U.S. patent application number 14/062059 was filed with the patent office on 2014-09-25 for incorporation of plant virus particles and polymers as 2d and 3d scaffolds to manipulate cellular behaviors.
The applicant listed for this patent is Lim Andrew Lee, Qian Wang. Invention is credited to Lim Andrew Lee, Qian Wang.
Application Number | 20140287507 14/062059 |
Document ID | / |
Family ID | 51569423 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287507 |
Kind Code |
A1 |
Wang; Qian ; et al. |
September 25, 2014 |
Incorporation of Plant Virus Particles and Polymers as 2D and 3D
Scaffolds to Manipulate Cellular Behaviors
Abstract
Methods are generally disclosed for attaching a cell binding
motif to a carboxy end of a coat protein of a Tobacco Mosaic Virus
particle to form a modified-TMV particle; and attaching a cell to
the cell binding motif of the modified-TMV particle.
Inventors: |
Wang; Qian; (Columbia,
SC) ; Lee; Lim Andrew; (Columbia, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wang; Qian
Lee; Lim Andrew |
Columbia
Columbia |
SC
SC |
US
US |
|
|
Family ID: |
51569423 |
Appl. No.: |
14/062059 |
Filed: |
October 24, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61795736 |
Oct 24, 2012 |
|
|
|
Current U.S.
Class: |
435/402 ;
435/395; 530/350 |
Current CPC
Class: |
C12N 7/00 20130101; C12N
2770/00031 20130101; C12N 2501/11 20130101; C12N 2501/15 20130101;
C12N 5/0068 20130101; C12N 2501/115 20130101; C12N 2533/50
20130101 |
Class at
Publication: |
435/402 ;
530/350; 435/395 |
International
Class: |
C12N 5/00 20060101
C12N005/00 |
Goverment Interests
GOVERNMENT SUPPORT CLAUSE
[0002] The present invention was developed with funding from the
National Science Foundation under award CHE-0748690 awarded by the
National Science Foundation. The government retains certain rights
in this invention.
Claims
1. A method, comprising: growing and cultivating a genetically
modified Tobacco mosaic virus coat protein, wherein the genetically
modified Tobacco mosaic virus coat protein comprises a cell binding
motif to promote cell attachment, outgrowth or differentiation.
2. The method of claim 1, wherein the cell binding motif comprises
an RGD tripeptide sequence derived from fibronectin.
3. The method of claim 1, wherein the cell binding motif comprises
an RGD tripeptide sequence derived from vitronectin.
4. The method of claim 1, wherein the cell binding motif comprises
an RGD tripeptide sequence derived from osteocalcin.
5. The method of claim 1, wherein the cell binding motif comprises
an PHSRN (SEQ ID NO 55) sequence derived from fibronectin.
6. The method of claim 1, wherein the cell binding motif comprises
an DGEA (SEQ ID NO: 4) peptide sequence derived from collagen.
7. The method of claim 1, wherein the cell binding motif comprises
a P15 peptide sequence derived from collagen.
8. The method of claim 1, wherein the cell attachment, outgrowth
and differentiation is further accelerated by using serum-free
environment supplemented with growth factors.
9. The method of claim 8, wherein the growth factors comprise a
fibroblast growth factor, a transforming growth factor-beta1, or an
epidermal growth factor.
10. The method of claim 1, further comprising: depositing the
modified TMV coat protein onto a substrate such that the coated
substrate is configured to be used to promote cell attachment,
growth or differentiation.
11. A method comprising: attaching a cell binding motif to a
carboxy end of a coat protein of a Tobacco Mosaic Virus particle to
form a modified-TMV particle; and attaching a cell to the cell
binding motif of the modified-TMV particle.
12. The method of claim 11, wherein the cell binding motif is
selected from the group consisting of GRGDSPG (SEQ ID NO: 1),
AVTGRGDSPASS (SEQ ID NO: 2), EDRVPHSRNSIT (SEQ ID NO: 3), DGEA (SEQ
ID NO: 4), and GTPGPQGIAGQRGVV (SEQ ID NO: 5).
13. The method of claim 11, wherein the cell is attached to the
mutated virus particle in a serum-free environment.
14. The method of claim 11, further comprising: prior to attaching
the cell, applying the modified-TMV particle onto a surface of a
substrate.
15. A method of forming a hydrogel, the method comprising:
incorporating a plurality of Tobacco Mosaic Virus particles into a
hydrogel precursor formulation; and polymerizing the hydrogel
precursor formulation to for a hydrogel matrix with the Tobacco
Mosaic Virus particles incorporated therein.
16. The method of claim 15, wherein the hydrogel precursor
formulation comprises a mixture of alginate in bicarbonate solution
and a mild acid.
17. The method of claim 15, further comprising: crosslinking the
hydrogel matrix.
18. The method of claim 15, wherein the Tobacco Mosaic Virus
particles comprise modified-TMV particles.
19. The method of claim 15, further comprising: prior to
incorporation into the hydrogel precursor formulation, attaching a
cell binding motif to a carboxy end of a coat protein of a Tobacco
Mosaic Virus particles to form the modified-TMV particles.
Description
PRIORITY INFORMATION
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 61/795,736 filed on Oct. 24, 2012 of
Wang, et al. titled "Incorporation of Plant Virus Particles and
polymers as 2D and 3D Scaffolds to Manipulate Cellular Behaviors,"
the disclosure of which is incorporated by reference herein.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jun. 13, 2014, is named USC-379_SL.txt and is 13,102 bytes in
size.
BACKGROUND
[0004] Plant viruses and other virus-like particles are utilized in
expanded roles as multi-facet nanosized building blocks for
directing cell growth and differentiation. Plant viruses are
isolated in high purity with batch to batch consistencies in
time-honored fashion at low costs. The surface properties of the
virus are adjusted through chemical or genetic modifications to
incorporate new biologically relevant functional groups.
Furthermore, the symmetrical arrangement of the viral proteins make
the viral particles attractive scaffolds for displaying identical
copies of the functional groups for applications in stem cell
cultures, electronics, catalysis, drug/gene delivery, imaging, and
immunotherapy.
[0005] An argument for using viruses as a biomaterial lies on the
premise that structurally ordered functional groups recruit
different cellular responses compared to unordered ligands. For
example, influenza virus attaches to erythrocytes through multiple
binding between hemagglutinin and sialic acid, and some animal
viruses display integrin binding sites in a pentameric motif to
promote cell internalization. The adhesion force associated with
the clusters of integrin binding motifs can be 7-fold stronger over
non-clustered ligand-receptor interactions. In cell signaling, the
integrin receptors, which are targets for the RGD peptide, form
dynamic clusters which are crucial in cell adhesion, motility, as
well as echano-transduction, which can all invariably affect stem
cell differentiation.
[0006] TMV is one of the simplest viruses known. Each viral
particle consists of 2130 identical protein subunits arranged in a
helical motif around a single stand of RNA to produce a hollow
protein tube. The internal and external surfaces of the protein
consist of repeated patterns of charged amino acid residues, such
as glutamate, aspartate, arginine, and lysine. The rod like TMV is
300 nm in length and 18 nm in diameter. The chemistry of TMV has
been studied extensively, and it has been previously demonstrated
that coating surfaces with TMV and another plant virus, Turnip
yellow mosaic virus (TYMV), enhanced mesenchymal stem cell
differentiation towards bone-like phenotype.
[0007] Studies have indicated that the coat protein of Tobacco
mosaic virus (TMV) can tolerate up to 25 amino acid insertions near
its carboxy terminus. By inserting cell-binding sequences to the
virus coat protein, specific bio-functionalities can be engineered
for use in tissue engineering. The advantages of using TMV or other
plant virus particles are multi-fold. First, the structural
features and the size range of the virus could be envisioned as
highly stabilized, macromolecular extracellular matrix mimetic. The
natural extracellular matrix (ECM) proteins play important roles in
guiding cell adhesion, migration, proliferation and stem cell
differentiation. However these ECM proteins difficult to obtain
high yields in cost-effective manner, but the virus-based ECM
mimics are routinely purified in high purity (>99%) in
time-honored manner. Second, previous studies with plant virus
coated substrates demonstrated that the coating of surfaces with
native and phosphate-modified virus particles accelerated stem cell
differentiation towards bone-like tissues from 21 days of culture
to 14 days. The accelerated differentiation process was marked by
increased expression levels of key osteogenic markers and an
important growth factor known as bone morphogenetic protein-2
(BMP-2). Although the role of TMV coated substrates and BMP-2 has
not been fully elucidated, these early studies indicate that the
virus-mediated differentiation is not simply based on virus-cell
interactions but multiple interactions between the osteoinduction
factors (dexamethasone, beta-glycerophosphate and ascorbic acid),
serum/cytokine soluble factors, and surface topography.
SUMMARY
[0008] Objects and advantages of the invention will be set forth in
part in the following description, or may be obvious from the
description, or may be learned through practice of the
invention.
[0009] Methods are generally disclosed for attaching a cell binding
motif to a carboxy end of a coat protein of a Tobacco Mosaic Virus
particle to form a modified-TMV particle; and attaching a cell to
the cell binding motif of the modified-TMV particle.
[0010] Other features and aspects of the present invention are
discussed in greater detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] A full and enabling disclosure of the present invention,
including the best mode thereof to one skilled in the art, is set
forth more particularly in the remainder of the specification,
which includes reference to the accompanying figures.
[0012] FIG. 1A shows a computer reconstructed image of the
structure of TMV from a top-down view.
[0013] FIG. 1B shows a computer reconstructed image of the
structure of TMV from a side cut-away view of the single disc of
TMV.
[0014] FIG. 1C shows a computer reconstructed image of a single
coat protein of TMV with highly rigid region and highly flexible
regions. The inside pore along the proximal is slightly flexible
with the alpha helices in the mid-section being highly rigid with
the carboxy end of the coat protein being highly flexible. This
region has been reported to tolerate up to 20-25 amino acid
insertions. Images were rendered using PyMol with coordinates
2TMV.pdb1 from Protein Data Bank.
[0015] FIG. 1D shows a transmission electron microscopy image of an
isolated TMV mutant, TMV-RGD1, with scale bar of 200 nm.
[0016] FIG. 1E shows an atomic force microscopy height image of
TMV-RGD1 dried on a surface (2.5.times.2.5 microns).
[0017] FIG. 2A shows a mass spectrometric analysis of the plant
virus mutants direct from plant sap. The high level of coat protein
expression in plants permitted the direct detection of the coat
protein from crude plant sap by MALDI-TOF MS.
[0018] FIG. 2B shows a plot of the RFU vs/ concentration taken by
modifying TMV particles with fluorescein to the interior surface
and then coating on high binding plates at various concentrations
(0.01, 0.05, 0.1, and 0.5 mg/mL). At 0.1 mg/mL, the coating density
reached a maximum. All coating experiments were done with n=8 with
three separate experiments. Significant values were based p values
less than 0.05 with two-tailed equal variance Student t-test.
[0019] FIG. 3 shows the mass spectrometric analysis of several
degraded plant virus mutants: (a) TMV-RGD1; (b) TMV-RGD7; (c)
TMV-PHSRN3; (d) TMV-DGEA; (e) TMV-P15.
[0020] FIG. 4 shows the phase contrast light microscopy images of
BHK cells prior and post centrifugation. The uncoated substrate
provided the highest level of attachment for the BHK cells in
serum-free conditions, whereas the wild type TMV coating blocked
cell attachment to the plate. 1% BSA coating was used to block all
non-specific interactions between the cells and substrate. The
cells on TMV-RGD1 coated substrate showed filopodial extensions, as
expected with the presence of RGD tripeptide sequence, as well as
for the RGD7 coated substrate. Both TMV-RGD1 and TMV-RGD7
substrates seem to support similar attachment levels with similar
long cell body extensions. The fibronectin synergy mimetic,
TMV-PHSRN3, provided weaker attachment with most of the cells
detaching at 30 g spin which is approximately 3.6 pN per cell.
Similar detachment forces were observed for on collagen mimetic,
TMV-DGEA coated substrates. Although the cells on the other
collagen mimetic, TMV-P15 coated substrates did not exhibit the
long filopodial extensions as on RGD1 or RGD7 substrates, higher
detachment forces were measured for the cells on P15. Scale bar is
50 microns.
[0021] FIG. 5A shows a the centrifugal adhesion assay to measure
detachment forces of several BHK cells. An inverted spin at 10 g
(.about.1.2 pN/cell) for 5 minutes resulted in cell detachment from
TMV and BSA coated substrates, whereas approximately 50% of the
cells were detached for TMV-RGD1, RGD7, and PHSRN3 coated
substrates. Only a fourth of the cells were remaining for the
TMV-DGEA coated substrates. TMV-P15 coated substrate, despite the
lack of filopodial extensions, had the highest percentage of cells
remaining on the substrate.
[0022] FIG. 5B shows that the centrifugal adhesion assay using a
higher centrifugal force of 30 g (.about.3.6 pN/cell) detached the
cells from PHSRN3 and DGEA (SEQ ID NO: 4) coated substrates,
whereas slight decrease in the number of cells was measured on
RGD1, RGD7 and P15. *p<0.05 (n=6, unpaired Student t-test) when
compared to TMV coated substrates from three separate experiments.
Error bars denote standard error of mean.
[0023] FIG. 5C illustrates the average cell body sizes prior to
subjecting the cells to inverted centrifugation. There appears to
be no clear relation between cell body sizes to adhesion forces.
*p<0.001; .sctn.p<0.005; **p<0.01 (n=50, unpaired Student
t-test) when compared to cells on TMV coated substrates. Error bars
denote standard error of mean.
[0024] FIG. 5D presents the results of integrin specific monoclonal
antibody inhibition studies for RGD1. Based on the results, cell
adhesion is only slightly reduced with anti-.beta.1 integrin
antibody for cells on TMV-RGD1.
[0025] FIG. 5E presents the results of integrin specific monoclonal
antibody inhibition studies for RGD7. Based on the results, the
cells on TMV-RGD7 show less attachment.
[0026] FIG. 5F presents the results of integrin specific monoclonal
antibody inhibition studies for P15 coated substrates. Cell
attachment on TMV-P15 is dramatically reduced when receptors are
inhibited with anti-.alpha.2 antibody. *p<0.05 (n=4, paired,
two-tailed Student t-test).
[0027] FIG. 6 shows a centrifugal adhesion assay to measure
detachment forces for CHO cells. An inverted spin at 10 g
(.about.1.2 pN/cell) for 5 minutes resulted in cell detachment from
majority of the substrates except for TMV-RGD1 and TMV-P15 coated
substrates *p<0.05 (n=6, unpaired Student t-test) when compared
to TMV coated substrates from three separate experiments performed
with six replicates. Error bars denote standard error of mean.
[0028] FIG. 7 shows the cell Morphology on TMV (left column) and
TMV-RGD1 (right column). The cells cultured on TMV were round and
poorly spread, but the cells on TMV-RGD1 possessed long and thin
filopodial extensions. The white arrow points to the extended
filopodia on TMV-RGD1. Such morphological features are not commonly
found on other surface coatings. Scale bar for phase contrast
images (top row) is 100 microns, and scale bars for SEM images
(bottom row) are 2 microns and 10 microns for TMV and TMV-RGD1,
respectively.
[0029] FIG. 8A shows results of plant viruses as substrates for
stem cell cultures. Shown is a schematic illustration of PEM
preparation by cleaning the glass substrate to generate hydroxyl
groups on the surface followed by coating with PAH, then PSS. The
last layer is the virus coating.
[0030] FIG. 8B shows AFM height image (2.5.times.2.5 microns) of
TMV on the top layer and indicates the viral particles are mostly
intact and laid flat on the surface.
[0031] FIG. 8C shows that the average cell metabolic rates based on
CellTiter Blue (Promega) indicate similar viability across all
substrates, indicating the plant viruses are not toxic to the
cells.
[0032] FIG. 9 shows the QCM measurements of PAH/PSS polyelectrolyte
membrane with TMV. Each layer of coating decreases the frequency in
a linear fashion. TMV coating was followed by incubation in
1.times.PBS solution for one hour. A slight shift in frequency was
observed, likely due to the osmolarity difference between TMV (100
mM K Phos pH 7.0) versus PBS. Error bars denote standard deviation
from the mean (n=9) and 3 separate experiments in triplicates.
[0033] FIG. 10 shows the mass density shift of polyelectrolyte
membrane. A linear mass shift is observed until TMV coating, where
the total amount of viral particles averaged 1.5.+-.0.3
.mu.g/cm.sup.2. Incubation with PBS solution allowed for slight
swelling of the PEM, increasing the density shift by 0.3.+-.0.1
.mu.g/cm.sup.2. Error bars denote standard deviation from the mean
(n=9) from three separate experiments performed in triplicates.
[0034] FIG. 11 shows stem cells aggregate after 48 hours of
osteoinduction. Phase contrast microscopy images of BMSCs (row A)
after one hour of seeding, the cells on PEM, hFN (human
fibronectin), TMV and TMV-RGD1 coatings on top of PEM exhibit
similar morphologies for all three samples, (row B) but after 2
days in serum-free osteogenic media, only the cells on TMV-RGD1
coating (labeled as RGD1 on figure) aggregate to large nodules.
Scale bar is 200 microns.
[0035] FIG. 12A shows RT-qPCR analysis for osteo-specific gene
expression of BMSCs under osteogenic conditions. For each growing
condition, the profiles showed two time points: 8 and 24 hours
after induction with osteogenic media. Gene expression in the cells
seeded on TCP, TCP with TMV in solution and TMV substrate under
osteogenic conditions. As shown, ALPL expression was upregulated in
cells grown on TMV at 24 hours.
[0036] FIG. 12B shows BMP2 expression at both time points was
significantly increased in cells grown on TMV substrates.
[0037] FIG. 12C shows IBSP was highly expressed in cells grown on
TMV substrates at both time points. In all graphs, the error bars
denote .+-.1 s.d. (**) and (*) represent p<0.05 and p<0.1,
respectively.
[0038] FIG. 13A shows the differential expression and localization
of BMP2 analyzed by ELISA and immunohistochemical staining. As
shown, the quantification of BMP2 protein expression at 8 and 24
hours was normalized to cell number by ELISA. The values are
expressed as fold change compared to cells on TCP before
osteoinduction. The error bars denote .+-.1 s.d. *p<0.01 based
on equal variance two-tailed Student t-test.
