20.109(F11): MLD

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WF Team Pink/Purple

  • Michelle Fung
  • Luis Juarez
  • Dorma Flemister

Contents

Project Overview

  • Fibrin and its uses as scaffold in tissue engineering and gene delivery.
  • Curing a Heart Disease through the use of Fibrin scaffolds.

Background Information

Title: Strategies for Tissue Engineering Cardiac Constructs to Affect Functional Repair Following Myocardial Infarction Ye, K. Y., & Black, L. D.,3rd. (2011). Strategies for tissue engineering cardiac constructs to affect functional repair following myocardial infarction. Journal of Cardiovascular Translational Research, 4(5), 575-591.

Fibrin is an attractive alternative biopolymer for cardiac tissue engineering because:

  • it can be easily formed into fibrillar networks
  • it can be autologous, since fibrin can be extracted from the patient’s blood, which reduces the chances of the body rejecting the scaffold as a foreign object
  • fibrin gel is very bioactive, stimulating the cells
  • it has been FDA-approved as a surgical sealant

Title: Fibrin gel – advantages of a new scaffold in cardiovascular tissue engineering

The field of tissue engineering deals with the creation of tissue structures based on patient cells. The scaffold plays a central role in the creation of 3-D structures in cardiovascular tissue engineering like small vessels or heart valve prosthesis. An ideal scaffold should have tissue-like mechanical properties and a complete immunologic integrity. As an alternative scaffold the use of fibrin gel was investigated.

  • Yuan Ye, K., Sullivan, K. E., & Black, L. D. (2011). Encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering. Journal of Visualized Experiments : JoVE, (55). pii: 3251. doi(55), 10.3791/3251.

Cardiomyocytes were cultured in a three dimensional hydrogel and studied Fibrin is a naturally occurring blood clotting protein. The paper describes the isolation of neonatal cardiomyocytes form three day old rat pups and their preparation for encapsulation in the fibrin gel constructs. Immunohistological staining was performed to examine the expression and morphology of some essential proteins.

  • Barsotti, M. C., Felice, F., Balbarini, A., & Di Stefano, R. (2011). Fibrin as a scaffold for cardiac tissue engineering. Biotechnology and Applied Biochemistry, 58(5), 301-310.

http://www.ncbi.nlm.nih.gov/pubmed?term=Barsotti%2C%20M.%20C.%2C%20Felice%2C%20F.%2C%20Balbarini%2C%20A.%2C%20%26%20Di%20Stefano%2C%20R.%20(2011).%20Fibrin%20as%20a%20scaffold%20for%20cardiac%20tissue%20engineering.%20Biotechnology%20and%20Applied%20Biochemistry%2C%2058(5)%2C%20301-310

This review shows some cardiac bioengineering uses of fibrin as a cell delivery vehicle and as an implantable biomaterial. Fibrin is great to be used since it is a natural biopolymer that has properties like biocompatibility, ease of processing, and a potential for incorporation of cells and cell mediators. Fibrin has found many applications in tissue engineering because it can be combined with cells, growth factors, or drugs.


Tissue Engineering Strategies for Cardiac Regeneration

"Functional vascularization – with the establishment of blood supply – remains a major unsolved problem of cardiac tissue engineering, and tissue engineering in general. Several different approaches are currently under investigation, ranging from the engineering of prevascularized tissues with capability for connection to the blood supply of the host, to the induction of vascularization by host cells using bioactive materials"


Title: Fibrin: A Versatile Scaffold for Tissue Engineering Applications [MF 04 Dec 2011]

"Fibrin as an ideal scaffold has a significant disadvantage: the gradual disintegration of the gel with subsequent loss of shape and volume before the proper formation of tissue-engineered constructs. This disadvantage can be overcome by optimizing the concentrations of fibrinogen, calcium ion (Ca2þ), and pH, by using a lower cell density or by adding specific protease inhibitors. Stability can also be enhanced by using highly denatured densely cross-linked FMBs or by combining fibrin with an artificial supporting polymer. Fibrin gel shrinkage and its low mechanical stiffness represent other disadvantages of fibrin scaffolds in some tissue engineering applications, which can be controlled by cross-linking or by combining fibrin with other artificial scaffolding material."


