

Ryan NagaoNov 03, 2010

Objective

• Create a construct that can be connected to the host vascular system for rapid anastomosis and tissue survival for large defects

Problem to Address

• Only successful tissue engineering technologies have been avascular (cartilage) or thin membranes (skin/bladder)

• Complex tissues require a equally complex system for nutrient distribution and oxygen transport, i.e., the vascular system

Brittberg, M., et al. New England Journal of Medicine, 1994.

Kirsner, R.S., et al. Trends in Biotechnology, 1998.

Previous Tissue Engineering Attempts

• Implanted constructs were reliant upon ingrowth of the host vascular system

• Natural angiogenesis and penetration of the construct is too slow (tenths of microns per day) to prevent necrotic core formation in constructs > 200 µm

Vascularization Methods

Current Solutions to Vascularization

Jain RK., et al. Nature Biotechnology, 2005

Use of Scaffolds

• Still relies upon host vasculature• Leads to necrotic core formation

Druecke, D., et al. Journal of Biomedical Materials Research Part A, 2004

Use of Angiogenic Factors

• Still relies on the host vasculature

• Faster vascular ingrowth than scaffolds alone

• Neovascularization is not mature and can lead to thrombi, and leaky vasculature

Prevascularization in vivo

• Construct seeded with cells is grown in benign region of the host by incorporating the host vascular system via a vascular axis

• Rapid anastomosis• Results in multiple surgeries and the loss of a vascular axis in the

host

Kneser, et al. Tissue Eng, 2006.

Bach, et al. J Cell Mol Med, 2006.

Prevascularization in vitro

• Construct is seeded with a co-culture system including tissue specific cells and endothelial cells

• Still requires host vascular ingrowth in the exterior of the construct

• Vascular system is not mature and might not be functional• Complex culture process

Unger, et al. Biomaterials, 2007.

Prevascularizing Acellular TissueEx

cisi

on

Decellularization

Perfusion culture

EC ce

ll se

eding

Aim 1• Preservation and

characterization of microvasculature using decellularization

Aim 2• Re-

endothelialization of a decellularized vascular construct

Aim 3• Microsurgical

anastomosis and patency of a re-endothelialized vascular construct in vivo

Specific Aims

Aim 1• Preservation and

characterization of microvasculature using decellularization

Aim 2• Re-

endothelialization of a decellularized vascular construct

Aim 3• Microsurgical

anastomosis and patency of a re-endothelialized vascular construct in vivo

Specific Aims

Specific Aim 1

• Preservation & Characterization of Microvasculature Using Decellularization– Evaluate the efficacy of different decellularization methods and

solutions• Cellular removal• ECM composition• Patency

– Test immunogenicity in vivo

Decellularizaton Methods

Thermal

Freeze-thaw•Non-specific•Cellular components persist•Damaging to ECM•Difficult to control

Sonication•Non-specific•Damaging to ECM•Difficult to control

Delivery• Diffusion• Perfusion

Detergent Selection• Ionic• Non-ionic• Amphoteric

Timing

• Clinically relevant

Chemical Decellularization

Detergent SelectionAmphoteric Detergents

SB-10

SB-16

Non-ionic Detergent

Selected for OA process

Triton X-200

Anionic Detergents

Sodium dodecyl sulfate (SDS)

Triton X-100

Optimized Acellular Protocol

1. Wash in ddH2O for 7 hrs

2. Wash in SB-10 solution for 15

hrs

3. Rinse 1 X 15 min in 100 mM Na/ 50 mM PBS

6. Wash in SB-10 solution for 7 hrs

5. Rinse 3 X 15 min in 100 mM Na/ 50 mM PBS

4. Wash in Triton X-200/ SB-16

solution for 24 hrs

7. Rinse 1 X 15 min in 100 mM Na/ 50 mM PBS

8. Wash in Triton X-200/ SB-16

solution overnight

9. Rinse 3 X 15 min in 50 mM

Na/ 10 mM PBS

•Lung tissue perfused with PBS following heparin injection (1000 U/kg) until blanched

•Left lung lobe isolated and decellularized using the following diffusion-based procedure:

Structure Composition Patency Organization Immunogenicity

neurophilosophy.files.wordpress.com/

Aim 1: Experimental Strategy

Tom WJ., et al. IEEE T Med Imaging, 2008

Structural Composition

Hematoxylin and eosin staining of fresh lung (top) and following OA process (bottom) at different magnifications (4x, 10x, 20x, 40x). All nuclei are removed from the OA process; however, an intact ECM persists.