[0039] FIG. 13B shows the cells on TCP control or TMV substrate
that were collected at 8 and 24 hours after osteogenic induction.
At both time points, cells on TMV-coated surface expressed more
BMP2 protein and the protein is highly expressed at cell
aggregates. Scale bar is 100 .mu.m.
[0040] FIG. 14A shows cytochemical analysis of bone differentiation
process of BMSCs on TCP, TCP with TMV in media, TMV substrate at 7,
14, and 21 days after osteogenic induction. Shown is alkaline
phosphatase activity of cells in three different conditions. Cells
on TMV substrate have an increase in enzyme activity at day 7 and
14, whereas the addition of TMV solution does not alter the enzyme
activity when compared to control. Alkaline phosphatase activity
drops to baseline at day 21 for all conditions.
[0041] FIG. 14B shows alizarin red staining of each sample at day
14.
[0042] FIG. 14C shows absorbance at 548 nm normalized to cell
number to indicate relative amount of calcium deposit at day 14
stained by alizarin red solution. The mineralization of cells on
TMV substrate doubles that of TCP and TMV in solution, suggesting
an improvement in osteogenesis (** p<0.05 based on equal
variance two-tailed Student t-test). The error bars denote
.+-.s.d.
[0043] FIG. 15 shows a comparison of cytokine profiles in culture
media of BMSCs on TCP control and TMV substrate for 24 hours, prior
to osteoinduction. An antibody array containing 29 different
cytokines was used to compare protein profiles produced by cells on
different substrates. TIMP-1 was detected in both cultures with
higher levels from TMV substrate. However, the productions of some
cytokines and growth factors were induced only by TMV-coated wafer,
including CINC-1, CINC-2, MIP-3.alpha., and VEGF.
[0044] FIG. 16 shows cytoskeleton immunochemical staining showing
actin polymerization and organization of cells on TCP control and
TMV substrate after 24 hours seeding, prior to osteoinduction, at
low- (top row) and high-magnification (bottom row). Similar fibrous
cytoskeleton organization was observed where cells spread out in
both cell cultures. However, in cell aggregates on TMV substrate
the actin intensity was higher (white circles) and well-defined
actin filament was absent (white arrow). Scale bars are 200 .mu.m
for top row and 25 .mu.m for bottom row.
[0045] FIG. 17A presents immunochemical staining showing the
difference in vinculin size of cells on TCP control or TMV
substrate for 24 hours. Shown are immunofluorescence images of
cells on different substrates for 24 hours prior to osteoinduction
(top panel). The bottom panel illustrates vinculin masking and
selection for size analysis. The selected vinculin spots as part of
focal adhesion complex are highlighted.
[0046] FIG. 17B shows average vinculin size of cells on either TCP
or TMV-coated surface. The error bars denote .+-.s.d. and *
p<0.05 based on equal variance two-tailed Student t-test.
[0047] FIG. 18A shows BMP-2 expressions are localized to the
nodules, most are found on TMV-RGD1 substrates (annotated as RGD1
on figure). Scale bar is 100 microns for phase contrast and 50
microns for DIC/fluorescence images.
[0048] FIG. 18B illustrates results of BMP-2 expression quantified
by probing with anti-BMP2 primary followed by anti-mouse goat
IgG-HRP with TMB solution. Error bars denote .+-.SEM (n=12).
*p<0.05 using two-tailed equal variance Student t-test comparing
TMV-RGD1 to all substrates.
[0049] FIG. 18C presents determination of focal adhesion kinase
(FAK) activity by probing with anti-FAK pY397 and anti-total FAK.
pY397 FAK intensity was normalized against total FAK and set
relative to hFN controls. Error bars denote .+-.s.d. (n=3).
*p<0.05 using two-tailed equal variance Student t-test.
[0050] FIG. 18D shows that the increase in focal adhesion kinase
phosphorylation at residue Y397 observed for cells cultured on
TMV-RGD-1 coated substrates was two-fold above the levels detected
for cells on human fibronectin.
[0051] FIG. 19A shows osteocalcin expression after 2 days of
induction. A canonical osteogenic marker, osteocalcin, is
exclusively found on cell aggregates growing on TMV-RGD1 coated
substrates. For cells on TMV, the isolated cells express low levels
of osteocalcin which was similar to uncoated PEM. Scale bars are
100 microns for FIG. 19A.
[0052] FIG. 19B: BMSCs undergo rapid nodule formation on TMV-RGD1
coated substrates only. Phase contrast and brightfield images of
BMSCs cultured in osteogenic media for 2 days then stained with
Alizarin Red S (red) for calcium. The cells on virus coated
substrates stain positive for calcium deposits faster than on cells
on human fibronectin substrates. The largest aggregates are
observed for the cells cultured on TMV-RGD1 coated substrates.
Scale bars are 200 microns for FIG. 19B.
[0053] FIG. 20 shows tobacco mosaic virus coating on high binding
plates. The coating was monitored by indirect ELISA, where the
virus is coated to the substrate, then probed with anti-TMV
polyclonal sera from immunized Rabbits (purchased from Sigma),
followed by anti-rabbit goat antibody conjugated with HRP. The
lower OD (optical density) indicates low density coating, with
higher OD indicating more virus on coated on substrates. At
concentrations above 100 .mu.g/mL, the signal appears to be
saturated. The two images (middle and right panels) are
representative atomic force microscopy height images of substrates
coated in low virus concentration (0.01 .mu.g/mL) and high virus
concentration (100 .mu.g/mL). Despite the near saturated signal
from ELISA, the AFM images indicate less than full coverage of the
surface with virus particles. Scan area for both images are
5.times.5 microns.
[0054] FIG. 21 shows water contact angle measurements of high
binding plates coated with Tobacco mosaic virus and mutants. Each
well was coated with 100 ul of virus solution at 0.1 mg/mL in pure
water. The surfaces were washed 3 times with pure water and cut out
from the plate. VCA Optima was used to measure the water contact
angle by sessile drop test.
[0055] FIG. 22A shows results from rat bone marrow derived
mesenchymal stem cells that were seeded on the virus coated high
binding plates. The cell attachment and spreading on the various
mutants were visualized by staining the live cells with calcein AM
within 24 hours of seeding (the live cells indicated by the green
fluorescence). Scale bar is 50 microns.
[0056] FIG. 22B: Fluorescence intensity (RFU--relative fluorescence
unit) is a measurement from the fluorescence plate reader for the
different samples. Higher fluorescence intensity correlates to
greater spreading of the cells on the substrates.
[0057] FIG. 22C: Cell spreading was measured from each microscope
image and averaged, then normalized to the total area of each
picture frame. This measurement matches the fluorescence
measurements from the plate reader.
[0058] FIG. 23A shows results of rat bone marrow derived
mesenchymal stem cells seeded on the virus coated high binding
plates that were differentiated under serum-free osteogenic media.
The alkaline phosphatase activity assay indicates progressive
differentiation of the cells in serum-free media over 21 days with
cells seeded on TMV-RGD1 and human fibronectin (hFN) exhibit high
enzymatic activity levels.
[0059] FIG. 23B: Calcium measurements also indicate progressive
increase in mineralization over the 21 day time frame with RGD1 and
hFN substrates having consistently higher calcium levels than the
other substrates.
[0060] FIG. 23C: Cells stained with Alizarin Red S were imaged with
brightfield inverted microscope.
[0061] FIG. 24A shows results of surface coating optimization using
single layer of polyelectrolytes. The illustrated results of quartz
crystal microbalance studies indicate that PAH deposition is
highest when surfaces are coated at pH near the pI value of PAH
(.about.9.5).
[0062] FIG. 24B: Virus deposition reaches maximum density for
quartz surfaces coated with PAH solutions prepared at pH 9-11
(virus concentration=1 mg/mL and 100 mM potassium phosphate pH 7
buffer).
[0063] FIG. 24C: Fluorescently labeled TMV particles were used to
coat tissue culture polystyrene and monitored using a fluorescent
plate reader (ex/em=490/520 nm). The results on plasticware
coincide with the QCM measurements on quartz wafers.
[0064] FIG. 24D: Atomic force microscopy height image of uncoated
surface.
[0065] FIG. 24E: Atomic force microscopy height image of surface at
pH 4.
[0066] FIG. 24F: Atomic force microscopy height image of surface at
pH 11.
[0067] FIG. 24G: Atomic force microscopy height image of surface at
pH 13. The scan areas for the AFM height images in FIGS. 24D-24G
are all 5 by 5 microns.
[0068] FIG. 25 shows surface coating optimization using single
layer of polyelectrolytes. Atomic force microscopy height images of
surfaces coated with TMV particles at different pH and buffer
strengths. The scan areas for the AFM height images are all 5 by 5
microns.
[0069] FIG. 26 shows surface coating optimization using single
layer of PAH. QCM measurements corroborate the results from the AFM
height images, where the pH 5 solutions provide highest depositions
of the viral particles on PAH. Increasing the pH to 8 decreases the
amount of virus deposited on the surface. Eliminating the salt and
using TMV in water further decreased the deposition, as indicated
by both AFM and QCM analyses.
[0070] FIG. 27 shows a schematic of a synthetic procedure of
generating virus functionalized porous composite hydrogels.
Functionalization of composite hydrogels for use in stem cell
cultures can be achieved through direct mixing of virus solution to
the foamy mixture of alginate. Step A: Alginate mixture comprised
of low viscosity alginate, Pluronic F108, sodium bicarbonate with
an equivalent amount of citric acid to generate gas template foamy
mixture. Step B: TMV was added 5 min before the foamy mixture was
frozen and lyophilized. The lyophilized sample was crosslinked with
CaCl.sub.2. Step C: Porous composite hydrogel was obtained after
dialysis against 0.1 M CaCl.sub.2 and lyophilization.
[0071] FIG. 28 shows Scanning electron micrographs of hydrogels and
TMV particles labeled A-F: (A) and (B) PAH at different
magnifications; (C) and (D) TMV-PAH at different magnifications.
The images of TMV-PAH reveal larger macropores and interconnecting
channels than PAH. Transmission electron micrographs of (E) intact
TMV particles released from TMV-PAH after decrosslinking with EDTA,
and (F) purified native TMV particles in buffer. Scale bars are 500
microns for (A) and (C), 100 microns for (B) and (D) and 200 nm for
(E) and (F).
[0072] FIG. 29A shows cell attachment and viability in different
types of porous composite hydrogels. Cell attachment was observed
from counting the floating cells in the media after initial cell
seeding for 8 h. Percent attachment of BMSCs in each type of
hydrogels is presented. Values expressed are mean (n=3).+-.S.D.
[0073] FIG. 29B: CellTiter Blue.RTM. metabolic activity assay of
BMSCs culture in different types of hydrogels at different time
points. Values expressed are mean (n=3).+-.S.D. of RFU that were
normalized against PAH for each time point. Samples were compared
using paired equal variance student t-test, **p<0.005.
[0074] FIG. 30A shows results of osteogenesis assays of BMSC in
porous composite hydrogels. ALP activity assay was performed on day
3, 7, and day 14 of osteogenic culture. Values expressed are mean
(n=3).+-.S.D., .noteq./*paired equal variance Student t-test,
p<0.05.
[0075] FIG. 30B: Calcium deposition quantified by ICP-OES on day 6
after background subtraction from control samples with no cells.
Values expressed are mean (n=3).+-.SEM, *paired equal variance
Student t-test, p<0.05.
[0076] FIG. 30C: Thin sections of hydrogels stained with Alizarin
red on day 14 cultures, scale bar=2 mm.
[0077] FIG. 31 shows confocal images of differentiated BMSCs in 3D
composite hydrogels stained for nucleus (1.sup.st column), actin
(2.sup.nd column), and osteocalcin (3.sup.rd column). Scale bar=50
microns.
DETAILED DESCRIPTION
[0078] Reference now will be made to the embodiments of the
invention, one or more examples of which are set forth below. Each
example is provided by way of an explanation of the invention, not
as a limitation of the invention. In fact, it will be apparent to
those skilled in the art that various modifications and variations
can be made in the invention without departing from the scope or
spirit of the invention. For instance, features illustrated or
described as one embodiment can be used on another embodiment to
yield still a further embodiment. Thus, it is intended that the
present invention cover such modifications and variations as come
within the scope of the appended claims and their equivalents. It
is to be understood by one of ordinary skill in the art that the
present discussion is a description of exemplary embodiments only,
and is not intended as limiting the broader aspects of the present
invention, which broader aspects are embodied exemplary
constructions.
[0079] Methods are generally provided for adding cell binding
sequences to the carboxy terminus of the TMV coat protein to
promote cell attachment and cell differentiation.
[0080] In addition, we have confirmed that other plant viruses,
including cowpea mosaic virus, turnip yellow mosaic virus, potato
virus X, can also be employed in the cell culture studies using
similar protocol listed in this invention disclosure. However, to
simplify the writing, we use TMV in the text and the protocol,
which can similarly be adapted to other virus systems.
EXAMPLES
[0081] Based on the above discussion, several genetically-modified
TMV mutants were designed with reported cell adhesion sequences to
expand the virus-based tools for cell studies. To assess whether
TMV exhibit any natural affinity towards mammalian cell types, the
cells were seeded in serum-free conditions on surfaces coated with
TMV and TMV mutant particles. The attachment strengths of the cells
were measured by an inverted centrifugation, and the results
suggest that native TMV exhibit no specific cell attachment
sequences. Among the cells cultured on glass substrates coated with
mutant TMV particles, the cells on substrates coated with mutant
virus expressing RGD tripeptide motifs, had formed filopodial
extensions with weaker attachment profile. The cells on TMV
expressing collagen I mimetic sequence (P15) displayed little
spreading but higher attachment strength. These differences in cell
morphologies and their associated spreading behaviors seem to
suggest that the cells are using different sets of receptors to
bind to the two substrates coated with the mutant viruses.
[0082] Among the reported TMV mutants that afforded higher binding
affinities, one mutant (TMV-RGD1) was selected as a prototype to
test whether substrates coated with the mutants enhanced stem cell
differentiation. Two polyelectrolytes were used to alternatively
coat the surface in order to stably integrate the virus to the
surface. A specific serum-free osteogenic cultures was formulated
to further enhance the cell differentiation from 14 days to 2 days.
The virus-based cell cultures along with chemically defined
serum-free cell cultures is a powerful model for studying how
accelerated bone healing process occurs and potential use as a
bio-scaffold in the medical community.
[0083] A step closer towards realizing the application of
virus-based scaffolds as a medical tool was by transitioning from
standard 2D coated substrates to 3D cultures. The transition from
2D to 3D is tantamount to studying cell behaviors in a more native
setting, as the cells are naturally embedded within a 3D matrix.
This transition from 2D to 3D alters many chemical and physical
properties, and often the studies from 2D systems do not equally
translate in 3D systems. The transition of TMV was realized by
generating hydrogels through a gas templated solid foam assembly
method reported by Barbetta et al. The primary materials were
biodegradable polysaccharide (alginate), a biocompatible polymer as
a surfactant (plurionic F-108), and virus particles. The virus
particles incorporated in the hydrogel were structurally intact and
surface accessible, as demonstrated by increasing cell affinity
towards hydrogels made with TMV-RGD1 mutant. Stem cell
differentiation and viability were measured within the hydrogel.
Alkaline phosphatase activity at early time points suggest more of
the stem cells commit towards osteogenic lineage when cultured in
3D systems with TMV particles. Calcium mineralization occurs
earlier for the stem cells in TMV and TMV-RGD1 hydrogels when
compared to no virus hydrogel controls.
[0084] Besides the unique nature of TMV, other biologically
relevant functional groups can be engineered to alter the scaffolds
(2D and 3D) for better therapeutic applications.
[0085] 1. Mutation of Tobacco Mosaic Virus
[0086] Biological functions were incorporated by inserting
sequences derived from extracellular matrix proteins to enhance
cell binding to the plant virus particles. The results herein show
that native TMV has no apparent cell binding ligands based on our
experimental conditions, and several mutants containing cell
binding amino acid sequence inserts were stably expressed in
tobacco plant host. Among the mutant CP sequences designed, five
allowed systemic infection and were purified with moderate virus
yields from the tobacco hosts. In order to screen the functionality
of the mutants, we adapted a centrifugal adhesion assay, in which
the average attachment strengths between large population of cells
and substrate could be quantitatively measured with simple and
reliable method. The method allowed for measuring the functionality
of the mutants in biological system in a low-cost and highly
reproducible manner, whereas previous studies required high
investment of specialized media, time and delicate bone marrow stem
cells harvested from animal hosts. Based on the centrifugal assay,
one particular mutant based on collagen I peptide mimic (P15),
provided the strongest attachment profiles, whereas the commonly
used RGD mutants exhibited much lower detachment forces. In
contrast, a peptide fragment derived from collagen I (DGEA (SEQ ID
NO: 4)) and the synergy site of fibronectin (PHSRN (SEQ ID NO: 55))
appeared to have slight benefit in promoting cell adhesion when
displayed on the viral particles. This reported centrifugal assay
will facilitate screening larger arrays of virus CP mutants prior
to transferring the platform for in-depth study with stem
cells.
[0087] TMV has been well characterized since its initial discovery
in the late 19th century. Recent studies with viruses as nanosized
materials have renewed interests in manipulating the surface
properties of the TMV capsid without disrupting its overall
structural integrity and morphology for the differentiation of
mesenchymal stem cell towards osteoblasts. The site of insertion
was based on other studies with TMV, where majority of the papers
reported to be near the carboxy terminus of the viral coat protein
(FIG. 1a, b). The genetic modification was based on previous
reports of TMV tolerating large inserts near its carboxy region
(FIG. 1c). The amino acid sequences were selected based on peptide
fragments reported to bind integrin receptors or partial sequences
derived from other integrin binding matrix proteins (i.e. collagen,
vitronectin, fibronectin, and integrin-binding sialoprotein) with
sequences ranging from 4 amino acids up to 15 amino acids. Majority
of the inserts were designed to not deviate from the original
isoelectric point of the CP to avoid issues with virus infectivity
in the tobacco plant hosts.