Tissue response to poly(ether)urethane-polydimethylsiloxane-fibrin composite scaffolds for controlled delivery of pro-angiogenic growth factors

"IHA of subcutaneously implanted samples showed that at 7 and 14 days the PEtU–PDMS/Fibrin + GFs scaffold induced a statistically significant increase in number of capillaries compared to bare PEtU–PDMS scaffold. IHA of ischemic hind limb showed that at 14 days the capillary number induced by PEtU–PDMS/Fibrin + GFs scaffolds was higher than that of PEtU–PDMS/Fibrin scaffolds. Moreover, at both time-points PEtU–PDMS/Fibrin scaffolds induced a significant increase in number of capillaries compared to bare PEtU–PDMS scaffolds."


Composite scaffold provides a cell delivery platform for cardiovascular repair

Development of a Fibrin Composite-Coated Poly(e-Caprolactone) Scaffold for Potential Vascular Tissue Engineering Applications

[02 December 2011]

Nanowired three-dimensional cardiac patches [JALC]

General Book of Info!!! Biodegradable Polymers in Clinical Use and Clinical Development

Agi's Reccommended book!!! Biomaterials science : an introduction to materials in medicine

Research Ideas on Problem and Goals

  • Identify a currently unsolved problem for fibrin as scaffold for use to treat cardiac disease and try to fix it.
  • Fibrin gels are ideal for use as a scaffold but they have two disadvantages. They shrink in size due to fibrin degradation and initially have low mechanical stiffness which do not allow for direct implantation of the new formed structures.
    • It is possible to use a combination of high porous biodegradable scaffolds with the fibrin gel as a cell carrier.
  • Composite/hybrid scaffold of fibrin + synthetic (or other) material?
    • Chitosan?
    • Polyurethane?

[LAJ/02 December 2011].

These four papers have a great idea. Let's explore and adjust to fit our needs. Please read one or more of these papers and post a summary as it relates to what we want to do.

I found a great paragraph explaining the use of Chitosan as the scaffold and Fibrin as a kind of glue to attach the heart cells to the scaffold. Assays at 3 weeks were: 1) survival, 2) vascularization, 3) organization, 4) functionality, 5) stability, 6) precondition to modulate contractile properties of transplanted myocardial cells.

EHT is Engineered Heart Tissue (a patch of cells). This is the paragraph I found in "Fibrin as a scaffold for cardiac tissue engineering."

"An EHT was also obtained with a miniaturized and automated method, by mixing neonatal rat heart cells, fibrin, and Matrigel [14]. This method may provide advanced in vitro models for drug testing and disease modeling. Cardiac tissue has also been successfully engineered using cardiac myocytes both in vitro and in vivo [47]. Engineering 3D cardiac tissue is usually limited by the difficulty of nourishment supply to cells in the center of the construct. In these articles, an intrinsic vascular supply was incorporated in a contractile 3D cardiac tissue, using a suspension of neonatal cardiac myocytes in fibrin gel. The suspension was used to cellularize porous scaffolds made of chitosan and opportunely molded. Fibrin gel was required to retain the cells within the constructs and resulted in the formation of contractile constructs. At 3 weeks, survival, vascularization, organization, and functionality of transplanted myocardial cells was demonstrated. The same authors further developed the engineering of this 3D heart muscle, demonstrating its stability in response to stretch protocols and engineering a functional cellbased cardiac pressure-generating construct [85]. The bioengineered heart muscle containing fibrin was also preconditioned, modulating its contractile properties for possible implantation onto injured hearts without cell shock [86]."


