Tissue Engineering

1
Tissue Engineering

• Goal and history
• Natural examples of tissue engineering
• Examples of tissue engineering
• Survey of applications
• Case study of small-diameter vascular grafts

2
Some Goals of Tissue Engineering

• donors ltlt potential recipients

Langer and J. Vacanti Tissue Engineering.
Science 260 920-6, 1993.
3
A Definition

• Tissue engineering is an interdisciplinary field
  that applies the principles of engineering and
  the life sciences towards the development of
  biological substitutes that restore, maintain, or
  improve tissue function.
• Langer and J. Vacanti Tissue Engineering.
  Science 260 920-6, 1993.
• But

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My definition

• Making or modifying a tissue for some useful
  purpose.
• But

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How to make or modify tissues

• In vitro tissue engineering

• In vivo tissue engineering
• Bone defects
• Ex vivo tissue engineering
• Extracorporeal liver support

Langer and J. Vacanti Tissue Engineering.
Science 260 920-6, 1993.
6
History of tissue engineering

• For modern history (1985-2003)
• NSF report The Emergence of Tissue Engineering
  as a Research Field.
• http//www.nsf.gov/pubs/2004/nsf0450/start.htm
• My summary of the recent history of TE

1980 1990 2000 2010
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Early tissue engineering
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Replicating Complex Biological Environments

• Cell culture
• Tissue culture
• Organ culture
• Organism culture
• Ecosystem culture

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Organ Culture A Sister of Tissue Engineering

• In practice - shared techniques.
• From a historic perspective - shared goals.

10
Alexis Carrel

• Pioneering work on cell culture
• 1912 Nobel Prize for Medicine
• Technique for vascular anastomosis
• Enabled vascular grafting and organ
  transplantation
• Early work on organ storage (e.g., cold storage)
• Organ culture

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Carrels Goals for Organ Culture

• A tool for scientific inquiry- phenomenon of
  regeneration, growth, nutrition, and internal
  secretions could be rendered more comprehensible
  by studies with prolonged organ perfusion.
• Autografts for clinical use if it were
  possible to culture whole organs, a diseased
  organ or part thereof could be removed, treated
  outside the body, and grafted back into the
  patient.
• Malinin TI and Lindergh CA Organ culture and
  perfusion by the Carrel method. In Alexis
  Carrel Papers of the Centennial Conference at
  Georgetown University.

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Carrels Goals for Organ Cultureare also the
goals of TE

• A tool for scientific inquiry- phenomenon of
  regeneration, growth, nutrition, and internal
  secretions could be rendered more comprehensible
  by studies with prolonged organ perfusion.
• Auto- or allografts for clinical use if it
  were possible to culture whole organs, a diseased
  organ or part thereof could be removed, treated
  outside the body, and grafted back into the
  patient.
• Malinin TI and Lindergh CA Organ culture and
  perfusion by the Carrel method. In Alexis
  Carrel Papers of the Centennial Conference at
  Georgetown University.

13

• Carrel noted that the culture of organs is, from
  a technical point of view, very difficult
• Contamination

14

• Carrel noted that the culture of organs is, from
  a technical point of view, very difficult
• Contamination! But why?

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• Carrel noted that the culture of organs is, from
  a technical point of view, very difficult
• Contamination! But why?
• Cell culture small distances diffusion OK
• Organ culture large distances diffusion
  inadequate
• Need a sterile perfusion system.

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Interdisciplinary BE Research

• Charles Lindbergh
• Engineer
• 1927
• 1930

1935
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Lindbergh Apparatus

• All glass
• Semi-closed system
• Pulsatile flow of liquid is generated by cyclic
  changes in gas pressures in several chambers.

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Carrel-Lindbergh Organ Culture

• 1935-9 used to conduct 898 organ perfusion
• Thyroid
• 3 Weeks
• Maintained normal histology
• Secreted hormone

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Carrel-Lindbergh Organ Culture

• Heart
• Heart removed from refrigerated primate carcasses
• 90 cadaver hearts responded to perfusion by
  contractions
• 1hr culture strong A/V contractions
• gt 1hr frequency and amplitude of contractions
  decreased
• Perfusion at decreased T ? longer survival ex
  vivo.
• 37C ? 1 day
• 22C ? 2 day
• 12C ? 10 days!

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Past as Prologue

• Prediction Organ culture and tissue engineering
  will remain entwined.

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Organ Culture ?Tissue Eng.

• Positive control
• Source of info technical and scientific
• Alternative route

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Organ Culture ?Tissue Eng.

• Organ culture a positive control for in vitro
  TE conditions.

Native tissue ?
Quality
Time
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Organ Culture ?Tissue Eng.

• Organ culture a positive control for in vitro
  TE conditions.

Native tissue ?
Quality
Time
24
Organ Culture ?Tissue Eng.

• Organ culture a positive control for in vitro
  TE conditions.

Native tissue ?
Quality
Time
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Organ Culture ?Tissue Eng.

• Organ culture a source of ideas

Native tissue ?
Quality
Time
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Organ Culture ?Tissue Eng.

