Prevascularized acellular lung construct for tissue engineering
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Prevascularized Acellular Lung Construct for Tissue Engineering. Ryan Nagao Nov 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.

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Prevascularized acellular lung construct for tissue engineering

PrevascularizedAcellular Lung Construct for Tissue Engineering

Ryan Nagao

Nov 03, 2010


Objective
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
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

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

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


Previous tissue engineering attempts
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


Current solutions to vascularization
Current Solutions to Vascularization

Jain RK., et al. Nature Biotechnology, 2005


Use of scaffolds
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
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
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

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

Kneser, et al. Tissue Eng, 2006.


Prevascularization in vitro
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 tissue
Prevascularizing Acellular Tissue

Decellularization

EC cell seeding

Excision

Perfusion culture




Specific aim 1
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




Detergent selection
Detergent Selection

Amphoteric Detergents

Selected for OA process

SB-10

Non-ionic Detergent

SB-16

Triton X-100

Anionic Detergents

Triton X-200

Sodium dodecyl sulfate (SDS)


Optimized acellular protocol
Optimized Acellular Protocol

  • 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:


Aim 1 experimental strategy
Aim 1: Experimental Strategy

neurophilosophy.files.wordpress.com/

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


Structural composition
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
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
Cellular Composition

A

B

C

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



Vessel patency
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).

B

A

  • 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
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
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




Criteria for implantation
Criteria for Implantation

Iterative process



Specific aim 2
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 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)


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