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Unformatted text preview: BioE10: Lecture 12 Professor Irina Conboy Tissue engineering/Regenerative Medicine
3D polymeric biomaterials for engineering tissues and organs ex-vivo Objectives:
To understand the current progress and limitations as well as future
strategies in biomaterials and tissue engineering. To understand the
effect of mechanical stretching and shearing on cells of interest.
g Biological fundamentals:
Integrins, growth factors, RGD; Extra-cellular matrix (ECM)
Cytoskeletal fillamets: actin, myosin, microtubules, microfilaments What are desirable properties of biomaterials (structural part) Lutolf and Hubbell, 2005 The native ECM is the prototypical hydrogel that
regulates cell function on many length scales. A:
Integrin-binding with ECM proteins (green ligands
and tan receptors), growth factor sequestration
within proteoglycans (red), and cell– cell contact
via cadherins (purple) occur on the scale of tens of
nanometers to microns.
B: Migration, which is critical in tissue
regeneration cancer metastasis, and wound healing,
initiates on the scale of tens to hundreds of microns.
Paracrine signaling that directs differentiation (pink
growth factors) and proliferation (red growth
factors) is also mediated on this length scale.
C: Tissue homeostasis, development, and wound
healing are regulated over hundreds of microns to
centimeters. Here, we illustrate neutrophils being
recruited to the site of a wound in the epithelium. Tibbitt and Anseth, 2009
Biotechnology and Bioengineering Synthetic–biologic hydrogels that incorporate several
well-defined and orthogonal chemistries serve as robust
ECM mimics for 3D cell culture. Depending on the
application, it may be advantageous to incorporate cell- or
user-defined regulation of the material properties to
emulate the native dynamic environment.
However, in many cases, synthetic hydrogels that
incorporate both cell- and userdefined chemistries will be
necessary. Here, we illustrate a cell cleaving MMP
degradable crosslinks (yellow circles) that allow it to
access sequestered growth factors ( ) and integrinq
binding sites, such as RGD (green circles). Ultimately,
this cleavage allows cell motility and the deposition of
ECM proteins (orange fiber).User-defined chemistries,
such as photodegradable crosslinks (blue ellipses) and
post-gelation attachment of RGD to the network
backbone, afford facile control of the dynamic
biochemical and biophysical properties of the gel, thereby
directing cell attachment and motility. Further, exogenous
application of enzymes (brown) can allow user-defined
release of sequestered growth factors. Professor Kevin Healy:sIPN hydrogels
Tibbitt and Anseth, 2009
Biotechnology and Bioengineering Let’s contrast Smart Biomaterials with General types of tissue analogs
Think of the advantages of smart biomaterials that are provided by genetics and
molecular biology toolkit 1. Single purpose acellular
transient graft BioBrane is a nylon material that contains a gelatin that interacts with
clotting factors in the wound. That interaction causes the dressing to
adhere better, forming a more durable protective layer.
Integra is a two-layered dressing. The top layer serves as a
temporary synthetic epidermis; the layer below serves as a
foundation for re-growth of dermal tissue. The underlying layer is
made of collagen fibers that act as a lattice thro gh which the bod
can begin to align cells to recreate its own dermal tissue. 2. Multi-purpose acellular graft;
Hydrogel Salubria for cartilage
and blood vessel replacement;
neuron guidance and drug
d li Electron micrograph of BD
P M t i P tid
Hydrogel Hydrogel called S l b i (d i d f
l ll d Salubria (derived from th L ti words f " f " and "h lth ”) G
d for "safe" d "healthy”). Georgia T h R
i Tech Research
Corporation; Dr. David Ku (Georgia Tech).
To date, tests in rats, dogs and sheep show that Salubria is biocompatible: Platelets do not adhere to it in
significant quantities, and thus the chance of blood clots is greatly reduced (the problem with Dacron, which
surgeons have used for artery replacement in the abdomen and legs since its development in the 1950s). In
addition these studies suggest Salubria can serve as a nerve guide to create a physical bridge that could
dramatically increase the speed at which severed nerves heal.
As an implantable drug delivery system, Salubria may work for many drugs, such as insulin and morphine, that
need to be injected. Its advantage for such is that it is hydrophilic (attracted to water), rather than hydrophobic
(resisted by water) like the silicone.
silicone 3. Living tissue equivalents Vascular?
Innervated? Organogenesis Inc. is selling its Apilgra, a living "human skin equivalent" to treat wounds and ulcers in Canada
and the company is seeking approval for sales in the U.S. Additionally, LifeCell Corporation makes implantable
human tissue for use in reconstructive surgery and burn treatment. Doctors are able to take a postage stampsized piece of skin from the patient and grow the skin under special tissue culture conditions. From this small
piece of skin, technicians can grow enough skin to cover nearly the entire body in just 3 weeks. Cultured skin has
been available in the U.S. for a decade. Artificial skins are only a temporary fix; the patient will still need skin
grafts. However, with the use of artificial skins means a thinner skin graft, which allows the donor site and the
patient to heal faster with fewer surgeries. The use of artificial skins has not yet been perfected and they are not
right for every burn patient. Additionally scarring still results, but it may be less severe. Q: why a sheet of skin cells does not suffice as tissue replacement? 4. Nucleic Acid and protein delivery systems=scaffolds Q: Could this approach be used for delivery of shRNA?