[0088] Nine RGD mutants, 3 fibronectin synergy mutants (PHSRN (SEQ
ID NO: 55)), and two collagen mimetics were designed in total
(Table 1). Of the 14 mutants designed, five allowed systemic
infection in tobacco hosts (Table 2). The maximal yields for the
mutants ranged from 3% to 50% of the unmodified virus particles
with varying stabilities (Table 3). The low efficiency could be
attributed to multiple factors, ranging from transfection
efficiency by mechanical abrasion, inability of mutant CP to form
the capsid, aberrant interactions with other integral proteins in
the host, poor propagation of the mutant virus in the plant host or
susceptibility of the host strain (N. benthamiana vs N. tabacum cv
Samsun). The low transfection efficiency observed with mechanical
abrasion can be circumvented by using agrobacteria-based
infiltration. Alternatively, a library-based passage and selection
scheme reported by Smith et al. could overcome some of the
limitations observed with single mutant infections reported here to
generate the mutants. In any case, the expression of recombinant
proteins with viral vectors in any host will be limited by host
tolerance.
TABLE-US-00001 TABLE 1 List of isolated TMV mutants (SEQ ID NOS
1-5, respectively, in order of appearance) Calculated Observed
Insert Mass Mass Mutants Sequence (m/z) (m/z) RGD1 GRGDSPG 18161
18161 RGD7 AVTGRGDSPASS 18621 18622 PHSRN3 EDRVPHSRNSIT 18927 18926
DGEA DGEA 17908 17905 P15 GTPGPQGIAGQRGVV 18910 18896
TABLE-US-00002 TABLE 2 List of TMV mutants (SEQ ID NOS 1, 6-10, 2,
11-14 and 3-5, respectively, in order of appearance) Calculated
Observed Insert Insert Mass Mass Mutants Sequence Site (m/z) (m/z)
RGD1 GRGDSPG Extended after T159 18161 18161 RGD2 GAGSGRGDSGA
Inserted between S155-G156 18408 n/i* (no infection) RGD3
TGRGDSPASS Deleted G156-T159 and 18124 n/i extended from S155 RGD4
GEPRGDTYRA Deleted G156-T159 and 18311 n/i extended from S155 RGD5
AGSGRGDSGA Inserted between S155-G156 18350 n/i RGD6 GEPRGDTYRAY
Inserted between S155-G156 18801 n/i RGD7 AVTGRGDSPASS Inserted
between S155-G156 18621 18622 RGD8 ENGPRGDNYRA Inserted between
S155-G156 18766 n/i RGD9 AGSGEPRGDTYRASGA Inserted between
S155-G156 18621 n/i PHSRN1 PHSRNG Extended after T159 18183 n/i
PHSRN2 EDRVPHSRNS Deleted G156-T159 and 18387 n/i extended from
S155 PHSRN3 EDRVPHSRNSIT Inserted between S155-G156 18927 18926
DGEA DGEA Inserted between S155-G156 17908 17905 P15
GTPGPQGIAGQRGVV Inserted between S155-G156 18910 18896
TABLE-US-00003 TABLE 3 Virus yields and stabilities Mutants Yields
(mg/g of leave) Stability.sup.[*.sup.] Wild type TMV 2-3 1 RGD1
0.5-1 1 RGD7 0.5-1 1 PHSRN3 0.1-0.2 3 DGEA 1-2 2 P15 0.1-0.2 4
.sup.[*.sup.]1--very stable (6 months at 4.degree. C.); 2--stable
(3-6 months at 4.degree. C.); 3--fairly stable (2-3 months at
4.degree. C.); 4--poor stability (<2 months at 4.degree. C.)
[0089] All five mutants were isolated in large quantities (>100
mg) using the standard purification methods used to purify wild
type TMV. The structural integrity of the mutant viruses was
verified by sucrose gradient centrifugation, transmission electron
microscopy (TEM) and atomic force microscopy (AFM) (FIG. 1b, c). In
addition, the final ultracentrifugation step in the purification
protocol eliminates any incomplete assemblies of the virus
particle. Therefore, obtaining any purified isolates of the mutants
can be seen as clear indication of its structural integrity. The
genetic insertions encoding for the peptide sequences fused to
virus CP were verified by RT-PCR sequencing of the extracted virus
particles and mutant protein expression was confirmed by MALDI-TOF
MS (FIG. 2a). The genetic stabilities of the mutants were also
confirmed by using the plant sap from infected plants to introduce
the mutants to new tobacco hosts, a process known as passaging.
RT-PCR sequencing and MALDI-TOF MS of the viral particles isolated
from newly infected hosts showed no deviation from the original
mutation, even after ten passages for all mutants. In previous
mutagenesis studies with Cowpea mosaic virus, mutants were found to
revert back to its original genotype, which raised concerns that
TMV mutants in older passages would revert back to their original
wild type sequence. However, we have yet to observe such reversions
in any of the mutants reported here.
[0090] 2. Initial Cell Culturing Studies
[0091] The functionality of the TMV mutants was screened by coating
the virus particles on high binding 96-well plates. The coating
density optimization was determined by fluorescently labeling the
carboxyl groups exposed to the interior surface by carbodiimide
chemistry. The maximal fluorescence intensity was achieved by
coating for 20 minutes with the virus solution concentration of 0.1
mg/mL (FIG. 2B). The virus coated plates were seeded with a large
population of BHK or CHO cells for one hour in serum-free media.
These two cell lines, unlike fibroblasts, express lower levels of
extracellular matrix proteins and allow for the direct observation
of the cell attachment to the substrate without adding inhibitors
such as cycloheximide. Furthermore, avoiding the use of serum
prevents proteins within the serum from adsorbing to the surface
and promoting cell attachment. In this study, the surfaces coated
with unmodified viruses behaved similarly to the negative controls
(1% BSA coated), suggesting that the TMV capsid lacks specific cell
binding motifs for the two cell lines tested here. Although in
previous stem cell cultures, rapid cell differentiation was
observed on the same wild type TMV capsids, several differences in
the culture methods make it difficult to directly compare these two
results. First, a different cell type was used. Some cells can
secrete large quantities of ECM proteins, which may displace or
interact with the underlying virus coatings. How these matrix
proteins may interact with the virus provide additional variables.
Furthermore, previous studies relied on extensive use of serum,
which contains many cell-binding proteins (fibrinogen, collagen,
growth factors). In this experimental system, we focused on the
ability of the native virus and its variants to interact with two
types of mammalian cell lines, baby hamster kidney (BHK) cells,
which are known to produce low levels of extracellular matrix
proteins and Chinese hamster ovarian (CHO) cells, which are
commonly used for suspension cultures and adhesion cultures.
Relative ease in culturing these cell lines and their low level of
matrix protein provided us with simple, yet reliable means of
screening the virus coated substrates. Based on these two cell
lines, no apparent cell binding was observed for the native TMV
capsid.
[0092] Prolonged storage at 4.degree. C. was detrimental to the
virus mutants with inserted peptide sequences often cleaved.
Partially cleaved products were observed by MALDI-TOF MS (FIG. 3),
which corresponded to loss of peptide fragments from the carboxy
end (Table 4). These cleaved mutants still appeared to be
structurally similar to that of wild type when observed under TEM,
but did not promote cell attachment (data not shown). The genetic
sequences, when analyzed by RT-PCR sequence, were unaffected and
despite the protein degradation, these viral particles still
infected and propagated in plant hosts. Non-degraded viral
particles were recovered from the newly infected plant hosts, which
suggest the observed degradations of the CPs are due to the
presence of trace amounts of plant proteases or a low level of
microbial contamination. Use of protease inhibitors during
purification and storage decreased degradation. Storage of the
viruses in -20.degree. C. with a single freeze/thaw cycle did not
change the structural features. Smaller rodlets (lengths <100
nm) were observed on TEM when virus underwent multiple freeze/thaw
cycles (>3 cycles).
TABLE-US-00004 TABLE 4 The corresponding peptide loss associated
with the truncated TMV coat protein. A mass difference of 17 is
associated to the loss of a hydroxy ion. (SEQ ID NOS 15-22,
respectively, in order of appearance) Mutants .DELTA.m/z Peptied
loss A. RGD1 18159-17801 = 358 DSPG (374) 18159-17429 = 730
TGRGDSPG (746) B. RGD7 18625-18123 = 502 SSGPAT (519) 18625-17754 =
871 DSPASSGPAT (889) C. PHSRN3 19001-17672 = 1329 RPHSRNSITGPAT
(1394) 19001-17399 = 1602 DVRPHSRNSITGPAT (1608) D. DGEA
17905-17372 = 543 EAGPAT (545) E. P15 18928-17485 = 1443
GPQGIAGQRGVVGPAT (1465)
[0093] 3. Cell Adhesion Depending on the Sequence of Peptide
Insertion on Tobacco Mosaic Virus
[0094] For BHK cells, the uncoated substrates promote strong
attachment with the expected pyramidal shaped spreading (FIG. 4).
These surfaces were used as positive controls. A small number of
unattached, rounded cells could also be observed on these
substrates, and these cells were immediately removed after the
first round of centrifugation at relative centrifugal force (RCF)
of 10 g (equals to detachment force of 1.2 pN/cell based on Eq.
1).[39]
F.sub.D=(.rho..sub.cell-.rho..sub.media)V.sub.cellRCF (Eq. 1)
[0095] Based on the study by Reyes and Garcia, the detachment force
(FD) imposed per cell can be calculated based on RCF multiplied by
the cell mass. For mass calculation, we assumed similar parameters
as stated in previous study, where .rho..sub.cell=1.07 g/cm.sup.3
and .rho..sub.media=1.00 g/cm.sup.3 with V.sub.cell.about.1700
cm.sup.3. The advantage of the inverted centrifugal assay is that
the system is simple yet provides several quantifiable variables.
By altering variables such as proteins, cell types, seeding times,
relative centrifugal forces, the assay can provide robust
statistics for quantifying cell adhesion as a standard method for
high-throughput screening of virus coated substrates.
[0096] For the cells seeded on TMV and BSA coatings, we observed a
large population of the rounded cells, which were immediately
removed upon the first round of centrifugation, which indicates
that the cells poorly adhere to native TMV (FIG. 4). The BHK cells
seeded on the TMV-RGD1 mutant exhibited a bipolar cell shape with
long spindle shape morphology. Similar morphology was also observed
on cells cultured on TMV-RGD7 mutant, which contains longer
flanking sequences derived directly from fibronectin. The cells on
the other mutants, TMV-PHSRN3, -DGEA, and -P15 remained rounded
with no filopodial extensions.
[0097] The cells on both RGD mutants had similar attachment
profiles after the first round of centrifugation, whereas the cells
on PHSRN (SEQ ID NO: 55) and DGEA (SEQ ID NO: 4) had much weaker
attachments (FIG. 5a). The mutant containing collagen I peptide
mimetic (P15) showed the highest level of cell attachment,
resembling similar cell attachment levels to positive controls
(FIG. 5a). The cells on TMV-P15 did not possess the pyramidal or
the spindle shape, which is traditionally associated with spreading
cells. Despite the lack of lamellopodial or filopodial extensions,
the cells on TMV-P15 exhibited the highest attachment strengths.
After a 30 g spin, approximately 50% of the cells detached from the
RGD mutants (TMV-RGD1 and -RGD7), and majority of the cells were
detached for cells on TMV-PHSRN and TMV-DGEA (FIGS. 4 and 5b). The
initial cell body sizes on the various mutants did not provide a
(FIG. 5c). Despite the cell shape, the attachment strengths for the
cells on TMV-P15 closely resembled to that of the uncoated wells
even after the highest centrifugal force tested.
[0098] The centrifugal adhesion assay of the TMV mutants had been
performed with another mammalian cell line, CHO cells. These cells
also secrete little to no extracellular matrix proteins, therefore
making it an ideal candidate to observe how the cells interact with
the substrates. At RCF of 10 g, approximately 60% of the cells
remained attached for the CHO cells for the positive controls, and
for cells on TMV-RGD1 and TMV-P15 (FIG. 7). Similar to the previous
results, the cells on uncoated surfaces exhibited pyramidal
morphology which is typically observed for cultures on standard
tissue culture polystyrene plasticwares. TMV and BSA blocked the
cell attachment and the RGD1 insertion restored cell attachment.
The cell spreading was again limited to a spindle shape with thin
filopodial protrusions, again similar to the morphologies observed
for BHK cells. However, TMV-RGD7 coated substrates did not promote
attachment as strongly as the RGD1 variant for the CHO cells (FIG.
6). The mutant containing fibronectin synergy site, TMV-PHSRN3, and
the short collagen I mimetic (TMV-DGEA) had lower attachment
strengths with no apparent cell spreading. TMV-P15, again showed
strong attachment profile, similar to that of the uncoated and RGD1
variant, but the cells still did not show long filopodial
protrusions. The mutants with collagen I peptide mimetics, TMV-DGEA
and TMV-P15, pose an interesting dilemma. Based on the weak
attachment profiles for TMV-DGEA, the short insert could be
sterically hindered and therefore it fails to bind to the
corresponding receptor, whereas the longer 15 amino acid insert of
P15 is more accessible and thereby allowing it to bind to cell
surface receptors.
[0099] 4. Specific Integrin Proteins can Interact with Mutant
Tobacco Mosaic Virus
[0100] Specific integrin receptors were blocked with monoclonal
antibodies (.alpha.2, .alpha.5, .alpha.V, .beta.1, .beta.2) for
three mutant substrates that showed the highest binding strengths
(RGD1, RGD7, P15). Cell attachment was unaffected by anti-.alpha.2,
.alpha.5, .beta.2 antibodies for cells seeded on TMV-RGD1 coated
substrates (FIG. 5d). Since alpha subunit of integrin receptors do
not possess the RGD binding pocket, it is reasonable that cell
binding is unaffected when blocked with anti alpha antibodies. Only
a minor drop (.about.25%) in attachment was observed for cells on
RGD1 when blocked with anti-.beta.1, which suggests that RGD1
mediates attachment through the vitronectin (.alpha.V.beta.3)
receptor rather than through fibronectin or collagen receptors
(.alpha.5.beta.1 and .alpha.2.beta.1, respectively). For the cells
seeded on RGD7, attached is drastically reduced with the
anti-.beta.1 antibodies (FIG. 5e). This specific reduction by
anti-.beta.1 antibodies suggests that RGD7 mediates cell attachment
more through the fibronectin receptor than through the vitronectin
receptors. However, additional studies will be necessary to
determine whether these mutants are truly specific for one integrin
receptor type. For the cells seeded on collagen mimic, TMV-P15,
anti-.alpha.2 and -.beta.1 antibodies reduced attachment to 30% and
55% of controls, whereas the cells treated with anti-.alpha.5 or
-.beta.2 showed no statistically significant reduction in
attachment (FIG. 5f). Interestingly, anti-.alpha.v treated cells
exhibited a drop in attachment. Ongoing studies involving receptor
binding affinity, downstream molecular events, and focal adhesion
complexes on these mutants will shed some light to how these
multivalent protein scaffolds affect cell surface receptors, their
downstream molecular signals, and ultimately the cell
behaviors.
[0101] The cell morphologies for two samples were closely inspected
by scanning electron microscopy (SEM) with the BHK cells cultured
on TMV and TMV-RGD1. To facilitate the imaging process under high
vacuum and sample handling for SEM, the cells were seeded on glass
coverslips and limited to two distinctive samples. The
morphological feature of cells on TMV and TMV-RGD1 were observed by
higher magnification (FIG. 7), where only on the TMV-RGD1 coated
substrates, the cells possess long filopodial extensions while
still retaining rounded cell bodies. The cells on TMV coated
surface remain rounded with short extensions around the cell body.
These long bipolar cell body shapes are typical for many of the BHK
cells cultured on the RGD variants (TMV-RGD1 and -RGD7).
[0102] 5. Bone Tissue Differentiation Studies
[0103] Based on models of infectious viruses and the importance of
ligand/receptor clusters, we rationalized that plant viruses can
function as non-infectious, multivalent probes for studying complex
ligand/receptor interactions. Moreover, the compatibility of
viruses for cell cultures has been well documented by several
groups. M13 bacteriophage engineered to display cell binding motifs
were assembled into tissue engineering matrices to support neural
cells and to control the directionality of cell growth. Previous
studies in our group demonstrated that substrates coated with plant
viruses supported mesenchymal stem cell growth and accelerated
differentiation. The stem cells cultured on modified phosphate
modified Tobacco mosaic virus (TMV) had increased calcium
mineralization and had higher expression levels of genes related to
bone formation, or osteoblastogenesis.
[0104] We hypothesized that cell differentiation on plant viruses
could be further improved by addressing three parameters. First, a
cell binding motif, GRGDSPG (SEQ ID NO: 1), was extended from the
carboxy end of TMV coat protein to promote attachment. The RGD
sequence is based on the well-known RGD tripeptide sequence to
target integrin receptors and the flanking sequences are derived
from the fibronectin sequence. As a control substrate, recombinant
human fibronectin (hFN) was used. Second, the cells were cultured
in chemically defined, serum-free media with growth factor
supplements. Bovine serum contains xenogeneic proteins and growth
factors, and therefore cannot be used for ex vivo implant models.
The selection of growth factors was based on previous studies shown
to enhance bone differentiation of mesenchymal stem cells. And
lastly, layer-by-layer assembly was used to ensure stable display
of the virus on the substrate (FIG. 8A). Poly(allylamine)
hydrochloride was selected due to its primary amines, which has a
positive charge upon protonation and can electrostatically bind the
viruses to the substrate, as verified by atomic force microscopy
(FIG. 8B). The cells were cultured with osteo-inducing factors
(dexamethasone, L-ascorbic acid 2-phosphate, and
.beta.-glycerophosphate), 1.times.ITS (insulin, transferrin, and
selenium) and growth factors8 (bFGF, TGF-.beta.1, EGF) in
serum-free media for 2 days. All substrates showed relatively
similar cell viability as determined by CellTiter Blue.TM. (FIG.
8C).
[0105] The average deposition density of virus (.about.1
.mu.g/cm.sup.2) on PEMs was measured by quartz crystal microbalance
(FIGS. 9 and 10).
[0106] BMSCs attached and spread on all substrates within one hour
of seeding (FIG. 11A), followed by aggregation of cells on TMV-RGD1
within 48 hours of culture (FIG. 11B). The cell aggregates on
TMV-RGD1 resembled the bone nodules observed in previous cultures.
