Intro Tissue Engineering E1 Study Guide

HW 1 Answers

Describe the differences between primary cells and cell lines.

Describe the different types of cell lines. List the benefits and
drawback from each type of cell.
Primary cells are obtained directly from the tissue of the animal and
primary cultures consist of a heterogeneous cell population. Cell lines are cells
that have been passaged from an initial cell culture to a subculture. Cell lines
tend to have a more homogeneous cell population and the number of cells
increases with number of passages.
The different types of cell lines are:
1. Finite: Cell lines that have a limited life span when after a certain number
of passages the cells lose their ability to proliferate.
2. Continuous: Cell lines that continue to proliferate through numerous
passages.
Primary cells are beneficial because they delineate accurate cell behavior
close to the in vivo system and they are widely accepted as a good predictor
of cell performance. However, the drawback is primary cells are limited in
supply, expensive and difficult to obtain (through elaborate isolations) and the
heterogeneous characteristics of such cultures makes it ambiguous to
interpret cell behavior from a particular cell type.
Cell lines have the benefit of being cost-effective, robust, homogeneous
and easily expandable in cell cultures with unlimited supply, requiring less
demanding technical procedures. However, cell lines have the capability to
mutate over time and do not show innate cell behavior accurately.

Describe two clinical scenarios: one in which you would use an

embryonic or induced progenitor stem cell and one in which you
would use a mesenchymal stem cell.
Embryonic stem cells (ESCs) or induced progenitor stem cells (iPSCs)
would be used for a tissue in which a biopsy would not be readily performed,
and when a tissue is being repaired that would not benefit from an MSC (ie.
Not a tissue that an MSC can be differentiated towards)
Mesenchymal stem cells (MSCs) are stem cells that can be derived
from adults/no-embryos and are harvested commonly from the bone marrow,
femoral defects, fat and muscles. Such cells can differentiate into multiple
tissue types (limited differential potential) but not any cell type like the
embryonic or induced pluripotent stem cells. MSCs would be used to repair a
tissue that arises from the mesenchyme (ie. Bone, ligament, cartilage,
muscle, tendon, etc)

Describe the process to test the stemness of a mesenchymal stem

cell.
In order to test the stemness, mesenchymal stem cells can be
isolated and differentiated into different tissue lineage such as bone, cartilage
and fat.
With appropriate growth factors, mesenchymal stem cells can be
differentiated into bone, cartilage or fat. Induced osteogenesis in
mesenchymal stem cells is indicated by an increase in alkaline phosphatase
and calcium deposition. In order to show the differentiation of MSCs into
chondrocytes, collagen type III upregulation for cartilage formation and
morphological changes of MSCs can be assessed.
MSCs can also be

differentiated into adipocytes and adipogenesis can be demonstrated by

staining with Oil red O that shows the formation of red lipid vacuoles.
Name two methods to separate cell types in a heterogeneous cell
population. Explain the fundamental approach each method uses
and give brief details describing each method.
Two methods to separate cell types in a heterogeneous cell population are:

Facs (Fluorescence-activated cell sorting): In this technique, fluorescently

labelled antibodies are attached to the target cells. The presence of the label
gives a precise measurement to sort out target cells.
Cell separation using cell density gradient is usually performed through test
tube centrifugation. When there is a centrifugal force field, cells of different
densities continuously separate out.
HW 2 Answers
Describe porogen leaching as a technique for scaffold synthesis. Give
two positive and two negative aspects about the technique.
Porogen leaching is a common fabrication technique used to make
porous scaffolds. Soluble particle such as porogen, is incorporated into a
polymeric or polymer/ceramic construct. After the construct hardens, it is
placed in an aqueous solution to remove the porogen which is soluble in water
(but the scaffold is not soluble in water). Removing porogen leaves pores in
place to make the scaffold porous.
The advantages of this technique are:
We can control the pore size by controlling the size of porogen.
We can control the extent of porosity by controlling the amount of
porogen used.
The disadvantages are:
May result in the lack of interconnected pore structure because
porogens do not necessarily bound to each other and therefore may
not form interconnected pores.
There may be chances that there is incomplete porogen removal and in
such cases, the porogen may be released in vivo once scaffold
degrades in vivo.

Describe thermally induced phase separation.