Paper 1: http://www.ncbi.nlm.nih.gov/pubmed?term=hansen%2C%20A.%2C%20Eder%2C%20A.%2C%20Bonstrup%2C%20M.%2C%20Flato%2C%20M.%2C%20Mewe%2C%20M.%2C pages 35-44

Paper 2: http://www.ncbi.nlm.nih.gov/pubmed/15998220 pages 803-813

Paper 3: http://www.ncbi.nlm.nih.gov/pubmed/18854950 pages 191-201

Paper 4: http://www.ncbi.nlm.nih.gov/pubmed/18500554 pages 1372-1382


TO DO: Make a list of Heart Tissue Properties and compare those properties to those of the following possible materials:

  1. - dextran
  2. - carboxyl methyl celulose
  3. - chitosan
  4. - elastin
  5. - silicon

[LAJ/02 December 2011].

Fibrin

[MF/04 December 2011]

  • natural biopolymer derived from fibrinogen
  • utilizes mammalian clotting mechanisms to form a stable, biodegradable polymer network

Fibrin glue

  • fibrinogen and thrombin solutions are mixed together during injection
  • thrombin enzyme converts fibrinogen into fibrin monomers
  • fibrin is crosslinked with Factor XIII to form network
  • crosslinking can occur in <1 min, depending on the concentration of thrombin

Fibrin glue clinical applications

  • adhesive or sealant
  • augmenting sutures, improving seals in cartilage repair

Domb, Abraham J. (Editor); Kumar, Neeraj (Editor); (). Biodegradable Polymers in Clinical Use and Clinical Development. Hoboken, NJ, USA: Wiley, 2011. p 640. http://site.ebrary.com/lib/mitlibraries/Doc?id=10466737&ppg=658 Copyright © 2011. Wiley. All rights reserved.

Uses for fibrin constructs

  • tissue engineering and drug delivery scaffolds
  • tissue engineering: cartilage, bone, vascular grafts, cornea, muscle, more

Challenges for using fibrin

  • weak mechanical strength
  • fairly rapid degradation
  • tendency to shrink upon culture with cells

Solutions

  • improve mechanical strength through enhanced crosslinking and composite scaffolds with synthetic and biological polymers
  • enhance the crosslinking and grafting of copolymers to reduce gel shrinkage and slow degradation
  • enhance the bioactivity of fibrin by covalently crosslinking synthetic peptides, growth factors, and other biomolecules to the fibrin scaffolds

Domb, Abraham J. (Editor); Kumar, Neeraj (Editor); (). Biodegradable Polymers in Clinical Use and Clinical Development. Hoboken, NJ, USA: Wiley, 2011. p 641. http://site.ebrary.com/lib/mitlibraries/Doc?id=10466737&ppg=659 Copyright © 2011. Wiley. All rights reserved.

Fibrin

  • excellent biocompatibility, biodegradability, and injectability

Use of Fibrin in Tissue Engineering: existing applications

  • microbeads of a fibrin (including a fibrinogen) that are biologically active and extensively crosslinked and consist of cells bonded to these microbeads
  • a composite skin material composed of the hypodermal cell layer and epithelial cell layer attached on fibrin scaffold
  • tissue-engineered autocorneal epithelium consisting of fibrin biostent and the patient’s autocorneal epithelium stem cells and corneal epithelium cells anchored on it
  • biocompatible material suitable for promoting cell growth, wound healing, and tissue regeneration: for use on implantable devices and tissue and cell scaffolding
  • use in surgical adhesive or sealant, as well as in peripheral nerve regeneration and angiogenesis

Domb, Abraham J. (Editor); Kumar, Neeraj (Editor); (). Biodegradable Polymers in Clinical Use and Clinical Development. Hoboken, NJ, USA: Wiley, 2011. p 690. http://site.ebrary.com/lib/mitlibraries/Doc?id=10466737&ppg=708 Copyright © 2011. Wiley. All rights reserved.