However, previous cultures required extended incubation time of up
to 14 days, whereas here we observe similar phenomenon within 48
hours (FIG. 11B). The nomenclature of the mutant was classified as
previously reported.
[0107] 6. TMV on Surface Alters BMP2 and IBSP Gene Expression
[0108] For the characterization of the cells cultured on the
various substrates, we probed for key osteogenic markers (BMP-2,
osteocalcin and calcium sequestration). Kaur et al reported that
unmodified TMV was a potent modulator of bone differentiation with
rapid increase in bone morphogenetic protein-2 (BMP-2) gene
expression within the first 24 hours of osteoinduction. The result
suggests that bone differentiation on the virus substrate is
mediated through the upregulation of BMP-2 expression.
[0109] We further examined the temporal changes in gene expression
involved in early differentiation (ALPL, BMP2 and IBSP) for cells
cultured on TMV coated substrates, cells supplemented with TMV in
solution, and cells cultured on conventional TCP (FIG. 12).
Although there was no significant difference in ALPL mRNA
expression for cells cultured on the three different substrates
after 8 hours, the expression significantly increased after 24
hours in cells cultured on TMV substrate. For cells cultured on TMV
coated substrates, BMP2 expression levels were six-fold higher than
uninduced cultures after 8 hours of osteoinduction. In comparison,
the cells in traditional cultures had only slightly increased BMP2
gene expression by two-fold (FIG. 12B). The initial increase in
BMP2 gene expression for TCP had diminished back to basal level
after 24 hours, whereas the cells cultured on TMV coated substrate
still maintained higher BMP2 gene expression levels, a three-fold
increase over TCP substrates (FIG. 12B). Although the addition of
TMV as a solution supplement to the culture media resulted in a
modest increase in BMP2 expression at 24 hours, it failed to
provide the same effects as the virus-coated substrates. The
initial results with TMV in solution indicate that the coating of
the virus to the solid support is necessary to affect early BMP2
gene expression and the virus itself does not act as a soluble
inducer. The results also suggests that TMV coated substrates
enhance osteogenic differentiation of BMSCs by increasing BMP2 mRNA
levels.
[0110] In addition to increased BMP2 gene expression, mRNA levels
for integrin-binding sialoprotein (IBSP), a secreted extracellular
matrix protein required for hydroxyapatite formation, increased
over time in both TCP and TMV samples. IBSP is an osteogenic marker
associated with mineralizing tissues, hence the increase in IBSP
gene expression within 24 hours is an important discovery in
explaining the role of TMV in osteogenic differentiation. IBSP gene
expression levels for cells grown on TMV were two-fold higher than
those of TCP after 8 hours of osteoinduction and the difference
further increased to five-fold after 24 hours (FIG. 12C). The
addition of TMV as a solution to the culture media did not provide
the similar increase in IBSP expression levels as TMV coated
substrates (FIG. 12C).
[0111] The apparent increase in BMP2 and IBSP mRNA levels for the
cells on TMV-coated substrates suggest that these two proteins are
involved in the enhancement of osteogenic differentiation (FIG.
12B, C). The observed differences in gene expression of BMP2 and
IBSP indicate that the surface coating with the virus moderates
BMP2 and IBSP expression levels within the first day, and the virus
itself does not act as a soluble chemical inducer, but rather as a
substrate with unique topographical features, or offering a rough
surface, at both nanometer and micrometer scales.
[0112] The increase in BMP2 mRNA expression level was corroborated
by ELISA, showing a similar trend of BMP2 increase at 8 and 24
hours in osteogenic media (FIG. 13A). In comparison to uninduced
controls, the cells on TMV coated substrates expressed four-fold
and three-fold higher levels of BMP2 at 8 and 24 hours,
respectively. For TCP culture, there was no significant increase in
BMP2 production at either time points (FIG. 13A). Clearly, among
the three growth conditions, only TMV coated surface induced a
significant increase in BMP2 mRNA and protein expression after 8
hours of osteogenic induction.
[0113] Immunofluorescence imaging for BMP2 revealed that the
morphogen is localized to the cell aggregates. As shown in FIG.
13B, BMSCs on TMV stained positive for BMP2 with higher
fluorescence intensity at both time points. BMP2 was observed
around the cells that formed nodules or aggregates. It is
hypothesized that too strong substrate binding may inhibit
osteogenic differentiation. Similar to the previous results, the
cells with TMV in solution stained poorly for BMP2. The
concentrations of secreted BMP2 in conditioned media from the three
culture conditions were measured by sandwich based ELISA. Higher
concentration of BMP2 was observed in media with cells on TMV
substrate, compared to cells on TCP with and without TMV
supplemented in the media. The concentrations ranged between
100-200 pg/mL (data not shown), which is consistent with that
reported in an ultrasound-induced BMP2 secretion within 24
hours.
[0114] 7. Enhanced Osteogenesis Requires TMV Coating on
Substrate
[0115] The enhancement in bone differentiation was also assessed by
measuring ALP activity and calcium deposition at day 7, 14, and 21.
ALP is an early marker of osteogenesis and its activity mediates
matrix mineralization. Although only minor upregulation of ALPL
mRNA levels was observed by RT-qPCR after 24 hours, ALP enzyme
activity assays over the course of 3 weeks showed significant
differences at day 7 and 14 (FIG. 14A). Similar to the earlier
results, the addition of TMV in solution does not increase ALP
activity, and only the cells on TMV coated substrates had
significantly higher enzyme activities compared to controls. The
enzyme activity at day 21 diminishes to the same level for all
samples, suggesting that the observed level may be a basal activity
level of ALP during mineralization stage.
[0116] Calcium deposition was determined at day 14 by staining with
Alizarin Red S. Small nodules of mineralized calcium were observed
for controls and cells supplemented with TMV in solution, whereas
the nodules were visibly larger for the cells on TMV-coated
substrates (FIG. 14B). UV-Vis absorbance measurements of the
extracted dyes indicated the cells on TMV substrate had double
content of calcium compared to control (FIG. 14C). The addition of
TMV solution does not enhance matrix mineralization. These results
confirm our hypothesis that TMV provides a topographical cue to
enhance osteogenic differentiation, likely by changing the surface
roughness. The combined results from RT-qPCR, ELISA and
immunostaining clearly indicate that BMP2 expression was
significantly induced within 24 hour of induction with a peak
expression level at 8 hours. The enhancement requires the virus as
a substrate rather than as a solution supplement, suggesting the
role of topography or surface roughness in TMV-mediated
osteogenesis.
[0117] 8. TMV Coating Alters Cytokine Expression, Actin
Depolymerisation and Focal Adhesion
[0118] We investigated the cells' response to the virus by
screening such cytokines or other relevant growth factors which
could affect early bone differentiation other than BMP2. Cytokine
antibody arrays for 29 cytokines were used to screen the
conditioned media from cells cultured on TCP and TMV coated wafers.
The media consisting of primary media only was collected after 24
hours of culture and examined. Tissue inhibitor of
metalloproteinases-1 (TIMP-1) was the only molecule secreted from
both culture conditions, while cytokine-induced neutrophil
chemoattractant 1 (CINC-1), CINC-2, MIP-3.alpha., and vascular
endothelial growth factor (VEGF) were detected from the conditioned
media for the cells on TMV wafers (FIG. 15). According to Kim et
al., CINC-1 and MIP-3.alpha. recruit neutrophils and monocytes to
induce osteogenesis. However, the previous studies did not
correlate these cytokines with enhanced BMP2 upregulation during
osteogenesis. Future studies on the osteogenic effects of these
individual cytokines may reveal their involvement in BMP2-mediated
bone differentiation. It could possibly lead to a discovery of new
biomaterials to elicit these cytokine productions in order to
improve bone healing.
[0119] The remaining 24 cytokines, CINC-3, ciliary neurotrophic
factor (CNTF), fractalkine, GM-CSF, sICAM-1, IFN-.gamma.,
IL-1.alpha., -1.beta., -1ra, -2, -3, -4, -6, -10, -13, -17, IP-10,
lipopolysaccharide induced c-x-c chemokine (LIX), L-selectin,
monokine induced by gamma interferon (MIG), MIP-1.alpha., RANTES,
thymus chemokine and TNF-.alpha. were below the detection limits of
the cytokine array.
[0120] Since there was no established direct correlation of other
cytokines and osteogenesis, cell morphologies were closely observed
prior to osteoinduction. It was demonstrated that BMP2 production
was highly localized around the cell nodules. The investigation of
actin polymerization and organization was carried out to compare
cells on TCP and TMV substrates prior to osteoinduction, at which
time cell aggregation was first observed. The actin intensity by
phalloidin staining was more intense in cells on TMV substrate,
indicating higher activity from actin polymerization which could
facilitate mobilization of cells to form nodules within 24 hours of
cell seeding (FIG. 16). At higher magnification, cells on TCP
surface maintained highly aligned sarcomeric striations, whereas
cells on TMV substrate displayed nonaligned striations. These
observations are similar to an experiment by Mendonca et al., which
demonstrated that cells on a rougher surface topography exhibit an
undefined long axis with thicker actin filaments leading to less
initial cell spreading followed by enhanced mineralization.
[0121] The interaction between cell and ECM is mediated by cell
surface receptors (i.e. integrins). Vinculins, part of focal
adhesion complexes (FACs), couple the integrins to the cytoskeleton
allowing crosstalk between ECM and intracellular signalling.
Previous studies illustrated that increased localization of
vinculin is associated with larger focal adhesion (FA) size and
strengthening of adhesion leading to reduced cell motility. Smaller
size of FAs suggests that BMSCs attached to TMV substrate weakly,
whereas larger size of FACs dictates stronger cell-substrate
adhesion. While BMSCs on standard TCP exhibited larger average
vinculin size, indicating stronger adhesion to the underlying
substrate, the cells on TMV substrate showed smaller FA sizes (FIG.
17A, B). The significantly smaller FA size for cells on is likely
to increase cell motility and facilitate the formation of cell
aggregates within 24 hours of seeding. Further studies are
necessary to further establish the correlation of the focal
adhesion of BMSCs on virus-coated substrates with the downstream
responses.
[0122] 9. TMV Mutants can Further Promote the BMP-2 Production and
Osteogenesis
[0123] To determine whether TMV-RGD1 coated substrates can mediate
similar response in BMSCs, the two day cultures were fixed and
probed with anti-BMP2 antibodies. Significantly higher levels of
BMP-2 were found for BMSCs on TMV-RGD1 (FIGS. 18A,B) and localized
only to the large cell aggregates which are only present on
TMV-RGD1 substrates (FIG. 18A). An advantage of the virus is the
multivalent ligand display capability. To probe for its effect on
cells, we probed for focal adhesion kinase activity.
Phosphorylation of FAK at Y397 is associated with increased
activity of FAK, and such increase in FAK activity has been
associated with multivalent ligands and increased osteogenesis
(bone formation). An increase in focal adhesion kinase
phosphorylation at residue Y397 was also observed for cells
cultured on TMV-RGD1 coated substrates (FIG. 18C), two-fold above
the levels detected for cells on human fibronectin (hFN) (FIG.
18D).
[0124] Expression of osteocalcin, a protein primarily expressed in
mature bone cells (osteoblasts and osteocytes), was observed only
in the cells grown on TMV-RGD1 substrates, whereas the cells on
other substrates had little to no detectable levels (FIG. 19A). The
cells were stained with Alizarin Red S, the cell aggregates stained
red for calcium minerals (FIG. 19B). These cell aggregates, which
resemble the typical nodules found in previous studies after 14
days of osteoinduction on TMV substrates, were formed within 2 days
and stained positive for calcium with Alizarin Red, a typical
cytochemical stain for calcium in cells undergoing osteogenesis. As
the stem cells undergo bone differentiation, calcium is sequestered
and mineralized with surrounding matrix proteins to form
mineralized bone tissues. Higher intensity of the cytochemical
stain is observed primarily for the cell clumps found on TMV-RGD1
coated substrates (FIG. 19B).
[0125] These adaptations significantly shortened the rate of our
previous differentiation time requirement from 2 weeks to 2 days of
osteoinduction. Previous studies indicate that mesenchymal stem
cells are induced for two to three weeks to observe canonical
osteoblast phenotypes. The results herein show that a TMV variant
can promote cell differentiation and show calcium sequestration and
expression of established osteogenic markers within two days of
osteoinduction. These results suggest that virus-based biomaterials
can function as alternative matrix mimetics to promote stem cell
differentiation.
[0126] The rate of stem cell differentiation on TMV coated
substrates was enhanced by manipulating three factors. The virus
was genetically modified with a cell binding ligand. A chemically
defined media and a stable display of the virus by layer-by-layer
assembly provided robust culture protocol that supported rapid cell
differentiation. In this study, we observed the typical bio-markers
for bone induction within two days of culture, which is
dramatically enhanced when compared to previous reports requiring
14 days for differentiation. The cells aggregated only on the
TMV-RGD1 substrates, whereas the cells on hFN remained spread and
stained poorly for calcium minerals. BMP-2 and osteocalcin, which
are found in cells undergoing bone differentiation, were only found
in cells cultured on TMV-RGD1 substrates. A study has shown that
the primary amino groups on substrates enhance osteogenesis, and
such groups are displayed on the polyelectrolyte (PAH), which is
the top-most layer. However, the amino group alone cannot account
for the increased number of nodules observed on TMV-RGD1 as shown
in the results (FIGS. 18, and 19). The enhanced rate of
differentiation could be due to the cell binding peptide, but
fibronectin coating does not provide the same rapid differentiation
(FIG. 19).
[0127] 10. New Coating Protocol for Preparing Homogeneous TMV
Surface for Cell Culturing
[0128] A major limitation of the layer-by-layer coating is the
required multiple coatings of polyelectrolytes. While an automated
process can be adapted to achieve a faster coating process, for
general laboratory use, repetitive coating process requires long
procedural time frames (20 hours to coat 5.times.24 well plates)
when performed manually. To address this issue, a preliminary study
was conducted using high binding plates without any polyelectrolyte
coating. The results have indicated that the virus can be directly
bound to the high binding 96-well plates by incubating 100 .mu.l of
the virus solution for 20 minutes at room temperature. Different
concentrations of the virus solution ranging from 0.01 .mu.g/mL to
100 .mu.g/mL were used to monitor the optimal coating density on
the high binding plates by ELISA (FIG. 20). The direct coating of
virus on high binding plates was initially used for cell adhesion
studies, but these initial studies indicate that surfaces are not
fully covered by the virus particles.
[0129] Coating the high binding plates with virus and the different
mutants slightly altered the hydrophobicity of the surfaces (FIG.
21). Original uncoated surfaces indicated a high water contact
angle (.about.90.degree.), and coating with wild type TMV increased
hydrophilicity as indicated in a decrease in the water angle
(.about.80.degree.). The mutants TMV-RGD1, PHSRN3, DGEA further
lowered the water contact, indicating increasing hydrophilicity.
TMV-P15 and human fibronectin coatings retained a slightly more
hydrophobic surface than the other three mutants.
[0130] The high binding plates coated with virus and the different
mutants, then the plates coated with virus particles were seeded
with rat bone marrow derived mesenchymal stem cells. Cell spreading
was measured after staining the cells with Calcein-AM for 1 hour at
37.degree. C. (FIG. 22). The cells were imaged by confocal
microscope using GFP filters (FIG. 22A) and spreading was
calculated by two different methods. In one method, the overall
fluorescence intensity of each sample was measured using a
fluorimeter (FIG. 22B), and the other method measured the area
coverage per image frame (FIG. 22C). To calculate percent coverage,
the area covered by the cells was divided by the total area of the
frame. Three images per well was taken from 4 different wells (n=12
per sample). Despite the incomplete coverage of the virus particles
on the high binding plates, the cells attached and spread on the
TMV-RGD1 mutant in a similar manner to human fibronectin coated
substrates.
[0131] rBMSCs seeded on the plates were treated with chemically
defined serum-free osteogenic media for 21 days to initiate
differentiation. Cell differentiation was monitored every 7 days by
measuring alkaline phosphatase activity and calcium deposition
(FIG. 23). Higher level of enzymatic activity was observed for the
cells cultured on TMV-RGD1 coated substrates in comparison to the
cells cultured on other mutants and on unmodified TMV (FIG. 23A).
This increased enzymatic activity indicating its progression
towards osteogenic differentiation. However in comparison to the
cells on human fibronectin (hFN) coated substrates, the cells
cultured on TMV-RGD1 had equal or lower levels of enzymatic
activity for the three time points examined. Since this particular
enzymatic activity is reported to increase during
mid-differentiation phase and to decrease during its final stages
of differentiation, it remains to be discovered whether the cells
on TMV-RGD1 had already reached maximal enzymatic activity prior or
post day 14 of differentiation time point. Calcium quantifies for
the differentiating cells were also calculated every 7 days on the
various mutants including unmodified TMV and hFN coated controls.
The increasing calcium levels suggest that the cells on TMV-RGD1
maintain its high rate of differentiation, similar to that of cells
cultured on hFN coated controls (FIG. 23B) and the progression of
osteogenic differentiation is similar to that of its alkaline
phosphatase activity levels. Calcium levels for the cells cultured
on the rest of the mutants increased on day 21 to the levels
observed on day 14 for the cells on TMV-RGD1 and hFN coated
substrates. The cells stained with Alizarin Red S dye for calcium
deposits were imaged after day 14. The differentiating cells were
imaged after 14 days of osteogenic differentiation (FIG. 23C). The
red colored regions represent higher levels of calcium deposits
around the stem cells. Based on these images, the cells appear to
have low levels of calcium deposits and do not show full
progression towards osteoblastic cells. Unlike the previous
layer-by-layer coatings, these surface coatings appear to delay the
cell differentiation rates.
[0132] Despite the potential use of the direct coating of virus on
high binding plates, there are several drawbacks to this
methodology. One drawback for using uncoated high binding plates is
the virus coating consistency. Another limitation is the slow
differentiation observed with chemically-defined serum free culture
conditions. In the previous layer-by-layer assemblies, osteogenic
differentiation was observed within 2 days of culture, whereas for
the high binding plates, the cells still required prolonged culture
periods (>21 days). Due to these limitations (slow
differentiation and uneven virus coatings), alternative coating
methods were sought. Surface coating optimizations to achieve even
surface coatings and more consistent surface depositions were
conducted.