Biochemical Composition (ECM)

Immunostaining against laminin (red) and fibronectin (green) with DAPI (blue) of native rat lung (left) and standard OA rat lung (right) reveals an intact ECM remains following the standard concentration OA procedure on rat lung. Scale bar = 200 µm

Cellular Composition

Immunostaining for CD-31 (green) with DAPI (blue) of fresh (A) and decell (B,C) lung following OA processing at low concentration (B) and standard concentration (C). Standard concentration removes all cellular components. Bar, 100 µm

A B C

Biochemical Composition

ECM

Target Antibody Optimized Ratio Permeablize

Laminin Anti-Laminin 1:100 NoFibronectin Anti-Fibronectin 1:500 NoCollagen-I Anti-Collagen 1:100 No

Cellular

Target Antibody Optimized Ratio Permeablize

Endothelial glycoprotein

CD-31 1:50 Yes

VEGF receptor FLK-1 1:300 NoSmooth muscle Anti-α-SMA 1:75 Yes

Vessel Patency

Cresyl violet injected into the pulmonary artery of the right lung of a rat following OA processing. Note: dye expanded to all the lobes of the lung except for the accessory lobe (arrow).

A B

SEM image of fresh lung reveals the presence of an intact vascular network down to the capillary scale. An acceleration voltage of 2 kV was used (Scale bars: A=10 µm, B=40 µm)

Fractal Analysis

• Way to measure geometries that are not of integer order

• Describes self similarity

• Describes complexity

http://www.proetcontra.com/wp-content/uploads/fractal_geometry.jpg

Fractal Dimensions are Present in Nature

• Trees, coastlines, and vascular beds have all been characterized using fractal analysis

– Vessels, Df = ~1.7– Capillaries, Df = ~2– Tumors, Df = ~1.9

• Implement a box-counting algorithm on a skeletonized figure

• This yields regressions lines whose slope will give the fractal dimension of your figure

• Can also get information about branching from the skeletonized image through an analysis algorithm

Fractal Image Processing

Implantation

Criteria

• Preservation of 85% ECM

• Removal of 90% cellular debris

• No statistical difference in vascular structure

Immunohisotchemistry

• Killer t-Cells• Macrophages• Excessive

inflammation (H&E)

• Isograft comparison

Criteria for Implantation

Iterative process

Aim 1• Preservation and

characterization of microvasculature using decellularization

Aim 2• Re-

endothelialization of a decellularized vascular construct

Aim 3• Microsurgical

anastomosis and patency of a re-endothelialized vascular construct in vivo

Specific Aims

Specific Aim 2

• Re-endothelialization of a Decellularized Vascular Tissue Construct – Determine extent to which hMSCs transdifferentiated toward

endothelial lineages will form functional endothelium in the lumen of an acellular vascular construct

– Seed cells in the vascular axes of construct• Penetration• Proliferation

– Apply pulsatile flow• Patency• Cellularity• Differentiation

Cell Source Extraction

Cell morphology in fibrin and PEGylated fibrin after 7 days of culture. Human MSCs in (A) 2D culture; (B) fibrin only; (C) NHS-PEG fibrin; (D) BTC-PEG fibrin; (E) SC-PEG fibrin; (F) SMB-PEG fibrin. Immunofluorescent staining for CD31 (G), VWF (H), VE-cadherin (I) and Flk-1 (J) of hMSCs embedded in BTC-PEG fibrin (as in D). Nuclear counterstain with DAPI. Bar, 10 μm (Zhang, 2010)

H

D

C

E

F

B

G

JI

A