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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