In thermally induced phase separation, polymer is put into a solution
and is slowly precipitated out of the solution by using a non-solvent and a
solvent exchange process. In this process, the non-solvent replaces the
solvent, forcing the polymer to precipitate out of solution. Appropriate
temperature and timing may yield a nanoscale scaffold structure.
Describe one method for functionalizing a scaffold.
One method of functionalizing a scaffold would be to slightly degrade
the outer layer of a polymer through hydrolysis. The slight degradation would
increase the available hydroxyl (OH) and carboxyl (COOH) groups on the
surface which in turn would change the surface charge of the scaffold to a
more negative state. This would encourage the deposition of calcium
phosphate on the surface.
Another method may be to add carbon nanotubes to the polymer that
would institute a charge that may have the same effect as degrading the
polymer by breaking down polymer chains to create more functional groups
with a net negative charge.
Describe selective laser printing (sintering) as a method for making
scaffolds.

Selective laser sintering (SLS) uses a focused laser beam to sinter

areas of a loosely compacted powder. In this method, a thin layer of powder is
spread evenly onto a flat surface with a roller mechanism. The powder is then
raster-scanned with a high-power laser beam. The powder material that is
struck by the laser beam is fused, while the other areas of powder remain
dissociated. Successive layers of powder are deposited one on top of another,
until an entire part is complete. Each layer is sintered deep enough to bond it
to the preceding layer. However, it poses significant material constraints for
scaffold fabrication and is currently mainly used to make calcium phosphatebased scaffolds for bone engineering. SLS has the disadvantage that
incorporation of sensitive biomolecules is difficult because of the need to
locally heat the powder layer so as to sinter it.

Quiz 1 Answers

From the paper entitled Intramuscular Delivery of 3D Aggregates of

HUVECs and cbMSCs for Cellular Cardiomyoplasty in Rats with
Myocardial Infarction presented by Nicole Piscopo, what were two
noted differences in the results from dissociated cells vs 3D
aggregates, in vitro or in vivo?
o The dissociated formed tubeular structures on day 1 then
regressed, while the 3-D aggregates kept adhering to each
other. The 3-D aggregates also had smaller defect sizes in one
experiment. 3-D aggregates showed better LVFS improvement,
better LC contraction, and inhibition of heart failure progression.
From the paper entitled Temporal and Spatial Changes in CartilageMatrix-Specific Gene Expression in Mesenchymal Stem Cells in
Response to Dynamic Compression presented by Jonathan Turner,
what was the overall conclusion from the paper and how could that
inform the clinical treatment of cartilage injuries?
o

From
the
paper
entitled
Hypoxia-induced
therapeutic
neovascularization in a mouse model of an ischemic limb using cell
aggregates composed of HUVECs and cbMSCs presented by Mark
McKenna, what were two experimental objectives of the study?
o

Experimental objectives: promote angiogenesis in critical limb

ischemia, and to test the capacity of hypoxia-induced 3D
HUVEC/cbMSC aggregates to secrete angiogenic GFs in a controlled
manner

From the paper entitled Utilization of Stem Cells in Alginate for

Nucleus Palposus Tissue Engineering presented by Stephanie
Knowlton, what was the purpose of the alginate hydrogel, and
what was its effect on tissue repair?
o

Using mechanical stimuli in cartilage regeneration applications are

useful only if used at appropriate stages of chondrogenesis. In the
clinical setting, the doctor would only use mechanical stimulation
during a certain stage of the healing cartilage

The alginate hydrogel serves as a good cell carrier. It was used at the
control and it was seen that alginate alone did not have any beneficial
effect on tissue repair.

Finite: Cell lines that have a limited life span when after a certain
number of passages the cells lose their ability to proliferate.
o

Continuous cells continue to proliferate

Lecture: Cells in Tissue Engineering

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Cell Types
o Organized into different tissue types (epithelia, CT,
nervous, musculoskeletal, blood, sensory, stem cells)
o Stem Cells: Cells with ability to copy themselves and form
other cell types of the body. Multiple pathways contribute
to proliferative and differentiation behavior (not completely
understood).
Types
Totipotent: any type of tissue
Pluripotent: any cell from the three germ layers
except placenta and umbilical cord.
Multipotent: MSCs, HSCs. MSCs are harvested
from bone marrow, fat, muscle can be tested
for their multipotency by isolating cells and
differentiating them (different stains for
Osteogenesis/adipogenesis/chondrogenesis.
Benefit of use: Good measure of in vivo
environment, aids in development of materials
that induce growth rather than purely repair
materials. Drawbacks are isolation difficulty
and maintenance
Induced pluripotent: any cell from the three
germ layers. De-differentiated or cell
reprogrammed back into pluripotent state
Drawback: Cells may turn into tumors and
grow uncontrollably