[0133] Based on the previous results, an amine displaying
polyelectrolyte appeared to be well suited for the cell
differentiations. To maintain that amino group, a single layer of
polyelectrolyte (polyallylamine hydrochloride--PAH) was used at
different pH ranging from 4 to 13 (FIG. 24). First, the densities
of PAH deposited at different pH were monitored by QCM (FIG. 24A).
The measurements indicated that raising the pH to the pI value of
PAH (.about.9-9.5) resulted in the greatest amount of
polyelectrolyte being deposited on the quartz surface (FIG. 24B).
TMV depositions increased on surfaces coated with PAH solutions
with decreasing acidity, with maximum depositions being reached
around pH 9-11, then decreasing when pH levels of PAH solutions
were further increased to 12 and 13 (FIG. 24C). These surface
coatings were visualized as uncoated (FIG. 24D), PAH coated at pH 4
(FIG. 24D), pH 11 (FIG. 24E) and pH 11 (FIG. 24F). The distinctive
shape of TMV (rod-like) is more distinct on pH 4 (FIG. 18E), but
the coverage appears to be less than 50% of the entire area. On the
other hand, the rod-like particles are not visible on the high
density coatings at pH 11 (FIG. 24F) and pH 13 (FIG. 24G). A
trade-off between high density coatings and even coatings of TMV
particles (high coverage with clearly visible side-by-side
assemblies) appear to exist.
[0134] To further explore this balance of various factors pH, salt
and virus concentrations, we examined virus depositions starting at
PAH coating solution at pH 5, then using two different buffer pH's
(5 and 8) and two different buffer concentrations (10 and 100 mM of
potassium phosphate). The desired result was to find the factors
that would allow for near complete coating of the surface with
virus particles and side-by-side assemblies with high
reproducibility.
[0135] Two major reasons behind this desired coating were;
[0136] 1) Complete surface coverage would be better correlated to
the changes in cellular behaviors, whereas partial coverage would
allow the underlying layer to interact and possibly overwhelm the
effects of the virus coatings;
[0137] 2) All of the cell binding sequences are inserted along the
longitudinal axis of the virus particle, therefore laying the virus
particles on its side was essential to maximize the exposure of
these ligands at its highest densities.
[0138] The deposition of the virus was monitored by AFM using TMV
in 10 or 100 mM buffer either in pH 5 or 8. All substrates were
prepared with PAH at pH 5 prior to coating with virus particles
(FIG. 25). Based on the results, coating the virus particles at pH
8 alone gave to the least amount of particles on the surface, and
lowering the pH of the virus solution to 5 increase deposition to
the PAH coated surfaces.
[0139] The AFM results were further validated by QCM to monitor the
deposition of the virus at the two different pH and buffer
concentrations (FIG. 26). All substrates were prepared with PAH at
pH 5 prior to coating with virus particles. Based on the results,
coating the virus particles at pH 8 alone gave to the least amount
of particles on the surface, and lowering the pH of the virus
solution to 5 increase deposition to the PAH coated surfaces.
Eliminating the salt, completely eliminated the inter particle
charge screening and reduced coverage and deposition, as indicated
by both QCM and AFM.
[0140] Testing these single PAH coatings for stem cell culture will
be essential and verifying that these single layer coatings can
provide the same rapid differentiation potential in chemically
defined serum-free media will become the critical next step.
Overall, we observe rapid stem cell differentiation in serum-free,
chemically defined osteogenic media on genetically modified plant
virus particles. This initial culture optimization in xeno-free
cultures on multivalent scaffolds is an important step for the
future development of ex vivo implant models. Furthermore, the
virus is a powerful molecular tool that can be isolated
consistently at low costs, with no observable cytotoxic effects,
and the potential for large libraries of ligands (single or
multiple) for dissecting some key regulating events in stem cell
differentiation.
[0141] The underlying mechanism involved for this enhanced will
require additional in depth analysis between the ligand sequence,
mock sequence, varied ligand densities, substrate chemistries. One
possible explanation is based on the interactions between virus,
substrate, and growth factors to provide additive effects on bone
differentiation to reduce the time required to observe
osteo-typical markers. Additional comparative studies with
collagen, other TMV variants, and structurally different viruses
will provide further clues to the role of ligand organization, its
effect on downstream signaling events (i.e. specific integrin
receptors, FAK) and controlling the enigmatic stem cell behavior.
The simplicity in altering the coat protein gene of TMV and the
high yield of these virus variants from the plant host make these
virus particles an attractive material for exploring how
bionanoparticles can be used to control cell differentiation. The
induction of rapid bone formation with autologous mesenchymal stem
cells is highly relevant for future clinical applications where
speedy ex vivo cultures of stem cells into bone tissues can
ameliorate patients in urgent need of bone defect replacements.
[0142] 11. Viral Particles can be Used to Functionalize
Biocompatible Porous Hydrogels
[0143] In regenerative medicine, synthetic extracellular matrix is
crucial for supporting stem cells during its differentiation
process to integrate into surrounding tissues. Hydrogels are used
extensively in biomaterials as such synthetic matrices to support
the cells. However, in order to mimic the biological niche of a
functional tissue, various chemical functionalities are involved.
We further developed a method of functionalizing highly porous
hydrogel with functional groups by mixing the hydrogel with plant
virus particles. Again, TMV and its mutants were used as the
prototypical particles, while the methods can be extended to
similar rodlike or spherical viruses.
[0144] Our approach is designed to be performed in a single pot
with the sequential additions of reagents to ultimately incorporate
biologically relevant functionalities in 3D scaffolds. TMV were
introduced into the hydrogel by adding the non-infectious virus
particles during the formation of gas template porous alginate
hydrogels (PAH) (FIG. 27). The process involving no covalent
modification and no further purification, which is contrary to
peptide or protein conjugated hydrogels that require extensive
dialysis to remove untethered peptides/proteins. The homogeneous
mixture of alginate in bicarbonate solution with low concentration
of surfactant (Pluronic F-108) is reacted with a mild acid (citric
acid) to generate CO.sub.2 in situ (FIG. 1A). The incorporation of
the virus solution pursues and the entire foamy material is flash
frozen and crosslinked. Additional procedures involving further
dialysis and lyophilization provided the final PAH functionalized
with the virus particles.
[0145] The initial characterization of the virus-incorporated PAH
by ESEM revealed that the addition of the virus did not hinder pore
formation with the macropore and interconnecting channels ranging
between 10-500 microns for both PAH and TMV-PAH. In a close up
magnification, PAH appears to have smaller pores and dense network
(FIG. 28B), whereas large well-defined spherical pores (100 to 500
microns) are formed in TMV-PAH (FIG. 28D). The ESEM images show
that the hydrogel formation is not hindered by the incorporation of
the virus, but rather facilitates the formation of large,
well-defined pores. The integrity of virus particles incorporated
in PAH was assessed by de-crosslinking with EDTA to chelate
Ca.sup.2+ ions. This resulted in the subsequent release of TMV from
PAH. TEM imaging of the dissolved TMV-PAH showed intact TMV
particles (FIG. 28E), which were similar in size when compared to
the TMV particles prior to incorporation (FIG. 28F). UV-Vis
absorbance measurement of the de-crosslinked TMV-PAH solution could
give the concentration of the TMV recovered that indicated that
nearly all of virus could be retrieved after treating the hydrogel
with EDTA.
[0146] The virus-incorporated PAH was also shown to have similar
physical properties that could potentially assist 3D cell culture
including swelling property, stability, and structural basis of
pore architecture. The hydrogels could absorb water up to 7 folds
of their initial dry weight indicating the extreme hydrophilicity
as tissue-like environment and capability to uptake medium for cell
culture. From the stability test, we observed no mass losses from
PAH and TMV-PAH during the 4-week period that could support the
entire cell culture experiments and suit for tissue engineering
applications. As well as in thermal analysis, while MDSC data
supported the incorporation of virus particles into PAH, we showed
that TMV did not significantly impact the thermo-stability of
original PAH as indicated by TGA. As there were no covalent
modifications on the polymer chain structure, alginate still
maintains their own properties and dominates as a bulk material.
The amount of TMV released from TMV-PAH into the aqueous solution
was measured and it confirmed that most TMV particles were
entrapped in the hydrogel matrices even upon long term incubation
in solution.
[0147] The incorporation of TMV in PAH did not at all impair the
pore formation or the pore architecture. Interestingly, TMV-PAH
(also RGD-PAH) gained larger extent of big pores. It is possible
that TMV can facilitate and stabilize the macropore formation due
to its coat protein amphiphilic property similar to other peptides
and proteins. TMV particles could help trapping larger hydrophobic
gas pockets upon the generation of CO.sub.2 to better mediate the
interfacial tension between aqueous and gas phases. The mechanical
properties of stem cell microenvironments are well-recognized as
one of essential determinants of differentiation fate. The
mechanical environments that promote certain differentiation fates
vary dramatically, with as much as 300-fold difference in elastic
modulus between matrices that favor soft brain tissue (0.1 kPa) and
decalcifying bone (>30 kPa). It is plausible that the alginate
bulk is mechanically reinforced by the inclusion of TMV particles
(resulting in increased stiffness at low strains), but the
macro-porous architecture begins to collapse at higher strains
causing a reduction in material stiffness. Considering the
cell-scaffold interaction that happens at a low strain range near
the original stage of compression, this increase in stiffness has
reinforced the advantage to the virus incorporated hydrogel
regarding bone tissue engineering.
[0148] 12. TMV-Hydrogel can Support the Cell Attachment and
Differentiation
[0149] To demonstrate the potential use in cell cultures, cell
attachment and viabilities were determined for the three types of
hydrogels. In cell attachment assay, RGD-PAH (96.+-.1%) had
significantly higher cell attachment after 8 h. incubation than for
PAH (85.+-.3%). The incorporation of TMV did not provide any
significant improvement to cell attachment when compared to the
control (85.+-.1%) (FIG. 29A). Based on the metabolic assay with
CTB reagent, the cell viabilities in all three different types of
hydrogels were determined for day 8 and day 14 cultures and
normalized to metabolic rates of cells in PAH (FIG. 29B). The
results indicate that BMSCs survived in both virus modified PAHs
(TMV- and RGD-PAH) and the cells cultured on RGD-PAH continued to
have the highest metabolic activity (FIG. 29B). However, there was
no further increase in metabolically active viable cells from day 8
to day 14.
[0150] We seeded BMSCs in hydrogels and cultured in osteogenic
media as an extension of our previous work on 2D substrates with
virus particles. Osteogenesis was initially assessed by alkaline
phosphatase (ALP) activity for day 3, day 7, and day 14 cultures
(FIG. 30A). Low levels of enzyme activity were detected for all
three samples for day 3 cultures, followed by markedly increases in
ALP activities on day 7 and 14. The incorporation of the virus did
not appear to hinder the differentiation potential as the same
elevation trends of overall ALP activities were measured among the
three groups from day 3 to day 14. Interestingly, RGD-PAH seemed to
accelerate an early osteogenesis of BMSCs as the ALP activity was
significantly higher among three samples on day 3. Although, on day
7, the higher level of ALP activity for PAH than the other two
samples was unexpected. Calcium deposition in three types of
hydrogels with BMSC osteogenic culture was quantified by ICP-OES
(FIG. 30B). The average amounts of calcium deposited in both
TMV-PAH and RGD-PAH after background subtraction from control
samples without cells were higher than in PAH (FIG. 30B). All three
types of hydrogels with BMSCs osteogenic culture were stained with
Alizarin red giving intense red color (FIG. 30C).
[0151] Lastly, osteocalcin, a well-known osteospecific marker, was
observed by immonostaining in cell-hydrogel cultures treated with
osteoinductive media in PAH, TMV-PAH, and RGD-PAH. FIG. 31
illustrated the accumulation of osteocalcin after day 10 (PAH) and
day 13 (TMV-PAH and RGD-PAH) and confirmed that virus incorporation
into 3D matrices did not impair the differentiation potential of
BMSC into osteogenic lineage.
[0152] The implication of this process resides with the three
important features of TMV: its well-defined genetic/chemical
modularity, multi-valency (its capsid is composed of 2130 copies of
identical subunits), and its well-defined structural features.
Previous studies utilizing the native TMV on 2-dimensional supports
accelerated mesenchymal stem cell differentiation and genetically
modified viral particles further enhanced cell attachment and
differentiation. We demonstrate that functionalization of porous
alginate scaffold can be achieved by addition of viral particles
with minimal processing and downstream purifications, and the cell
attachment and differentiation within the macro-porous scaffold can
be effectively manipulated by altering the peptide or small
molecule displayed on the viral particles.
[0153] General Procedure:
[0154] Virus Purification:
[0155] Wild type TMV and its mutant variants were purified
according to previously reported method with slight modification.
The infected leaves were collected and stored in Ziploc.RTM. bags
(.about.100-150 g of leaves per bag) and stored in -80.degree. C.
freezer for later processing. For purification, the frozen leaves
were crushed by hand inside the plastic bag, then transferred to a
1.5 L blender (Oster). Approximately 3 volumes of phosphate buffer
was added to the crushed leaves with an addition of 0.2-0.3%
2-mercaptoethanol. The leaves were blended for 2 minutes at low
setting then switched to highest setting for an additional 3
minutes. The blended plant sap was filtered through two layers of
cheesecloth, then flow through was centrifuged at 13,500 rpm
(Sorvall SLA1500) at 4.degree. C. for 30 minutes. The resulting
supernatant was then pooled together and mixed with
n-butanol/chloroform at a ratio of 2:1:1 (plant
sap:n-butanol:chloroform). The homogenate was stirred for 30
minutes on ice, then centrifuged at 12,500 rpm (SLA 1500) for 20
minutes at 4.degree. C. The aqueous layer was transferred to a
beaker and the virus was precipitated by adding 0.2 M NaCl and 8%
(wt/vol) PEG-8000. The mixture was stirred on ice for 60 minutes,
and centrifuged at 13,500 rpm (Sorvall SLA1500) for 20 minutes. The
white precipitant was then resuspended with 10 mM K phos buffer (pH
7) supplemented with PMSF protease inhibitor (10 .mu.g/mL final
concentration). The solution was centrifuged at 9,500 rpm for 10
minutes at 4.degree. C. to remove excess PEG. The supernatant was
transferred to ultracentrifuge tubes (Beckman 50.2 Ti) and the
virus was pelleted at 42,000 rpm at 4.degree. C. for 2.5 hours. The
virus pellet was resuspended in 100 mM K phos buffer (pH 7)
overnight at 4.degree. C. The virus solution was centrifuged again
at 9,500 rpm for 10 min at 4.degree. C. to remove insoluble debris.
UV absorbance was measured at 260 and 280 nm wavelengths to
determine the concentration of the virus. The virus concentration
of 0.1 mg/mL has an absorbance of 0.3 at 260 nm and exhibits a
characteristic 260/280 ratio of 1.26-1.27. The purified virus
solutions were aliquoted and stored at -20.degree. C. freezer.
[0156] Atomic Force Microscopy and Scanning Electron
Microscopy:
[0157] Tapping-mode atomic force microscope (AFM) images were
obtained at ambient conditions using Nanoscope IIIA MultiMode AFM
(Veeco). Si tips with a resonance frequency of approximately 300
kHz, a spring constant of about 40 N m-1, and a scan rate of 0.75
Hz were used. For scanning electron microscopy, the cells were
washed three times in 1.times.PBS at ambient temperature. Cells
were then fixed using 4% paraformaldehyde for 15 minutes at room
temperature. After which the cell samples were fixed with 0.1 M
cacodylate buffer (pH 7.2), 1% OsO.sub.4 for an hour at 4.degree.
C. The samples were washed several times in cacodylate buffer, and
dehydrated in a series of ethanol solutions (50%, 70%, 95%) for 10
minutes each. The samples were immersed in 100% ethanol for the
final dehydration step. The cells on the cover-slips were mounted
on a stub and sputter coated with a thin layer of gold prior to
imaging under FEI Quanta 200 ESEM.
[0158] Generation of TMV Mutants:
[0159] TMV coat protein gene was mutated using overlap-extension
PCR. The TMV coat protein gene sequence for PCR was obtained by
digesting U3/12-4 plasmid (TMV U1 strain) with NcoI and KpnI
restriction enzymes. The digested fragment from the TMV U3/12-4
plasmid contained the entire TMV coat protein gene plus the 3' UTR
of the viral genome. The fragment was inserted into cloning vector
pBluescript SK(+) II. This plasmid was designated pCP-NK. Two
complementary mutagenic oligos were made with the following list of
primers. For the overlap extension mutagenesis, two PCR reactions
were performed in parallel, M13F primer was paired with the
mutagenic forward primer while the M13R primer was paired with
mutagenic reverse primer. The products of the two PCR reactions
were gel purified and combined in a third PCR reaction using the
M13F and M13R primers with the two previous PCR products as
templates. This final PCR product was digested with NcoI and KpnI
and cloned into the TMV U3/12-4 plasmid, which was designated
TMV-RGD1. The mutated coat protein ORF was confirmed by sequencing.
The plasmid was cut at the unique KpnI site and the linearized
plasmid was transcribed using the MEGAscript T7 kit (Ambion)
following the manufacturer's instructions. Ten micrograms of RNA
per tobacco leaf was used for inoculation. For the rest of the
mutants, the entire TMV genome was inserted 3' of a CaMV 35S
promoter within a plasmid previously digested to remove the
downstream TMV sequences. The original plasmid construct was
designed in a pALTER-1 vector (pU1R 35S), which exhibited some
instability. Therefore the entire TMV genome with the CaMV 35S
promoter was transferred to a BlueScript vector.
