BMED/MSE 4751
Homework 4
Due March 26, 2009
1. Stem Cell X matures into Cell Type A without specific environmental cues, but differentiates
into Cell Type B if Ligand α is bound to its Membrane Receptor β.
You have a solution of Protein α, which contains Ligand α, and a series of biomaterials that you
need to test. You test 4 different materials, measuring the amount of protein found on the
surface, the amount of Ligand α accessible on the surface, the amount of cell binding on the
surface, and the differentiation state of the cells on the surface.
A) (10 pts.) Name the assay/technique that you would use to measure each of the
parameters listed below and describe briefly how each assay would work to give you the
information needed (3 sentences or less each):
a. Amount of protein on surface [2 pts]
Several techniques could be used to measure the amount of protein on the surface.
HPLC/RP-HPLC—elute the protein off the surface and run it through the column. (The area
under the curve is indicative of the amount of protein present.)
Colorimetric/Fluorescent Assays— Add a substrate that will change color in the presence of the
protein (colorimetric assay) or label the protein with a fluorescent dye before incubating it with
the material.
ELISA—antibody binds to the protein and a subsequent reaction will produce a measurable
color change (FYI: The antibodies used in the ELISA will only react with specific protein sites in
their native conformation, thus one would not measure the amount of denatured protein or
proteins oriented such that the antibody does not bind. This is not the best assay for
quantitatively measuring the total amount of protein on the surface. (i.e. a material that binds
the protein irreversibly, causing protein denaturation may actually look like it binds less protein
than the material for which the protein has less affinity, but does not cause unfolding of the
protein.))
b. Amount of functional Ligand α accessible on the surface [2 pts.]
Modified ELISA assay—Use an antibody that binds to functional Ligand α.
c. Amount of cell binding on the surface [3 pts.]
Incubate cells with the material, allow them to attach for a set amount of time then wash away
nonadhered cells. Count either the cells attached to the material or the number of cells that
were washed away.
d. Differentiation state of the cells on the surface [3 pts.]
Differentiation state could be measured in a number of different ways depending on the cells
types involved. All methods must be able to distinguish between different phenotypes. Some
examples:
Morphology—if Cell Type A and Cell Type B display different morphologies, then one should be
able to distinguish between the two cell types using light microscopy.
Cell Types A and B likely show some differences in their gene expression. RT-PCR could be used
to see how much specific genes are being expressed in a cell population.
Immunostaining—If Cell Types A and B show differences in their protein expression, then one
could distinguish the two cell types using antibodies that bind to a protein that is expressed in
one cell type, but not in the other. Either indirect immunostaining could be used, or the
technique could be used to fluorescently label proteins and image them with a fluorescent
microscope.
B) (14 pts.) Complete the table below indicating the qualitative results that you expect
from your study. (Answer choices are indicated in parenthesis in the top row of each
column.)
Biomaterial
Relative Amount of
Protein on
Cell
Ligand α on Surface?
surface?
Binding?
(None, Low, Medium,
(Yes/No)
(Yes/No)
High)
Hydrophobic
Yes
Surface
Surface treated to Yes
Cell Differentiation (Not
applicable, All A, Mostly
A with some B, Mostly B
with some A, All B)
Low
(c) Yes
Mostly A with some B
High
Yes
(d) All B
specifically bind a
region of Protein
α (Region γ)
Surface
with
covalently(a) No
tethered PEG
Hydrophilic
Yes
Surface
None
No
N/A (or All A)
(b) Medium
Yes
Mostly B with some A
C) (4 pts.) Two boxes were already filled in, indicating that there was a low level of
availability of Ligand α on the hydrophobic surface and a medium level of availability of
Ligand α on the hydrophilic surface. What could these experimental results indicate
about the hydrophilicity of Protein α?
The protein may contain a high number of hydrophobic residues, as it appears that adsorption
to the hydrophobic surface did cause some structural rearrangement of the protein (less
availability of Ligand α). It appears that the protein also bound the hydrophilic material, but
with less denaturing of the protein (ligand α still intact).
D) (14 pts.) For each of the boxes in the table that contains a letter, justify the answer to
that slot. [Questions are clarified somewhat below.]
a. Is there protein adsorbed to the tethered-PEG surface—why or why not? [2 pts.]
Steric hinderance prevents the adsorption of proteins to the surface.
b. Explain why a medium (rather than high or low) amount of Ligand α is seen on
the surface of the hydrophilic biomaterial. [4 pts.]
A medium level of Ligand α activity would be seen when there is high adsorption of the protein
to the biomaterial surface, but the orientation of the protein is random, so not all adsorbed
proteins present Ligand α for the cell receptors (or the antibodies in the ELISA assay) to bind to.
[Alternatively, adsorption could cause partial degradation of the protein, so that some Ligand α
is able to bind the ELISA antibody, but not all.]
c. Describe the cell binding to the hydrophobic surface based on the protein
adsorption you predict, as well as Ligand α availability. [4 pts.]