Cell Sources
o Primary Culture: Cells isolated from tissue and plated to
confluence for the first time. Heterogenous cell population
because theyre taken directly from tissue
Benefit: Very accurate assessment of cell behavior,
closest to in vivo as you can get. Use of primary cells
gets you more approval from the science crowd.
Drawback: Expensive, time-consuming to do, cell
populations are heterogeneous, meaning it will be
difficult to isolate individual cell responses.
o Cell Line: Given to cells once primary culture has been
passaged or subcultured.
Finite Cell Line: Will eventually senesce (age),
therefore losing the ability to proliferate after a while.
Telomeres shorten
Continuous cell line : Can be virally induced, can
occur spontaneously will the cell line and examples
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include cell lines that dont senesce such as tumor

cells
Benefits: Cost-effective, quick, easily expandable,
less technical demand
Drawbacks: Less-indicative of natural in vivo
behavior, cells can possibly mutate over time
Cell isolation/culturing/maintenance
o Primary Culture: Can filter hetereogenous population by
controlling duration of adhesion, FACs, or density-gradient
separation via centrifugation (percoll gradient)
o Cell
acquisitionexpansion/maintenancedifferentiation
Culture medium for expansion (salts, AAs, serum,
GFs)
Differentiation: marked by a slowing of proliferative
stage. Induced by:
adding growth factors/chemical stimuli
co-culturing with another cell feeder
Physical means (mechanical
stimulation/culturing topography/bioreactors)

Lecture: Scaffolds in Tissue Engineering

Materials
o Polymers
Degradable: Erosion, breakdown, disintegration,
resorption. Can be highly tuned by controlling the
rate, mechanism, nature of degradation. The nature
of degradation depends on the application. Bulk
degradation (hydrophilic). Rate of water entry
exceeds rate at which material breaks down, material
maintains general shape, then collapses from within.
Surface degradation (hydrophobic): Surface
erodes and exposes inner layers, ideal for drug
delivery apps. Degradation occurs through hydrolysis
(cleavage of molecule by water) and enzymatic
degradation.
Polylactide: Degradation time>2yrs. Drug dlvry
potential. Degrades to lactic acid. Hydrophobic
Polyglycolide: Hydrophilic, degrades to glycolic
acid
PLGA: Can vary degradation rate by altering
PLA:PGA ratio. The smaller the ratio, the more
crystalline and shorter the degradation.