TABLE-US-00005 TABLE 5 List of TMV mutants (SEQ ID NOS 1, 6-10, 2,
11-14 and 3-5, respectively, in order of appearance) Calculated
Observed Insert Insert Mass Mass Mutants Sequence Site (m/z) (m/z)
RGD1 GRGDSPG Extended after T159 18161 18161 RGD2 GAGSGRGDSGA
Inserted between 18408 n/i* (no S155-G156 infection) RGD3
TGRGDSPASS Deleted G156-T159 and 18124 n/i extended from S155 RGD4
GEPRGDTYRA Deleted G156-T159 and 18311 n/i extended from S155 RGD5
AGSGRGDSGA Inserted between 18350 n/i S155-G156 RGD6 GEPRGDTYRAY
Inserted between 18801 n/i S155-G156 RGD7 AVTGRGDSPASS Inserted
between 18621 18622 S155-G156 RGD8 ENGPRGDNYRA Inserted between
18766 n/i S155-G156 RGD9 AGSGEPRGDTYRASG Inserted between 18621 n/i
S155-G156 PHSR PHSRNG Extended after T159 18183 n/i N1 PHSR
EDRVPHSRNS Deleted G156-T159 and 18387 n/i N2 extended fro S155
PHSR EDRVPHSRNSIT Inserted between 18927 18926 N3 S155-G156 DGEA
DGEA Inserted between 17908 17905 S155-G156 P15 GTPGPQGIAGQRGVV
Inserted between 18910 18896 S155-G156
TABLE-US-00006 TABLE 6 The corresponding peptide loss associated
with the truncated TMV coat protein. A mass difference of 17 is
associated to the loss of a hydroxy ion. (SEQ ID NOS 15-22,
respectively, in order of appearance) Mutants .DELTA.m/z Peptide
loss A. RGD1 18159-17801 = 358 DGPG (374) 18159-17429 = 730
TGRGDSPG (746) B. RGD7 18625-18123 = 502 SSGPAT (519) 18625-17754 =
871 DSPASSGPAT (889) C. PHSRN3 19001-17672 = 1329 RPHSRNSITGPAT
(1394) 19001-17399 = 1602 DVRPHSRNSITGPAT (1608) D. DGEA
17905-17372 = 543 EAGPAT (545) E. P15 18928-17485 = 1443
GPQGIAGQRGVVGPAT (1465)
TABLE-US-00007 TABLE 7 TMV mutant primers (SEQ ID NOS 23-50,
respectively, in order of appearance) Mutant Primer Sequence RGD1
Forward TCT GGT CCT GCA ACT GGA AGA GGA GAC TCT CCA GGA TGA GGT AGT
CAA GAT Reverse ATC TTG ACT ACC TCA TCC TGG AGA GTC TCC TCT TCC AGT
TGC AGG ACC AGA RGD2 Forward AGC GGC AGA GGC GAC AGC GGC GCC GGT
CCT GCA ACT TGA GGT AGT Reverse GCT GTC GCC TCT GCC GCT GCC GGC ACC
AGA GGT CCA AAC CAA ACC RGD3 Forward AGA GGC GAC AGC CCC GCC AGC
AGC TGA GGT AGT CAA GAT GCA TAA Reverse GGC GGG GCT GTC GCC TCT GCC
GGT AGA GGT CCA AAC CAA ACC RGD4 Forward GGC GAG CCC AGA GGC GAC
ACC TAC AGA GCC TGA GGT AGT CAA GAT GCA TAA Reverse GGC TCT GTA GGT
GTC GCC TCT GGG CTC GCC AGA GGT CCA AAC CAA ACC RGD5 Forward AGC
GGC AGA GGC GAC AGC GGC GCC GGT CCT GCA ACT TGA GGT AGT Reverse GCT
GTC GCC TCT GCC GCT GCC GGC AGA GGT CCA AAC CAA ACC AGA RGD6
Forward CCG AGA GGA GAT ACA TAC AGA GCA TAC GGT CCT GCA ACT TGA GGT
AGT Reverse TCT GTA TGT ATC TCC TCT CGG CTC TCC AGA GGT CCA AAC CAA
ACC AGA RGD7 Forward GGA AGA GGA GAT TCA CCG GCA TCA TCA GGT CCT
GCA ACT TGA GGT AGT Reverse CGG TGA ATC TCC TCT TCC TGT CAC TGC AGA
GGT CCA AAC CAA ACC AGA RGD8 Forward AAT GGA CCG AGA GGA GAT AAT
TAC AGA GCA GGT CCT GCA ACT TGA GGT Reverse TCT GTA ATT ATC TCC TCT
CGG TCC ATT CTC AGA GGT CCA AAC CAA ACC RGD9* Forward CGC GGT GAT
ACG TAC CGT GCG AGC GGC GCC GGT CCT GCA ACT TGA Reverse GTA CGT ATC
ACC GCG TGG CTC TCC GCT GCC GGC AGA GGT CCA AAC CAA PHSRN Forward
TCT GGT CCT GCA ACT CCA CAC TCT AGA AAT GGA TGA GGT AGT CAA GAT 1
Reverse ATC TTG ACT ACC TCA TCC ATT TCT AGA GTG TGG AGT TGC AGG ACC
AGA PHSRN Forward AGA GTG CCC CAC AGC AGA AAC AGC TGA GGT AGT CAA
GAT GCA TAA 2 Reverse TCT GCT GTG GGG CAC TCT GTC CTC AGA GGT CCA
AAC CAA ACC PHSRN Forward GTG CCG CAC TCA AGG AAT TCA ATA ACG GGT
CCT GCA ACT TGA GGT AGT 3 Reverse ATT CCT TGA GTG CGG CAC TCT ATC
CTC AGA GGT CCA AAC CAA ACC AGA DEGA1 Forward TGG ACC TCT GAC GGC
GAG GCC GGT CCT GCA ACT TGA GGT Reverse AGC AGG ACC GGC CTC GCC GTC
AGA GGT CCA AAC CAA ACC P15 Forward AGC GGC AGA GGC GAC AGC GGC GCC
GGT CCT GCA ACT TGA GGT AGT Reverse GCT GTC GCC TCT GCC GCT GCC GGC
AGA GGT CCA AAC CAA ACC AGA *RGD9 created using U3/RGD5 plasmid as
template.
[0160] Interior Modification of TMV:
[0161] An interior surface of TMV was modified with fluorescein
amine by using EDC/HOBT coupling reported by Schlick et al. The
fluorescently labeled virus particles were purified by
ultracentrifugation at 45,000 rpm for 1.5 hours (4.degree. C.)
(Beckman 70.2 Ti). The pellet was resuspended in 100 mM K phos
buffer (pH 7) and used to determine surface coating density by
measuring fluorescence on plate reader (ex/em 490/520 nm, Molecular
Devices, SpectraMax M2e).
[0162] Mass Spectrometry Analysis of TMV Mutants:
[0163] An infected tobacco leaf of each TMV mutant was flash frozen
in liquid nitrogen and ground up by using a mortar and pestle. The
ground-up leaves were transferred to a 15 mL conical tube and
re-suspended in 1-3 mL of 0.1 M K phos buffer (pH 7). The samples
were spun down at 13,500.times.g (Sorvall RC5, SLA-1500 rotor) at
4.degree. C. for 15 minutes, and the resulting supernatant (1
.mu.L) was added to matrix solution (9 .mu.L of saturated MS grade
sinapic acid in 70% acetonitrile and 0.1% TFA). The sample mixture
was then spotted on the MALDI plate, air-dried and analyzed using a
Bruker Ultra-Flex I TOF/TOF mass spectrometer. Alternatively,
purified virus solutions were mixed (1 .mu.L of 0.1 mg/mL) with 9
.mu.L of matrix solution (saturated sinapic acid in 70%
acetonitrile, 0.1% TFA) and 1 .mu.L of the mixture was spotted on
the MALDI plate. The dried co-crystal was analyzed by MALDI-TOF MS
using a customized setting (Bruker Daltonics Ultraflex I TOF/TOF).
An average of 1000 shots was taken for each of the mutants and wild
type virus solutions.
[0164] Cell Culture:
[0165] BHK cells were maintained in high glucose DMEM containing 4
mM L-glutamine, 1 mM sodium pyruvate and supplemented with 10%
fetal bovine serum (FBS). CHO cells were cultured in DMEM/F12 media
containing 4 mM L-glutamine, 1 mM sodium pyruvate, and supplemented
10% FBS. All cell culture reagents were purchased from HyClone.
[0166] Rat bone marrow stromal cells (rBMSCs) were harvested
according to protocol approved by IACUC. Passages from 5-8 were
used in the study. Rat BMSCs were isolated from the bone marrow of
young adult male Wister rats (80 g, Harlan Sprague Dawley, Inc.).
The procedures were performed in accordance with the guidelines for
animal experimentation by the Institutional Animal Care and Use
Committee, University of South Carolina. The isolated BMSCs were
maintained and expanded for an additional passage in DMEM with 10%
FBS. All cell culture reagents were purchased from HyClone. BMSCs
were induced with DMEM/F12 without serum and had been supplemented
with osteogenic factors (10 nM dexamethasone, 50 .mu.g mL-1
ascorbic acid, 10 mM .beta.-glycerophosphate) and growth factors
(10 ng mL-1 recombinant human bFGF, 2.5 ng mL-1 recombinant human
TGF-.beta., 10 ng mL-1 recombinant human EGF) and 1.times.ITS
(insulin, transferrin, selenium). Dexamethasone, ascorbic acid and
.beta.-glycerophosphate were purchased from Sigma-Aldrich.
Recombinant human bFGF and EGF were purchased from Stemgent,
recombinant human TGF-.beta. was purchased from StemRD.
100.times.ITS stock was purchased from VWR/Mediatech. The cells
were cultured to near 80% confluence and harvested by
trypsinization. 4 mL of 0.25% Trypsin/EDTA (Hyclone) was used to
dislodge the cells from the flasks and 3.times.105 cells were
seeded in each well.
[0167] Adhesion Assay:
[0168] For centrifugal adhesion assay, high binding 96-well plates
(Greiner Bio-one) were cleaned under UV light for 20 minutes at
room temperature. Then, the plates were coated with virus solutions
at concentrations of 1, 0.1 and 0.05 mg/mL-1 for 20 minutes at room
temperature. The wells were rinsed three times with water, blocked
with 1% BSA solution in PBS. The cells were pre-labelled with
Calcein-AM (BD Bioscience) and each well was seeded with 104 BHK
cells in serum-free DMEM. The cells were incubated at 37.degree. C.
for one hour, and imaged under an inverted phase contrast
microscope with 10.times. lens (Olympus CKX41). The plate was read
using a fluorimeter (Molecular Devices, SpectraMax M2e) with
excitation wavelength at 490 nm, and emission cut-off at 520 nm.
The wells were filled to the top with serum-free media and sealed
with microplate film (Bio-Rad). The plate was inverted and spun in
a swinging rotor (Beckman Coulter, SX4850) at specified RCF (g)
ratings for 5 minutes at room temperature. The solution was
carefully removed and fresh media (100 .mu.l) was added.
Afterwards, the fluorescence was measured and the post-spun
intensities were normalized against pre-spin intensities to
determine the fraction of adhered cells. The same adhesion assay
was performed for CHO cells.
[0169] Adhesion Inhibition Assays:
[0170] The plates were prepared as described in adhesion assay. The
cells were pre-labelled with Calcein AM for 30 minutes at
37.degree. C. in humidified incubator. The cells were washed twice
with 1.times.DPBS and blocked with 10 .mu.g of monoclonal
antibodies (anti-.alpha.5, anti-.alpha.2, anti-.alpha.V,
anti-.beta.1, anti-.beta.2 from BioLegend) per 105 cells in 1 mL of
serum-free DMEM. The cells were incubated at 37.degree. C. for 15
minutes, and then seeded on virus coated 96-well high binding
plates (Greiner). The cells were incubated at 37.degree. C. for an
additional 30 minutes. The media was removed and replaced with new
media prior to fluorescence measurements (Molecular Devices,
SpectraMax M2e) with excitation wavelength at 490 nm, and emission
cut-off at 515 nm. The fluorescence intensities were normalized
against unblocked virus coated controls to determine the fraction
of adhered cells in the presence of antibodies.
[0171] Cell Imaging and Analysis:
[0172] The cells were observed under a phase contrast microscope
(Olympus CKX41). For each cells sample, the pre-spin, post 10 g
spin, and post 30 g spin images were captured using Infinity2
(Lumenera). The plate was read using a fluorimeter (Molecular
Devices, SpectraMax M2e) with excitation wavelength of 490 nm, and
emission cutoff at 525 nm. The fluorescence data of the plate were
measured for pre-spin, post 10 g spin, and post 30 g spin of BHKs
cells. The post spin fluorescence values were normalized against
the pre-spin fluorescence intensities. Then, the average values
(n=6) and standard errors of mean were obtained for each TMV
mutants. All experiments were conducted at least three separate
times with multiple replicates. For CHO cells, the pre-spin, and
post 10 g spin fluorescence data were obtained. The post 10 g spin
data was normalized against the pre-spin data. The average values
and standard errors of mean were also calculated for each TMV
mutant. Significant values were based on p values <0.05 based on
two-tailed equal variance Student t-tests.
[0173] Layer by Layer Assembly and Substrate Characterization:
[0174] For tissue culture plasticware preparation, the 6-well plate
was cleaned with O.sub.2 plasma cleaner for 15 minutes. The first
coating was for 30 minutes with poly(allylamine) hydrochloride
(PAH, Mw .about.56,000, Aldrich) at a concentration of 1 mg/mL in
150 mM NaCl, filtered through 0.2 micron membrane (PALL). The wells
were washed three times with dH.sub.2O (Millipore 18.2 MO) to
remove unbound polyelectrolytes. Then each wells were incubated for
15 minutes with 1 mL of poly(styrene sulfonate) (PSS, Mw
.about.70,000, Sigma) at a concentration of 2 mg/mL in 150 mM NaCl,
filtered through 0.2 micron membrane (PALL). The polyelectrolyte
solution was removed and wells were washed three times with water.
The coating of polyelectrolytes, alternating between PAH and PSS,
was repeated until a total of 7 layers were coated. The final layer
was then coated with TMV or TMV-RGD1 (prepared at 1 mg/mL and
filtered through 0.45 micron HT Tuffryn Membrane Syringe Filter,
PALL life sciences). The final coating was washed with water three
times and dried overnight. Prior to seeding the cells, the
substrates were treated with UV irradiation for 15 minutes. All
substrates were prepared within one day prior to seeding of
cells.
[0175] For quartz crystal microbalance measurements, the silver
electrodes were washed three times with dH.sub.2O (18.2 M.OMEGA.),
dried under a stream of N.sub.2. The dried electrodes were measured
(9 MHz). The electrodes were then coated with PAH for 30 minutes at
room temperature. The electrodes were washed with water and then
dried under a stream of N.sub.2. The dried electrodes were measured
and the shift in oscillation frequencies was recorded. The coating,
washing, and drying steps were repeated as each new layer of
polyelectrolytes was added to the silver electrodes. All
measurements were conducted in triplicates and in three separate
experiments. The frequency shifts were converted to mass densities
using Sauerbrey's equation.
.DELTA. f = - 2 .DELTA. m f o 2 A .rho. q .mu. q ##EQU00001##
[0176] Uncoated PEM and TMV coated PEM substrates were further
characterized by water contact angle measurements. The piranha
solution treated microscope coverslips (VWR 18 mm diameter, No. 2
thickness) measured approximately 10.degree..+-.1.degree. and the
final PEM averaged 28.degree..+-.5.degree.. Coating of the virus
did not alter the water contact significantly, but could be well
visualized by atomic force microscopy and mass density shift
(QCM).
[0177] Cytochemical Staining and Immunofluorescence:
[0178] Batches of BMSCs culture on TMV coated substrates were
terminated post 2 days of osteoinduction. Cells were fixed in 4%
PFA in 1.times.D-PBS pH 7.4 at room temperature for 15 minutes.
Each of the samples was then permeablized for 5 minutes with
1.times.D-PBS, 0.05% Triton X-100 and blocked with 3% bovine serum
albumin (BSA, Rockland), 1.times.D-PBS for 1 hour at room
temperature. After blocking, the cells were incubated overnight
with primary antibodies targeting the osteo-specific marker
osteocalcin. The secondary antibodies goat anti-rat FITC (VWR
Scientific) were used for osteocalcin (BGLAP) at 1:100 dilutions in
buffer at 4.degree. C. TRITC-phalloidin (1:200 in 1.times.D-PBS)
was used to stain filamentous actin. Nuclei were stained with DAPI
(4,6-diamidino-2-phenylindole, 100 ng/ml). Images of the stained
substrates were collected using Olympus IX81 fluorescent microscope
with 60.times. oil lens (UPlanSAPO, NA=1.35). Negative control for
staining included only secondary antibodies. After 2 days in
osteogenic cultures, BMSCs seeded on hFN, PEM, TMV and TMV-RGD1
substrates were stained with Alizarin red calcium rich deposits.
The cells were fixed in 4% paraformaldehyde at room temperature for
5 minutes, washed with water and then stained with 0.1% solution of
Alizarin red (Sigma-Aldrich) pH 4.1-4.5 for 60 minutes at room
temperature. Since the reaction was highly light sensitive, the
substrates were wrapped in aluminum foil during the entire time of
incubation.
[0179] ELISA and Western Blot:
[0180] Cells were fixed in 4% paraformaldehyde/1.times.D-PBS for 5
minutes at room temperature. The cells were washed and blocked with
1% BSA/1.times.D-PBS/0.05% Triton X-100 for 30 minutes. The primary
antibody against BMP-2 was incubated in three wells overnight at 4
degrees celsius at a dilution of 1:100. The samples were rinsed
with PBS, 0.05% Triton X-100, three times and the secondary
antibody (anti-mouse Goat polyclonal with HRP conjugate) was
incubated for 1 hour at room temperature. The samples were washed
three times and incubated with TMB solution for 30 minutes and the
reaction was stopped with concentrated sulfuric acid. The solution
was read with a UV-Vis spectrophotometer at 450 nm.
[0181] For western blots, cells were cultured on 6-well plates. The
media was aspirated and the cells were directly lysed by adding 100
.mu.l of Laemmeli buffer (Bio-rad) containing 2-mercaptoethanol and
protease inhibitor, PMSF (1 .mu.g/mL). The lysate was scrapped and
transferred to a clean microfuge tube and heated at 95.degree. C.
for 15 minutes, then immediately cooled on ice prior to loading on
a 12% SDS-PAGE. The gel was run for 60 minutes at 200 V in a
Mini-Protean 3 Gel electrophoresis rig (Bio-rad). The gel was
transferred to PVDF membrane then block with 2% BSA in PBS with
0.05% Triton X-100 (PBS-T) for one hour at room temperature. The
primary antibody (total FAK or pY397 FAK, Cell Signaling
Technology) was added to the block solution at 1:1000 dilution and
incubated overnight. The membrane was washed three times with
PBS-T, then probed with secondary antibody (anti-rabbit goat IgG
with HRP conjugate) at 1:5000 dilution for one hour at room
temperature in 2% BSA in PBS-T. The membrane was washed three
times, then imaged with enhanced chemiluminesence kit (Pierce ECL)
according to manufacturer's protocol. The resulting blots were
scanned and intensity plots were measured by ImageJ. The average of
three separate images were used. The intensity of pY397 FAK was
normalized to total FAK for all samples and the normalized
intensities were plotted relative to cell lysates from hFN
substrates.