• Organ culture a source of ideas

Native tissue ?
Quality
Time
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IGF in TE cartilage
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Tissue Engineering

• Goal Tissue replacement/repair Science
• Approaches (in vitro, in vivo, and ex vivo)
• History
• Next Natural examples of tissue engineering

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Natural examples of TE
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Normal development
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Alternative development

• Transgenic animals

gene
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Alternative development

• Cloning
• Tells about the potential of DNA

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Alternative development

• Tells about the potential of early embryo cells

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Embryonic stem cells are totipotent

• Have potential to differentiate to any cell type
• Great proliferation potential
• Can develop teratomas when implanted in vivo

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Later in development, most/all cells loose this
potential

• Cell culture
• Cell transplant studies
• Implications for tissue engineering
• Origin of cells
• Precursor / stem cells
• Simple division
• Permanent cells

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Differentiation and determination

• Differentiation when a cells expresses outward
  markers of a given cell type. (e.g., mature beta
  cell insulin)
• Determination - when a cell has committed to a
  particular differentiation fate.

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Test for determination

• Figure 21-7. The standard test for cell
  determination. MBC

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Determination and positional value
http//www.ncbi.nlm.nih.gov/books/bookres.fcgi/mbo
c4/ch21f8.gif
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Normal development

• Increase in organism size

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The four essential processes by which a
multicellular organism is made

• http//www.ncbi.nlm.nih.gov/books/bookres.fcgi/mbo
  c4/ch21f1.gif

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The four essential processes by which a
multicellular organism is made

• http//www.ncbi.nlm.nih.gov/books/bookres.fcgi/mbo
  c4/ch21f1.gif

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Specialization
Genetic
Environment

• http//www.ncbi.nlm.nih.gov/books/bookres.fcgi/mbo
  c4/ch21f1.gif

http//www.ncbi.nlm.nih.gov/books/bookres.fcgi/mbo
c4/ch7f72.jpg
43
Cell interaction
Induction - when one cell/tissue alters the
developmental fate of an adjacent cell or tissue
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Cell interactions
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Natural examples of TE
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Regeneration Liver as a model
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• Homeostasis Regulates blood sugar, lipids and
  cholesterol, amino acids.
• Detoxify remove hormones, hemaglobin, and toxins
  from the blood.
• Forms of red blood cells in the young embryo.
• Makes plasma proteins including albumin and
  clotting factors.
• Stores and makes vitamins

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Structure of liver

• Homeostasis Regulates blood sugar, lipids and
  cholesterol, amino acids.
• Detoxify remove hormones, hemaglobin, and toxins
  from the blood.
• Forms of red blood cells in the young embryo.
• 9. Making heparin this is a substance that
  prevents the blood from clotting as it travels
  through the blood system.
• Makes plasma proteins including albumin and
  clotting factors.
• Stores and makes

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The livers regeneration potential

• Resection studies
• Transplant studies
• Dogs
• Humans
• Gene defect and rescue studies
• Mechanism local factor or systemic factor?

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Symbiotic circulation
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Hepatocyte growth factor

• Stimulates hepatocyte proliferation in culture
• Increased following partial resection of liver
• A simplified conceptual model
• Body tissue makes HGF and delivers to circulation
• Liver tissue removes HGF from circulation
• Liver grows if HGF gt some value
• Simple test of conceptual model?

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Does HGF stimulate liver growth in vivo?

• HGF ?
• HGF Collagenase ?

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Implications of liver regeneration for Tissue
engineering

• Why even need TE?
• Massive acute problems
• Chronic insults

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Implications of liver regeneration for Tissue
engineering

• Type of system needed?
• Massive acute problems
• Chronic insults

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Extracorporeal device

•  Algenix Inc.'s LIVERX2000 System
• http//biomed.brown.edu/Courses/BI108/BI108_2002_G
  roups/liver/webpage/liverxpg.htm

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Natural examples of TE

• Soluble factor (HGF)
• ECM
• Negative factors for regen
• Potential of DNA
• Differentiation and determination / Plasticity of
  cells.
• Role of TF (myoD)
• Role of cell shape
• Role of cell-cell interactions

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Wound healing skin as a model system
Scar
Not healing
For a good review of this topics, see Wound
Healing--Aiming for Perfect Skin Regeneration by
Paul Martin http//www.sciencemag.org/cgi/content
/full/276/5309/75
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Structure of skin
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Stages of wound healing

• Fibrin Clot
• Recruitment of Inflammatory cells
• Reepithelialization

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Fibrin Clot

• Stops bleeding
• Formation of provisional matrix
• Source of mitogens and chemotaxic factors
• PDGF

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Recruitment of Inflammatory cells

• Recruited by factors derived from clot and
  infections.
• Recruitment mediated by endothelial cells.
• Neutrophils
• Monocytes
• Secrete factors that stimulate next step.

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Reepithelialization

• Epithelial cells migrate (crawl) into wound
• Alter integrin expression
• Secrete proteases
• t-PA
• MMPs
• As horizontal surface area covered, a striated
  epithelium is formed.

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Better healing

• Embryonic wounds heal without scar.
• Why? (environment, tissue, ?)
• Different mechanism?

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Embryonic vs. Adult

• Different mechanism?

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Molecular mediators of wound healing

• Transforming growth factor beta
• Stimulates fibroblasts in the wound to become
  myofibroblasts.

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Examples of TE skin (science)
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Tissue engineered skin (clinic)
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Tissue-engineered skin (clinic)
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Blood vessel