Polyanhydride: Surface eroding polymer (good

for drug dlvry)
Polyphosphazenes: Phosphorus backbone
allows for secondary functionality: conjugation
with amino acid ester, glyceryl, and glycolic
side groups.
Non-degradable
Polyethylene: (Low density: flexible vs. UHMWP:
high mechanical properties. UHMWP found in
acetabular cup liners, joints in prosthetic knees
o Hydrogels: Water-swollen cross-linked polymeric
structures held together by covalent bonds and H-bonding.
Made of natural (collagen, gelatin, agarose) material
Drug Delivery: Drug put in matrix, then swollen, drug
is eluted as the hydrogel swells. The hydrogel
collapse releases the drug
Cell Delivery: Encapsulating the cell, degradation to
let ECM take over
o Ceramics
Degradable vs. Non-degradable: Usually see
resorption or dissolution of materials into
body/incorporation of the material into the body
tissue.
Resorbablity: SA of material increaseCrystallinity
decreasescrystal size decreasesionic
substitutions of molecule are made
Calcium Phosphate (HA): Highly crystalline,
poor resorbablility, comes in powder, coating,
blocks.
Bioactive Glass: Releases ions which interact
with neighboring bone tissue, creating a
bioactive layers that bonds permanently to
surrounding new bone.
Bioactive Surfaces: Rich in reactive, negatively
charged groups (Si-Oh, Ti-OH, PO4-H2). These
negative groups attract cations (Ca+), making
the surface more positive, which then attract
anions (PO4), forming the apatite layer
Bone Cements: Ceramic powder (HA, TCP or
Calcium Carbonate) + Sodium phosphate
solution. Non-porous, brittle, strong in
compression but weak in tension.
Critical Aspects of designing a scaffold
o Aspects independent of tissue
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Biocompatible
Biodegradable
Tunable (Mechanical, structural, topographical,
degradable)
o Aspects dependent on tissue
Mechanical properties
Degradation time
Pore structure
Surface functionality
Bulk shape
Scaffold Techniques
o Sintering
Heating spheres leads to fusion
Smaller spheresmore contact surface
areastronger structures. A tradeoff between cell
occupancy and strength of sintered scaffold
o Porogen Leaching: Particle template (any particle) is
immersed into polymer solvent. Polymer cures, and particle
template is leached out using chemical solution. Cheap but
does not make interconnected pore structures which are
crucial for cell trafficking and integration for regenerative
applications
o Rapid prototyping (3-D printing, SolidFreeForm,
bioprinting): Building structures layer by layer, allowing for
greater control over architecture (pore size, shape, volume)
Soft Lithography
Photolithography
3-D/ bioprinting: laser binds powder into 3-D
structure. Building layer-by-layer constructs to
contain multiple types of conduits which will give rise
to vessels composed of multiple cell types
o Microfluidics: Manipulating fluids at them micron scale.
Laminar flow dominates over turbulent flow at this scale.
Used mainly for analysis tools (diagnostic, examining
cell/tissue interface behavior). Ex. Making a microfluidic
device that models capillaries.
Nanofiber scaffolds: Created using electrospinning or selfassembled networks.
o Benefit of a nanostructured surface: Can affect cellbiomaterial interactions (cell-adhesion, orientation,
mobility, specificity). General notion that cell adhesion
increases with surface roughness
Alignment: Aligned vs. random orientation : affects
strength

o Thermally Induced Phase separation: forcing a polymer to

precipitate out of solution
o CNTs: Very strong, light, conducts.
Doping sintered matrices with CNTs: Improves
mechanical properties and cell integration.
Bladder acellular matrix conjugated with BFGF for bladder
regeneration
Question 1: Why is bladder a good candidate organ for the
decellularization process?
o Simple structure, SMCs and other bladder-associated cells
are more likely to enter the tissue. Decellularizing the
bladder also leaves behind the ECM intact as well as some
residual growth factors that werent washed away. Bladder
cells will naturally recognize the bladder structure and
seed there
Question 2: What purpose did cross-linking the bladder ECM
have? Was it beneficial?
o The purpose of crosslinking the BAM/bFGF scaffold was to
achieve a controlled release of bFGF since it is so hard to
retain in a scaffold.
o Crosslinking the bladder was beneficial because it allowed
for the growth factors within in the scaffold to be retained
more easily. It increased biostability, mechanical strength
and the crosslinking promoted cell migration, viability and
differentiation.
Tough and flexible CNT-polymeric hybrid scaffolds for
engineering cardiac constructs
Question 1: what advantage does having aligned nanofibers
have for cardiomyocyte development?
o Allows better alignment for cells
o Better signal propagation as a result of more direct cell-cell
signaling since the cells are aligned
o Microenvironment is more natural
Question 2: which is a more preferable outcome, requiring a
higher or lower excitation threshold? Why? Was this goal
achieved?
o A lower excitation threshold is preferred because a lower
excitation threshold mean less stimulation is required for
the heart to beat synchronously
o This was achieved with the CNT-PG hybrid scaffold-it
mimicked natural tissue the best

Microfluidic techniques for development of 3D vascularized

tissue
Question 1: what was one advantage of prevascularization
over vasculogenesis/angiogenesis techniques?
o An advantage of prevascularization over angiogenssis
techniques is that channels made under this method can
be immediately perfused with growth media/blood. In
addition, oxygen delivery and waste removal can be
performed continuously as in the body.
Question 2: compare and contrast the subtraction method to
the addition method for generating vascular networks.
o Additive: pre-formed 2D layers of hydrogels with imprinted
channels are stacked; these layers are bonded together by
partial melting or hydrogel fusion giving a tightly sealed 3D
network.
o Subtractive: the subtractive method works by forming a
hollow structure or network of channels by removing
sacrificial material from a hydroge
Effects of biologic scaffolds on human stem cells and
implications for CNS tissue engineering
Question 1: what types of ECM were used and why would this
influence stem cell growth?
o Brain, urinary bladder, spinal cord. ECM influences stem
cell growth based on ECM properties that coordinate cell
signaling, chemotaxis, differentiation, etc.
Question 2: why were the cells passaged below 100%
confluence?
o Cells were passaged below 100% confluence so that cells
can be examined to test proliferation of cells and not only
their differentiation behavior.
Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue
Engineering
Question 1: what are two benefits and two drawbacks of using
bone marrow mesenchymal stem cells (BM-MSCs)?