[0182] Detailed Protocols for Purification, Handling and Use of
Tobacco Mosaic Virus and Mutant Variants are incorporated by
reference herein from U.S. Provisional Patent Application No.
61/795,736 filed on Oct. 24, 2012 of Wang, et al.:
[0183] Infecting Plants by Mechanical Abrasion Using Purified Virus
Stock Solution:
[0184] Method:
[0185] 1. Infections occur best when the plants are infected near
its dark cycle. The artificial sunlight may reduce infections.
[0186] 2. Spray water on the leaves using a mist/spray
[0187] 3. Sprinkle carborundum (silicon carbide) on the leaves.
Wear a mask to prevent accidental inhalation. Carborundum is an
abrasive material.
[0188] 4. Dilute the virus stock solution to 0.05 mg/mL in buffer
solution. Typically use 1 mg in 20 mL of buffer for one tray of
plants (24 containers).
[0189] 5. Cut cheesecloth to 4'' square piece and soak the
cheesecloth in the virus solution.
[0190] 6. Wrap the cheesecloth around the index finger and gently
rub the cheesecloth along the tobacco leaves. The presence of
carborundum on the leaves will introduce micro-abrasions and the
virus infection will occur once it penetrates through the upper
layer of the leaf.
[0191] 7. Repeat steps 6 and 7 for every 3-4 plants.
[0192] 8. Residual carborundum should be washed off by watering the
plants the next morning.
[0193] Infecting Plants by Mechanical Abrasion Using Genetic
Material:
[0194] Method:
[0195] 1. Infections occur best when the plants are infected near
its dark cycle. The artificial sunlight may reduce infections.
[0196] 2. Spray water on the leaves using a mist/spray (10
microgram of genetic material is sufficient for 2 leaves from one
plant).
[0197] 3. Sprinkle carborundum (silicon carbide) on the leaves.
Wear a mask to prevent accidental inhalation. CAUTION: Carborundum
is an abrasive material.
[0198] 4. Cut cheesecloth to 4'' square piece and gently wipe the
cheesecloth along the tobacco leaves. The presence of carborundum
on the leaves will introduce micro-abrasions.
[0199] 5. Pipette droplets of the genetic material around the
leaves.
[0200] Post-Infection Plant Monitoring:
[0201] NOTE: Wild type TMV infected plants exhibit dark green
patches around the leaves.
[0202] 10 to 14 days post-infection, a leaf is cut. The leaf should
weigh 0.5-2 g and be one of the two primary leaves used for
infection. The cut leaf is flash frozen in liquid nitrogen and
ground to fine powder using a mortar and pestle. A portion of the
leaf is used for protein analysis by MALDI-TOF MS and another
portion for mRNA extraction and sequencing.
[0203] Viral Coat Protein Analysis by MALDI-TOF MS:
[0204] 500 mg of leaf material is transferred to a 2 mL eppendorf
tube, and 1 mL of chilled buffer (100 mM potassium phosphate pH 7)
is added to the tube. The mixture is vortexed for 30 seconds, then
centrifuged at 16,000.times.g for 10 minutes at 4.degree. C. The
supernatant is transferred to a new eppendorf tube. 2 .mu.l of the
supernatant is mixed with 8 .mu.l of matrix solution (saturated
solution of sinapic acid in 70% acetonitrile, 0.1% trifluoroacetic
acid). The mixture is vortexed, centrifuged and 2 ul is spotted on
an aluminum MALDI plate. Sample is analyzed using Bruker Daltonics
MALDI-TOF MS. Expected m/z is 17,535 for singly charged wild type
TMV coat protein, which matches M+H+. A peak at m/z 8,768
corresponds to a doubly charge. A peak around m/z of 10,477 is
typically observed for plant lysate samples. For TMV-RGD1 mutant,
the expected peak is 18,151. Save plant sap in -20.degree. C.
freezer for later use.
[0205] The mass of the viral coat protein is calculated by adding
the mass of an acetyl group to the N-terminal amino acid (serine).
The first methionine is cleaved during post-translational
processing in plant hosts.
[0206] Viral Coat Protein Gene Sequencing:
[0207] Total plant mRNA is isolated using Qiagen plant RNA
extraction kit and used for cDNA synthesis using the specified
primer.
TABLE-US-00008 cDNA primer/Reverse: (SEQ ID NO: 51) 5' CGT GCC TGC
GGA TGT ATA TGA AC 3'
[0208] The cDNA is used as a template for PCR amplification of the
virus coat protein gene. The two specific primers used are:
TABLE-US-00009 CP Sequencing/forward: (SEQ ID NO: 52) 5' CGT TAT
CGT ACG CAC CAC GTG TG 3' cDNA primer/Reverse: (SEQ ID NO: 51) 5'
CGT GCC TGC GGA TGT ATA TGA AC 3'
[0209] The PCR product should be 756 by for wild type TMV. The PCR
product is isolated and sequenced using the CP sequencing/forward
primer.
[0210] Propagating the Mutant Virus from Single Plant Host:
[0211] (Only Necessary if Genetic Material was Used to Infect One
Plant)
[0212] 1. After verifying the infection has occurred in the
original plant, the frozen plant supernatant/sap saved from
previous method (MALDI-TOF MS) can be used to infect 20 plants (2
leaves per plant) by diluting 1 mL of the sap in 20 mL of buffer
and following the method under infections by mechanical abrasions
with purified virus stock.
[0213] 2. If the plant sap is not available, a fresh cut leaf can
be flash frozen in liquid nitrogen, ground to fine powder using
mortar and pestle. For every 1 g of plant material, add 2 mL of
pre-chilled buffer. Dilute the supernatant with buffer to 20 mL
final volume and use to infect 20 plants following the methods
under mechanical abrasions with purified virus stock.
[0214] Tobacco Mosaic Virus and Mutant Variant Purification
Protocols
[0215] (Chloroform/Butanol Extraction):
[0216] EQUIPMENT: Standard Kitchen brand blender (4 L) or a
Laboratory grade tissue homogenizer; Sorvall RC-5C plus centrifuge
and rotor models SLA-1500 and SLC-4000; 250 mL and 1 liter Nalgene
centrifuge bottles (withstand up to 20,000.times.RCF); Beckman
Ultracentrifuge, 50.2 Ti rotor and screw cap tubes
[0217] MATERIALS: Organic solvents--Chloroform and n-butanol; Large
Kimwipes; Cheesecloth; Beakers (1 and 2 liters); Funnels; 100 mM
and 10 mM potassium phosphate buffer (pH 7); Polyethylene Glycol
8,000; .beta.-mercaptoethanol; PMSF, aprotinin, leupeptin (for
mutants with long inserts)
[0218] 1. All buffers are 0.1 M potassium phosphate pH 7 unless
specified, and should be pre-chilled prior to use. For TMV1Cys
mutant, additional .beta.-mercaptoethanol is used (0.3 to 0.5% of
volume). For mutant strains with long peptide inserts (>4 amino
acids), add plant protease inhibitors.
[0219] 2. Check the bag of leaves used for the purification in the
logbook.
[0220] 3. The frozen bag of leaves is manually pulverized prior to
transferring to a 4 L blender (Oster or any kitchen brand is used
to further homogenize the plants; however a laboratory grade tissue
homogenizer can be used).
[0221] 4. For every 100 g of plant material, add 300 mL of
pre-chilled buffer and 0.1% .beta.-ME (vol % of buffer).
[0222] 5. Slowly start the blender from lowest setting and
gradually increase to highest setting to homogenize the leaves.
Leave the blender on for 5 minutes.
[0223] 6. Place a large beaker (1 L for 100 g of leaves, or 2 L for
200 g of leaves) on ice. Place a clean funnel on top of the
beaker.
[0224] 7. Place two layers of cheesecloth on top of a funnel and
collect the flow-through in the large beaker.
[0225] 8. Transfer the flow-through to Nalgene centrifuge tubes
(250 mL tube for SLA-1500 rotor used in Sorvall RC-5C plus floor
centrifuge).
[0226] 9. Weigh out the tubes and balance in pairs prior to
centrifuging at 10,500 RPM for 20 minutes at 4.degree. C.
[0227] 10. Prepare another beaker (same volume as step 5) on ice
and a funnel on top of the beaker.
[0228] 11. Place one layer of Kim-wipe on top of the funnel.
[0229] 12. The clarified supernatant is filtered through one layer
of Kim-wipe.
[0230] 13. Add equal volume of n-butyl alcohol and chloroform
(organic solvent) to the clarified plant supernatant at volume
ratio of 1:1:2, respectively. (Make sure mix n-butyl alcohol and
chloroform first; always pour n-butyl alcohol first so the
solutions can be mixed before used; always pour those solutions
under the fume hood). Stir the mixture on ice for 30 min.
[0231] 14. Pour the solution into Nalgene centrifuge tubes and
balance the tubes. Centrifuge at 4.degree. C., 10,500 RPM, for 10
min in SLA-1500 rotor (Sorvall RC-5C plus)
[0232] 15. For large volumes (>1 L), use the SLC-4000 rotor and
centrifuge at 6,000 RPM for 20 minutes at 4.degree. C.
[0233] 16. Transfer the aqueous layer to a clean beaker.
[0234] 17. Add PEG 8000 to 8% wt/vol and 0.2 M NaCl. (Ex: 8% wt/vol
of PEG 8000 for 450 mL final volume requires=0.08*450 mL=36 g and
0.2 M NaCl*0.450 L*59 g/mol=5.31 g).
[0235] 18. Stir the mixture on ice (or at 4.degree. C.) for 1
hour.
[0236] 19. Transfer to 250 mL Nalgene centrifuge bottles, and
pellet the viral particles at 4.degree. C., 13,500 RPM, for 20
minutes using SLA-1500 rotor in RC-5C plus Sorvall centrifuge.
[0237] 20. Discard the liquid, and resuspend the pellet with 10 mM
buffer pH 7. The pellet contains purified virus particles with PEG
and trace contaminants.
[0238] 21. Transfer the resuspension to a 50 mL conical vial and
centrifuge at 9,500 RPM for 15 min at 4.degree. C. using SLA-1500
rotor. NOTE: The conical tubes should resist up to 13,000 RCF.
[0239] 22. Transfer the supernatant to ultracentrifuge tubes (For
50.2 Ti rotor use Beckman Centrifuge Bottles, Polycarbonate,
Catalog Number 355654 with cap assemblies, 26.3 mL volume; for 70.1
Ti rotor, use polycarbonate centrifuge bottles with cap assemblies,
catalog number 355603, 10.4 mL volume capacity) NOTE: Adhere to the
vendor's instructions regarding solvent compatibilities and
sterilization strategies.
[0240] 23. Fill the tube up to about 2-3 cm from the top. Then,
layer the sucrose cushion (30% sucrose in 10 mM buffer) at the
bottom of the tube. Balance the tubes in pairs
[0241] 24. Centrifuge with the 50.2 Ti rotor at 42,000 RPM for 2.5
hours at 4.degree. C. CAUTION: Check the O rings in the rotor and
the rotor lid before using it. Grease the O-rings with vacuum
grease.
[0242] 25. Resuspend the pellet in 100 mM buffer overnight at
4.degree. C.
[0243] 26. Transfer the solution to conical vials and centrifuge at
4.degree. C., at 9500 RPM, for 15 minutes using Sorvall RC-5C plus
centrifuge.
[0244] 27. To determine the concentration, measure the UV (260 nm,
280 nm). TMV has a typical 260/280 ratio of 1.2 and 1 mg/mL
solution has absorbance value of 3 at 260 nm.
[0245] 28. Transfer the supernatant to a clean tube and store the
purified virus solution in aliquots at -20.degree. C. or
-80.degree. C. for long term storage. Do NOT flash freeze in liquid
nitrogen.
Alternative Virus Purification Methods:
[0246] Tobacco Mosaic Virus and Mutant Variant Purification
Protocols (Butanol ONLY):
[0247] EQUIPMENT: Standard Kitchen brand blender (4 L) or a
Laboratory grade tissue homogenizer; Sorvall RC-5C plus centrifuge
and rotor models SLA-1500 and SLC-4000; 250 mL and 1 liter Nalgene
centrifuge bottles (withstand up to 20,000.times.RCF); Beckman
Ultracentrifuge, 50.2 Ti rotor and screw cap tubes
[0248] MATERIALS: Organic solvents--n-butanol; Large Kimwipes;
Cheesecloth; Beakers (1 and 2 liters); Funnels; 100 mM and 10 mM
potassium phosphate buffer (pH 7); Polyethylene Glycol 8,000;
.beta.-mercaptoethanol; Triton X-100
[0249] 1. All buffers are 0.1 M potassium phosphate pH 7 unless
specified, and should be pre-chilled prior to use. For TMV1Cys
mutant, additional .beta.-mercaptoethanol is used (0.3 to 0.5% of
volume).
[0250] 2. Check the bag of leaves used for the purification in the
logbook.
[0251] 3. The frozen bag of leaves is manually pulverized prior to
transferring to a 4 L blender (Oster or any kitchen brand is used
to further homogenize the plants; however a laboratory grade tissue
homogenizer can be used).
[0252] 4. For every 100 g of plant material, add 300 mL of
pre-chilled buffer and 0.2% .beta.-ME (vol % of buffer).
[0253] 5. Slowly start the blender from lowest setting and
gradually increase to highest setting to homogenize the leaves.
Leave the blender on for 5 minutes.
[0254] 6. Place a large beaker (1 L for 100 g of leaves, or 2 L for
200 g of leaves) on ice. Place a clean funnel on top of the
beaker.
[0255] 7. Place two layers of cheesecloth on top of a funnel and
collect the flow-through in the large beaker.
[0256] 8. Add 8 mL of n-butanol for 100 mL of plant sap.
[0257] 9. Transfer to Nalgene centrifuge tubes (250 mL tube for
SLA-1500 rotor used in Sorvall RC-5C plus floor centrifuge).
[0258] 10. Weigh out the tubes and balance in pairs prior to
centrifuging the plant debris at 12,000 RPM for 15 minutes at
4.degree. C.
[0259] 11. Prepare another beaker (same volume as step 5) on ice
and a funnel on top of the beaker.
[0260] 12. Place one layer of Kim-wipe on top of the funnel.
[0261] 13. The clarified supernatant is filtered through one layer
of Kim-wipe.
[0262] 14. Add 4 g of PEG 8000 and 4 g of NaCl for 100 mL of
supernatant.
[0263] 15. Stir the mixture on ice for 1/2 hour.
[0264] 16. Transfer to 250 mL Nalgene centrifuge bottles, and
pellet the viral particles at 4.degree. C., 12000 RPM, for 20
minutes using SLA-1500 rotor in RC-5C plus Sorvall centrifuge.
[0265] 17. Discard the liquid, and resuspend the pellet with 10 mM
buffer pH 7, 1% Triton X-100.
[0266] 18. Transfer the resuspension to a 50 mL conical vial and
centrifuge at 9,500 RPM for 15 min at 4.degree. C. using SLA-1500
rotor. NOTE: The conical tubes should resist up to 13,000 RCF.
[0267] 19. Transfer the supernatant to ultracentrifuge tubes (For
50.2 Ti rotor use Beckman Centrifuge Bottles, Polycarbonate,
Catalog Number 355654 with cap assemblies, 26.3 mL volume; for 70.1
Ti rotor, use polycarbonate centrifuge bottles with cap assemblies,
catalog number 355603, 10.4 mL volume capacity) NOTE: Adhere to the
vendor's instructions regarding solvent compatibilities and
sterilization strategies.
[0268] 20. Fill the tube up to about 2-3 cm from the top. Then,
layer the sucrose cushion (30% sucrose in 10 mM buffer) at the
bottom of the tube. Balance the tubes in pairs
[0269] 21. Centrifuge with the 50.2 Ti rotor at 42,000 RPM for 2.5
hours at 4.degree. C. CAUTION: Check the O rings in the rotor and
the rotor lid before using it. Grease the O-rings with vacuum
grease.
[0270] 22. Resuspend the pellet in 100 mM buffer overnight at
4.degree. C.
[0271] 23. Transfer the solution to conical vials and centrifuge at
4.degree. C., at 9500 RPM, for 15 minutes using Sorvall RC-5C plus
centrifuge.
[0272] 24. To determine the concentration, measure the UV (260 nm,
280 nm). TMV has a typical 260/280 ratio of 1.2 and 1 mg/mL
solution has absorbance value of 3 at 260 nm.
[0273] 25. Transfer the supernatant to a clean tube and store the
purified virus solution in aliquots at -20.degree. C. or
-80.degree. C. for long term storage. Do NOT flash freeze in liquid
nitrogen.
[0274] NOTE: Use of Triton X-100 will increase yield but can affect
downstream use of the viral particles. If the trace amount of the
detergent needs to be removed, additional ultracentrifugation step
is required.
[0275] Coating Virus on Surfaces for Cell Cultures:
[0276] Layer by Layer Coating on Plasticwares:
[0277] EQUIPMENT: Harrick's Plasma Cleaner with O.sub.2 regulator;
Bio-safety cabinet for aspetic operations
[0278] MATERIALS: Polyallyamine Hydrochloride (Alfa Aesar, Cat. No.
43092)--1 mg/mL in 18.2 M.OMEGA. MilliQ ddH.sub.2O; Poly(sodium
4-styrenesulfonate) (Aldrich, Cat. No. 527483)--2 mg/mL diluted in
18.2 M.OMEGA. MilliQ ddH.sub.2O; Tissue culturewares (Corning,
Bio-Greiner One have been tested from 6-well plates to 96-well
plates); 0.45 micron syringe filters PALL HT Tuffryn low protein
binding membrane; Syringe; Sterile conical tubes; 10 mM potassium
phosphate buffer pH 7; Sterile water solution
[0279] 1. Tissue culture plasticware is cleaned with oxygen plasma
for 15 minutes (Harrick's plasma cleaner). Oxygen plasma is a white
pink glow. Purple glow note air plasma, and oxidation will be
sub-optimal. Plasma treatment should be for 30 minutes for 96-well
plates.