[MF/04 December 2011]

[MF/05 Dec 2011]

Title: Fibrin: A Versatile Scaffold for Tissue Engineering Applications

Materials:

  • commercially available fibrinogen and thrombin combined to form fibrin hydogels
    • commercial fibrinogen contains other plasma proteins that can contribute to rapid degradation
  • autologous fibrin

Fibrin hydrogel as scaffold advantages:

  • high seeding efficiency
  • uniform cell distribution
  • adhesion capabilities
  • possibility to be autologous

Fibrin as scaffold disadvantages:

  • gradual disintegration of the gel, causing loss of shape and volume before proper construct formation
  • gel shrinkage and low mechanical stiffness

Solutions:

  • optimizing the concentrations of fibrinogen, calcium ion (Ca2+), and pH
  • lower cell density
  • adding protease inhibitors specific for plasmin and matrix metalloproteinases
  • using highly denatured densely cross-linked FMBs (fibrin micro beads) or by combining fibrin with an artificial supporting polymer
  • cross-linking or by combining fibrin with other artificial scaffolding material


Title: An Introduction to Biomaterials

Fibrin advantages

  • natural role in wound healing
  • availability relative ease of isolating and purifying fibrinogen and thrombin


Changing fibrin properties

  • fibril thickness, homogeneity, porosity
  • salt influences porosity
  • Factor XIII affects elasticity
  • fibrinogen concentration influences breaking strength and adhesiveness
  • microstructure correlates to ratio of fibrinogen to thrombin concentration
    • lower fibrinogen and thrombin concentration -> more open, homogeneous
    • higher fibrinogen and thrombin concentration -> dense and nonhomologous


Fibrin pore size and sizing:

More reading:

[MF/05 Dec 2011]

Elastin

[DCF/04 December 2011]

C. Barbié, C. Angibaud, T. Darnls, F. Lefebvre, M. Rabaud, M. Aprahamian. Some factors affecting properties of elastin-fibrin biomaterial.

http://www.sciencedirect.com/science/article/pii/0142961289900847

The benefit of a Fibrin-Elastin material is that it can present three different mechnical qualities:

  • tense and very strong,
  • very elastic and less strong,
  • very elastic and strong.

As a consequence, this material can be used in any situation, since the sulphur derivative concentration allow to prepare artificial matrix very closed to in vivo tissue.

F. San-Galli, G. Deminière, J. Guérin, M. Rabaud. Use of a biodegradable elastin—fibrin material, Neuroplast®, as a dural substitute.

http://www.sciencedirect.com/science/article/pii/0142961296859084

Project Details and Methods

  • Immunofluorescence

Calcium Imaging (for Electroconductivity Study)

  1. http://www.springerlink.com/content/w2762812hpn670nu/#section=830833&page=1
  2. During each heart beat, a time-dependent transient increase in intracellular Ca2+ concentration (“[Ca2+]i transient”) occurs and is responsible for activating contraction in a process called excitation–contraction coupling. The [Ca2+]i transient is triggered by the cardiac action potential (AP) and spreads through the heart as the AP is propagated.


H&E - Can be used to image living cells and see the thickness of the tissue caused by amount of blue stain present. Nanowires will appear as black dots since they wil not be imaged/ produce a stain.

TEM

AFM

Live/Dead Assay (for cell viability). ==> The materials we are using are not toxic to the cell.

To test for elasticity analysis and success:

  1. DMA - Dynamic Mechanical Analysis.

Predicted Outcomes

  • If all goes well, we’ll have increased efficacy in Cardiac Cell Therapy
  • If nothing goes well, we’ll end up knowing more potential uses of Fibrin, and more of its properties for use in Tissue Engineering.

Necessary Resources

  • $$
  • Cells, materials, knowledge of assays
  • Eventually animal trials (Heart Surgeons)
  • Eventually Human trials (more $)
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