It appears that Protein α does adsorb to the hydrophobic surface, but the low Ligand α
availability indicates that either the protein preferentially binds with the Ligand α portion of the
protein buried on the surface, thus not exposed for binding by the antibody used in the ELISA or
the cell, or (more likely) the protein denatures on the surface of the hydrophobic material,
preventing Ligand α from binding the antibody in the ELISA or the cell receptors. The cells will
be able to bind to the protein-coated material surface (but they will not be stimulated to
differentiate into Cell Type B by Ligand α).
d. How can the differentiation state of the cell on this material be explained in
terms of the other properties that were measured? [4 pts.]
Because the surface was modified to specifically bind Region γ of Protein α, the protein did
definitely coat the surface and did so in a way that left a high density of Ligand α exposed and
functional on the surface. Thus, cells were able to bind to the surface specifically using their cell
membrane receptor βs, inducing differentiation into Cell Type B.
2. (16 pts.) You have developed a new ceramic coating to be used on hip implants and test it in
a series of in vitro assays. For each of the parameters listed below, describe 1) how you would
measure the characteristic and 2) what outcome you would hope for in that particular assay to
demonstrate that you have the ideal biomaterial for bone tissue engineering. (Limit the answer
to each section to 3 sentences.)
a) Cell cytotoxicity
Cells could be cultured directly on the material (Direct Contact Assay). After 1-2 days the cells
would be stained with a dye that indicates whether the cells are alive or dead (like the MTT
assay) and imaged microscopically. An ideal biomaterial would cause minimal cell cytotoxicity,
so we would hope to see the majority of the cells surviving.
b) Cell proliferation
To study cell proliferation, cells would be cultured on the material for several days and the
number of cells on the surface would be counted at several different timepoints. If cell numbers
are increasing over time, we know that the cells are dividing. Cell proliferation could be
advantageous for integrating the hip implant into surrounding tissues.
c) Cell migration
A capillary tube assay could be used to measure the cell migration on the surface of the
ceramic. Cell migration could be beneficial in producing a uniform tissue or more even
integration of the hip implant.
d) Cell differentiation
Cell differentiation should be measured by looking for cell characteristics known to be present
in desired cell types. This can include cell morphology or the expression of proteins known to be
markers for the desired cell type. Alternatively, bone tissue can be detected by looking for
excretions typical of osteoblasts (i.e. mineral deposition). The ideal hip implant would promote
the differentiation of cells into osteoblasts, which would then produce the desired mineralized
tissue for integration into the surrounding bone structure.
3. a) (8 pts.) Describe the body’s normal wound healing response to a gash on the arm from
immediately after the cut through 2 weeks after injury. (Limit your answer to 5 sentences.)
The first response following injury is blood clotting and the formation of a fibrin network to
prevent fluid loss. This is followed by acute inflammation, which is characterized by removal of
tissue debris and deposition of hyaluronic acid in the extracellular matrix. The inflammatory
response triggers an influx of fibroblasts, which proliferate and deposit extracellular matrix,
and mark the beginning of granulation tissue formation. New blood vessels are formed at this
time. As new extracellular matrix is deposited, the fibrin clot is dissolved via release of specific
enzymes and phagocytosis from the remaining macrophages. Remodeling starts about one
week after injury as the collagen type III in the extracellular matrix is degraded and replaced by
collagen type I.
b) What would happen differently at the injury site if non-degradable stitches were used and
not removed? (4 pts.)
Extrusion or encapsulation of the sutures.
5. (10 pts.) Describe 2 phagocytic complications that can occur after biomaterial implantation
and briefly explain the impact on the surrounding tissue. (5 sentences or less)
1. Frustrated phagocytosis occurs when the size of the nondegradable material is much larger
than that of the cell. This results in the release of lysosomal enzymes and other products into
the environment around the foreign material since the particle is too large to be taken into the
cell and degraded intracellularly.
2. Small, undigestible particles: If particles are small enough to be phagocytosed by a
machrophage, but resists degradation, they can be sequestered in the macrophage until the
cell dies and lyses, releasing the particle and other cell contents back into the environment.
Both of these reactions can cause tissue damage or scarring as the chemicals released by the
cells cause tissue damage or recruit other cells to the site, prolonging the reaction to the
biomaterial.
6. (20 pts.) You’ve developed a polymeric scaffold for cartilage tissue engineering that has been
shown to be promising in reconstruction of facial features, such as the nose. You now believe
that the same material could also be very effective in repairing cartilage in the knee. Describe
two in vitro assays and one in vivo assay that you would have to do to assure biocompatibility of
the polymer for the facial reconstruction and explain why each is important. Do you have to
repeat the same in vitro and in vivo assays for the new application of the material (in the knee)?
In each case (in vitro and in vivo), explain why or why not. (10 sentences or less).
A variety of answers were accepted. In vitro assays should include something for
inflammation/toxicity and in vivo tests should include something about assaying for integration
and the inflammatory response. The answer to the second part depends on what was chosen
for the first, however, the in vivo tests need to be redone in the knee to account for different
responses based on implantation site variables (vascularization, mechanics, etc).