o Two benefits of using BM-MSCs include that they can survive/proliferate in

vivo and that they provide rapid pain relief and water content elevation in
DDD patients. Two drawbacks of using BM-MSCs include a limited cell
source and low cell acquisition rate.

Question 2: What was the purpose of the Alginate Alone group

of implantations? That is, what was the purpose of having one
group dedicated to just implanting the cell carrier with no cells?
o The alginate hydrogel serves as a good cell carrier. It was used at the
control and it was seen that alginate alone did not have any beneficial
effect on tissue repair

Temporal and Spatial Changes in Cartilage-Matrix-Specific Gene

Expression in Mesenchymal Stem Cells in Response to Dynamic
Compression
Question 1: what is the overall conclusion about compression
and chondrogenesis?
o The overall conclusion about compression and
chondrogenesis is that compression can have a beneficial
effect on chondrogensis, if applied at the right time
(appropriate stages). This right time to compress would be
useful in helping to support or maintain a chondrogenic
phenotype.
Question 2: what was the rationale behind these studies? What
clinical question(s) would this study help address?
o
The rationale behind these studies is to test whether or
not mechanical stimuli would be ideal for the chondrogensis
of Mesenchymal Stem Cells. Clinical questions that this study
would help address is whether or not this would allow stem
cell use to be effective because the differentiation of the stem
cells into chondrocytes must be controlled (can compression
do this?) This study was done to determine the relationship
between the time and location that dynamic loading is
applied to the differentiation of the stem cells. This could be
applied so that stem cells can be used as a treatment for
articular cartilage damage.
Hypoxia-induced Therapeutic Neovascularization in a Mouse
Model of an Ischemic Limb using Cell Aggregates Composed of
HUVECs and cbMSCs
Question 1: What state of tissue was described as a promoter of
Hypoxia Induced Factors (HIFs)? How did this promoter of HIFs
tie into the goal of this paper.
o The state of tissue that was described as a promoter of HIFs
was angiogenesis. This promoter was able to tie into the goal
of this paper because this is a key factor to promote the
survival of cells in oxygen comprised environments. Increased
activation of HIF-1-mediated pro-angiogenic pathways led to
increased tubular formation in vitro.

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Question 2: Describe the difference between neovascularization

and angiogenesis. Which one were the authors looking for?
o The difference between neovascularization and angiogenesis is that
neovascularization is mainly a formation of functional microvascular
networks and angiogenesis is characterized from previously existing blood
vessels. In this article, the authors were looking for neovascularization
through HUVEC/cbMSC aggregates.

Intrasmuscular Delivery of 3D Aggregates of HUVECs and cbMSCs

for Cellular Cardiomyoplasty in Rats with Myocardial Infarction
Question 1: what was the overarching conclusion of the paper
in terms of 3-D aggregates vs dissociated cells?

o The overarching conclusion of the paper in terms of 3-D aggregates vs

dissociated cells was that in vitro, 3D cell aggregates of HUVEC and cbMSC
cells provided the best cell-ECM and cell-cell interactions which stabilized
the tubular networks; in addition, in vivo, they induced considerable
neovascularization, reducing the size of the infarct zone and restoring cardiac
function the tissue. Dissociated cells were not capable of providing this level
of aid at all whatsoever.

Question 2: where are HUVECs and cbMSCs harvested from and

how were they to be used to recover ischemic myocardium?
o The HUVECs and cbMSCs are each harvested from umbilical vein and the
cord tissue, respectively. They were used to recover ischemic myocardium by
collectively being characterized into 3D HUVEC/cbMSC aggregates which
allowed for oxygen diffusion and myocardial perfusion.
o These 3-D aggregates were small enough to be injected via syringe. The cell
aggregates recovers ischemic myocardium by facilitating the best cell-ECM
and cell-cell interactions,

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