(A) Inductive factors (red circles) may be embedded or encapsulated within hydrogels, which may, in turn, be used to form 3-D
scaffolds that are capable of releasing factors at the site of implantation. The highest factor concentration exists within the scaffold,
with lower concentrations found within the surrounding tissue. Delivered factors target a variety of different cell populations (e.g.,
y y ,
g) ( )
myocytes, neurons, fibroblasts or osteoblasts) for various applications (e.g., muscle, nerve, bone, wound healing). (B) Substrate
immobilization is characterized by factors being bound to a biomaterial. The interaction between the factor and biomaterial can be
(top): (1) K on > K off, such that the factor is effectively bound irreversibly; (2) Kon=Koff, where the factor associates and dissociates
from the surface at roughly equal rates; and (3) Kon < Koff, such that the factor is loaded onto the biomaterial and dissociates to
serve as a delivery vehicle. Cells interact with the immobilized factors upon interaction with the biomaterial (bottom). Mechanical forces applied to a cell will propagate via cytoskeletal filaments
stretching C.elegans C.elegans Shear stress Cellsalive.com Figure 7-31 Actin filaments in mammalian cell Intermediate filaments in mammalian cell Microtubules in mammalian cell Figure 7-30-Table 7-3
http://www.sumanasinc.com/webcontent/animations/content/intermediate_filaments.html http://www.wiley.com/college/pratt/0471393878/student/animations/actin_myosin/actin_myosin.swf http://www.sinauer.com/cooper/4e/animations1203.html Why mechanical integrity is important for a cell? Why did cytoskeleton evolve? C.elegans C.elegans All the elements form one mechanical
network that functions as a prestressed
scaffold that can withstand mechanical
stress. The main cytoskeletal components
(microfilaments, microtubules and
intermediate filaments) are key elements in
linking the cellular surface to the nucleus. Cell
adhesion molecules (
l (such as cadherins and
integrins) bind to the cytoskeleton through
linker proteins, in this way forming
connections to adherent cells or to the
extracellular matrix. On the other side, the
cytoskeleton is linked to the nuclear
membrane and the underlying nuclear lamina. Q: Why would you predict and how
would you check for a non-specific
response to stretch (consider that
there are particular stress response
genes, e.g. hsp-70). Correct signal transduction, cell
division and cell metabolism,
including ATP production can
only occur with undisturbed
cytoskeleton and cell shape Integrating natural and synthetic components in structural part of biomaterial.
Specific example: Am J Physiol Cell Physiol 288: C30–C38, 2005. Laminin YIGSR How this biomaterial could be optimized?
Polymer chemistry, peptide density, peptide
composition. Cell stretching in tissue engineering and regenerative medicine What is the main difference between
embryonic cells (mesoderm)
dedicated to make lung tissues (or
those, making heart valves and
arteries) and many adult cells (e.g.
hMSCs, liver cells, etc.)?
Evolutionary adaptation to use
“stretching” as a relevant signal (of
relatively limited modulation). Schematic map of the mouse tip gene structure.
Coding (black) and noncoding (green) exons are
shown as vertical boxes. The initiation codon
(ATG) is also shown. Approach:
1. Get rid of common mRNAs expressed by both stretched and non-stretched cells
2. DNA microarray or similar methods
3 Analyze gene structure
4. Candidate gene approach:
A) Expression in correct place/time/condition
B) Over-expression enhances function: Over-expression substitutes for stretching;
Tip 1 myogenesis; Tip 3 adipogenesis; Tip 2 is not involved in this
C) RNAi inhibits function: Tip1 and Tip3 RNAi targeting inhibits myogenic and
adipogenic cell fates (respectively) Q: What is the significance of Figures F and G? Generally speaking, gradients
of biochemical molecules
(proteins mostly) and
signal transduction networks
control cell-fate determination
of stem cells and orchestrate
tissue and organ formation,
maintenance and repair. These
biochemical cues work best in
the physiological range of the
matrix rigidity and mechanical
recapitulating these molecular
signals is the key for creating
smart biomaterials (Next
module: Stem Cell
Technologies) Q: What cells-tissues are
likely to recognize
stretching as a
(rather than a trauma)?
Q: Would you use stretching
for in vitro
A. Beta cells from pancreas
(lost in type I diabetes)?
B. Heart valves?
C. Liver cells?
D. Arteries? What is the best source of cells for tissue engineering?
Current approach: align cells using patterned substrate (e.g. smooth muscle cells aligned on
collagen grooves; hMSC-derived cells on pattered substrates, etc). Thakar et al., Biochem Biophys Res Comm (2003) Q: What will likely happen to the engineered tissue after transplantation in vivo:
A. To differentiated cells?
B. To di idi
B T dividing precursor cells?
C. Is self-renewing required for successful tissue transplantation? What is the best source of cells for engineered tissues? Myofiber: a unit of regeneration in adult muscle
Basal lamina Satellite cell Laminin/PI Myonucleus Myofiber
Plasma membrane Deciphering and reconstructing
the biochemical and structural
components of muscle ECM Using cells produced in culture by muscle stem (satellite) cells Testing this principle for engineering regenerating tissue. Work in progress. Skeletal muscle fibers (green) that are capable of self-assembly and of continuous
regeneration (dividing cells are in red, BrdU incorporation), are manufactured in
biochemically defined 3D micro-environment.
H. Silva and I. Conboy; unpublished Conlusions:
An ideal engineered tissue has chemically defined ECM that is embedded
with biologically active molecule enabling predictably regulate tissue
regeneration and/or function.
The optimal source of cells, as well as the optimal rigidity and mechanical
stress conditions are determined based on the physiological ranges of
signals that operate during normal organogenesis and tissue remodeling. Check your understanding:
You should be able to compare the functional properties of several
classes of biomaterials discussed in this lecture;
You should be able to propose strategies for optimal control of cell
adhesion and cell functionality in an engineered tissue
You should understand the criteria for selection of cells for tissue
You should understand the function and structure of cytoskeletal
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