[0280] 2. Substrates are immediately coated with PAH (1 mg/mL,
polyallylamine chloride) solution for 15 minutes. The solution
should be sufficient to cover the entire well (2 mL per well for
6-well plates, 1 mL per well for 12-well plates, 0.5 mL for 24-well
plates and 0.1 mL per well for 96-well plates).
[0281] 3. The polymer solution is removed and the surfaces are
washed with water (18.2 M.OMEGA. MilliQ ddH2O) three times, and
then coated with PSS (2 mg/mL, polystyrene sulfonate) solution for
15 minutes.
[0282] 4. The polymer is removed and washed three times with water.
Steps 2-4 are repeated until 5 total layers of polymers are coated
on the surface (PAH/PSS/PAH/PSS/PAH). The final top layer should be
PAH.
[0283] 5. The polymer coating is UV-irradiated for 15 minutes, then
air-dried in the sterile hood overnight. CAUTION: Failure to dry
the polymer coating will result in loose coating which will peel
off during cell culture.
[0284] 6. Filter sterilize the virus solution with a syringe filter
(PALL HT Tuffryn low protein binding membrane 0.45 micron) and
verify the virus concentration by measuring UV-Vis absorbance at
260 and 280 wavelengths. Concentration should be adjusted to 0.1
mg/mL in 10 mM potassium phosphate buffer (pH 7).
[0285] 7. Pipette the virus solution to the wells and incubate for
20 minutes at room temperature.
[0286] 8. Remove the virus solution and wash three times with
water
[0287] 9. UV-irradiate for 15 minutes and let the substrate air-dry
overnight.
[0288] NOTE: The final substrate as dried film is very stable at
room temperature as long as contaminants are avoided by keeping a
lid or a tight seal around the plates. For coating the glass
coverslips or other silica based substrates, the glass is treated
with piranha solution (70% sulfuric acid, 30% hydrogen peroxide)
for 10-20 minutes. After cleaning glass with piranha solution,
rinse with copious amounts of water and proceed with polymer
coatings (steps 2-4).
[0289] Preparing the Piranha Solution
[0290] Chemicals: Sulfuric Acid (H2SO4 96%), Hydrogen Peroxide
(H2O2 30%).
[0291] Note: These percentages are the dilutions as they arrive in
the bottles from the chemical supplier. Just use the solution
straight out of the bottle.
[0292] Single Polymer Layer Coating on Plasticwares:
[0293] EQUIPMENT: Harrick's Plasma Cleaner with O2 regulator;
Bio-safety cabinet for aseptic operations
[0294] Materials:
[0295] Polyallyamine Hydrochloride (Alfa Aesar, Cat. No. 43092)--1
mg/mL in 18.2 MO MilliQ ddH2O with pH adjusted to 5 with 1 M NaOH;
Tissue culturewares (Corning, Bio-Greiner One have been tested from
6-well plates to 96-well plates); 0.45 micron syringe filters PALL
HT Tuffryn low protein binding membrane; Syringe; Sterile conical
tubes; 1 M NaOH; 10 mM potassium phosphate buffer pH 5; Sterile
water solution
[0296] 1. Tissue culture plasticware is cleaned with oxygen plasma
for 15 minutes (Harrick's plasma cleaner). Oxygen plasma is a white
pink glow. Purple glow note air plasma, and oxidation will be
sub-optimal. Plasma treatment should be for 30 minutes for 96-well
plates.
[0297] 2. Prepare PAH (1 mg/mL, polyallylamine chloride) solution
by dissolving the polymer in water (18.2 M.OMEGA. MilliQ ddH2O) and
adjust to pH 5 with 1 M NaOH.
[0298] 3. The polymer solution is used to coat the surfaces for 15
minutes.
[0299] 4. The solution is removed and the surfaces are washed with
water (18.2 M.OMEGA. MilliQ ddH2O) three times.
[0300] 5. The polymer coating is UV-irradiated for 15 minutes, then
air-dried in the sterile hood overnight. CAUTION: Failure to dry
the polymer coating will prevent virus from binding to the
substrate.
[0301] 6. Filter sterilize the virus solution with a syringe filter
(PALL HT Tuffryn low protein binding membrane 0.45 micron) and
verify the virus concentration by measuring UV-Vis absorbance at
260 and 280 wavelengths. Concentration should be adjusted to 1
mg/mL in 10 mM potassium phosphate buffer (pH 5).
[0302] 7. Pipette the virus solution to the wells and incubate for
20 minutes at room temperature.
[0303] 8. Remove the virus solution and wash three times with
water
[0304] 9. UV-irradiate for 15 minutes and let the substrate air-dry
overnight.
[0305] NOTE: The final substrate as dried film is very stable at
room temperature as long as contaminants are avoided by keeping a
lid or a tight seal around the plates. Glass substrates can also be
coated by cleaning with piranha solution and rinsing with large
amounts of water.
[0306] Direct Virus Coating on High Binding Plasticwares:
[0307] EQUIPMENT: Bio-safety cabinet for aseptic operations
[0308] MATERIALS: Tissue culturewares (high binding 96-well plates
from Corning, or Bio-Greiner One); 0.45 micron syringe filters PALL
HT Tuffryn low protein binding membrane; Syringe; Sterile conical
tubes; Sterile water solution
[0309] 1. Virus solution is directly pipette to the high binding
plates at concentration of 0.1 mg/mL, 100 mM potassium phosphate
buffer pH 5.
[0310] 2. Remove the virus solution and wash three times with
water
[0311] 3. UV-irradiate for 15 minutes and let the substrate air-dry
overnight.
[0312] Follow-Up Quality Control Assessments on Virus Coated
Substrates:
[0313] EQUIPMENT: Veeco Atomic Force Microscope; Water contact
angle measurement using static sessile drop method with goniometer
(VCA Optima contact angle)
[0314] Procedure:
[0315] 1. Samples were cut out from the plasticware with a box
cutter knife. Tapping-mode atomic force microscope (AFM) images
were obtained at ambient conditions using Nanoscope IIIA MultiMode
AFM (Veeco). Si tips with a resonance frequency of approximately
300 kHz, a spring constant of about 40 N m-1, and a scan rate of
0.75 Hz were used.
[0316] 2. Three different substrates are cut out and measured with
static sessile drop method with goniometer (VCA Optima contact
angle). Two measurements per drop and three different areas on each
substrate are recorded (total measurements equals 6 per
substrate).
[0317] Bone Marrow Derived Mesenchymal Stem Cell Cultures on
Virus-Coated Surfaces:
[0318] MATERIALS: DMEM/F12 (HyClone or Mediatech) without phenol
red indicator; 10.times.ITS (Mediatech); Basic FGF (Stemgent)--100
.mu.g/mL stock solution in 5 .mu.l aliquots and stored at
-80.degree. C.; TGF-beta (Stemgent)--100 .mu.g/mL stock solution in
2 .mu.l aliquots and stored at -80.degree. C. EGF (Stemgent)--100
.mu.g/mL stock solution in 5 .mu.l aliquots and stored at
-80.degree. C.; Trypsin/EDTA solution (HyClone)--0.25%;
1.times.D-PBS sterile; Sodium beta glycerophosphate (Sigma);
Ascorbic 2-phosphate (Sigma)--Prepare a 25 g/mL stock solution in
sterile water; Dexamethasone (Sigma)--prepared by dissolving 1 mg
in 1 mL absolute ethanol and adding 49 mL of sterile media.
[0319] Procedure:
[0320] 1. To prepare 150 mL of serum-free osteogenic media, add
0.46 g of sodium beta glycerophosphate, 0.3 mL of 25 g/mL ascorbic
2-phosphate stock solution and 30 .mu.l of dexamethasone (20
.mu.g/mL stock solution) to DMEM/F12.
[0321] 2. Transfer 40 mL of the media to conical vial and add 0.4
mL of 10.times.ITS (Mediatech)
[0322] 3. Add 1 .mu.l of 100 .mu.g/mL TGF-beta, 4 ul of 100
.mu.g/mL bFGF, and 100 .mu.g/mL EGF.
[0323] 4. The media with growth factors can be used for 3-7
days.
[0324] 5. Bone-derived mesenchymal stem cells are cultured to near
confluence in standard media (DMEM/F12 supplemented with 10% FBS),
then harvested by trypsinization.
[0325] 6. The cells are centrifuged at 300 rpm for 5 minutes and
washed twice in pre-warmed serum-free media (DMEM/F12).
[0326] 7. The cells are centrifuged and resuspended in osteogenic
serum-free medium at cell density of 100,000 per mL.
[0327] 8. For controls, use human fibronectin (hFN, BD Biosciences)
coatings. Cells will attach and spread on hFN coated
substrates.
[0328] Synthesis of Virus-Incorporated Porous Alginate Hydrogels
and Cell Culturing
[0329] The synthesis of gas template solid foam was based on the
methodology reported by Barbetta et al. 20 Briefly, 5% w/v low
viscosity sodium-alginate (Protanal.RTM. LF 10/60 FT, 30-60 mPas
for 1%, kindly provided by FMC Biopolymer UK Ltd.), was dissolved
in 2% w/v sodium bicarbonate (NaHCO3) and 4% w/v pluronic F108
solution. Molar equivalent of citric acid with respect to NaHCO3
was added to the mixture while stirring. Stirring was continued for
15 min to allow CO.sub.2 to fully develop. Afterward, the foamy
alginate solution was flash frozen in liquid nitrogen and
freeze-dried. The resulting solid foam was soaked in 2 M of
CaCl.sub.2 for 24 h to induce the formation of the calcium-based
physical gel and then dialyzed against large volume of 0.1 M
CaCl.sub.2. Finally, the solid foam was freeze-dried, resulting in
porous alginate hydrogel (PAH). For the TMV modified PAH (TMV-PAH),
or mutant TMV-RGD1 modified PAH (RGD-PAH), 0.1% w/v of virus was
added to the solution 10 min after adding citric acid. The mixture
was stirred for another 5 min.
[0330] TMV-PAH was de-crosslinked and dissolved in 0.5 M EDTA, and
the solution was analyzed under transmission electron microscopy
(TEM) (Hitachi H-8000 microscope) on 300 mesh copper grids coated
with carbon and stained with 2% uranyl acetate. The absorbance at
260 nm was measured by UV-Vis spectroscopy to confirm the
concentration of TMV in de-crosslinked alginate solution.
[0331] Lyophilized PAH, TMV-PAH, and RGD-PAH were cut to
3.times.3.times.2 mm3 cubes. In each set of experiment, six
hydrogels per type of sample were used for these studies. Hydrogels
were sterilized in 70% ethanol for 15 min, ethanol was drained on
sterile filter paper and the hydrogels were further sterilized
under UV-light for 60 min in laminar airflow hood. Hydrogels were
then saturated in complete primary media at 37.degree. C. for 60
min, blotted excess media with sterile filter paper, and placed in
12-well non-adhesive plate (6 hydrogels in each well). Rat BMSCs
were harvested from 80% confluent culture flask using 0.25%
trypsin/EDTA for 5 min and counted the number of cells and
re-suspended in complete primary media. 1.4.times.103 BMSCs were
seeded per one volume of hydrogel. Rat BMSCs loaded hydrogels were
initially incubated with 200 .mu.L of complete primary media in
each well to prevent hydrogels from drying out. After 6 h of an
initial incubation, hydrogels were completely submersed in 1 mL of
complete primary media in each well. The hydrogels seeded with
rBMSCs were cultured at 37.degree. C. and 5% CO2.
[0332] Attachment percentages were determined for different types
of hydrogels on day 1 after cell seeding by counting floating cells
in each well with a hematocytometer. Three separate experiments
were carried out in this study.
Attachment percentage = total seeding cell number - floating cell
number total seeding cell number ##EQU00002##
[0333] Cell Viability:
[0334] CellTiter-Blue.RTM. (CTB) cell viability assay (Promega)
after day 8 and day 14 of culture was performed for each type of
hydrogel. The culture media in each well was replaced by pre-warmed
with 1 mL of media containing 10% CTB and incubated for 1 h at
37.degree. C. and 5% CO.sub.2. The media containing CTB was used
without cells as negative controls. The measurement of the CTB
product was taken at two time points. First, to detect the cells
that attached to the outer surface of hydrogel, the solution was
collected and replaced with 1 mL of culture media. The incubation
was continued for another 2 h to allow diffusion of the fluorescent
product from the inner part of hydrogel. After 2-hour incubation,
the media solution was collected again and replaced with 1 mL of
culture media in each well to continue cell culture. The 1:1
mixture of solution from first collection and second collection was
measured fluorescence intensity at 560/590 nm (Ex/Em) using
SpectraMax M2 Multi-Mode microplate reader (Molecular Devices).
Also, three separate experiments were conducted with each type of
sample comprised of 6 hydrogels.
[0335] Osteogenic Culture:
[0336] After 1 day of cell seeding, complete primary media was
replaced by osteogenic media consisting of DMEM supplemented with
10% FBS, 10 mM sodium .beta.-glycerol phosphate, l-ascorbic acid
(50 .mu.g/mL), 10-2 .mu.M dexamethasone, penicillin (100 units/mL),
streptomycin (100 .mu.g/mL), and amphotericin B (1 .mu.g/mL). The
osteogenic media was changed every 2 days. The culture was
incubated at 37.degree. C. and 5% CO.sub.2.
[0337] Alkaline Phosphatase (ALP) and Mineralization Assays:
[0338] Alkaline phosphatase (ALP) activity was determined using
pNPP (p-nitrophenyl phosphate) assay (Sigma Diagnostics). After 3,
7 and 14 days, the hydrogels with cells were pre-washed with TBS
then incubated with pNPP solution at room temperature for 2.5 h.
Absorbance was read using a M2 SpectraMax plate reader at 405 nm
indicating ALP activity levels. The enzyme activity was calculated
from Beer-Lambert law as follow,
Enzyme activity ( moles / min / g ) = V ( l ) .times. OD 405 nm (
cm - 1 ) .times. incu bation time ( min ) .times. enzyme ( g )
##EQU00003##
where .epsilon. is the molar extinction coefficient
(M-1.times.cm-1). For p-nitrophenol, .epsilon.=1.78.times.104
M-1.times.cm-1. OD405 nm (cm-1) is the absorbance at 405 nm divided
by the light-path length (cm). V is the final assay volume (100
.mu.L).
[0339] Alizarin Red S (ARS) staining for Ca.sup.2+ was performed on
day 14 cultures. The hydrogels with cells were washed 3 times with
TBS and fixed in 10% buffered formalin overnight and sliced into 1
mm thick. The sections were then stained with 0.1% solution of ARS
(Sigma-Aldrich) pH 4.1-4.5 for 30 min, washed with 1:1
xylene:acetone solution and placed with toluene mounting solution
on glass slides. Control experiments without cells were conducted
for PAH, TMV-PAH, and RGD-PAH.
[0340] Calcium concentrations were quantified using Varian 720-ES
ICP-OES (Inductively Coupled Plasma--Optical Emission Spectrometer)
elemental analysis. Standards were created using 0-0.4 mg/L calcium
standard solutions. PAH, TMV-PAH and RGD-PAH with similar size and
weight were seeded with BMSCs and cultured with osteogenic
supplement media until day 6. Samples were washed with sterile
Milli-Q water and completely lysed with dissolving solution (0.1%
HNO3, 0.01% TritonX-100, in 100 mM sodium citrate). The dissolved
samples were diluted to fit the range of the standard curve.
Approximately 4 mL of each diluted sample was fed into the
instrument per run. The experiment was performed in triplicates.
Control experiments without cells were also performed for PAH,
TMV-PAH, and RGD-PAH in the same manner.
[0341] Immunostaining:
[0342] Immunostaining was performed to observe the presence of
osteocalcin. rBMSCs culture in PAH, TMV-PAH, and RGD-PAH were
terminated after culturing 10 days for PAH and 13 days for TMV-PAH
and RGD-PAH hydrogels in osteogenic media. Hydrogels were washed
three times with 100 mM cacodylated buffer and then fixed in 2.5%
glutaraldehyde in cacodylated buffer overnight. The fixed samples
were then permeablized for 15 min in TBST (0.05% Tween20) and
blocked in 2% w/v bovine serum albumin (BSA, Sigma-Aldrich) in TBS
for 20 min with gentle agitation at room temperature. After
blocking, the hydrogels were incubated for 2 h with primary
antibody targeting the osteospecific gene osteocalcin (Santa Cruz
Biotechnology). Secondary antibody detection Alexa Fluor.RTM. 546
(Molecular Probes, Invitrogen) was used for osteocalcin at 1:100
dilutions in blocking buffer for 1 h at room temperature. In the
end, the hydrogels were washed with TBS and counter-stained with
DAPI (nuclear staining) and FITC-Phalloidin (actin staining).
Images of the stained samples were obtained using Olympus IX81 with
DSU confocal mode under 20.times. oil lens (NA=0.85). Exposure
times were kept constant for all samples (500 ms for DAPI and
phalloidin, 1000 ms for osteocalcin).
[0343] V. Summary
[0344] The viruses are well-tolerated as underlying substrates by
various mammalian cells (murine and human cells), and have
surprisingly exhibited some tremendous potential in guiding cells
for outgrowth and even for osteogenic differentiation. Previous
studies had primarily focused on the role of native TMV U1 strain
and its chemically modified variants to promote cell attachment and
direct cell outgrowth. Other groups have demonstrated the
possibility of inserting cell binding motifs to the coat proteins
of bacteriophages to promote cell differentiation, redirect the
natural targeting motifs towards specific cell types, or repackage
the inner contents with imaging agents or therapeutic agents. For
this study, longer cell binding peptide sequences were inserted
near the carboxy end of the viral coat protein (up to 15 amino
acids), which is not permissible for the major coat protein of M13
bacteriophages. Two of the RGD mutant containing different flanking
sequences, a 12-mer fibronectin synergy site, and two collagen
peptide sequence variants were successfully expressed on the TMV
coat protein. A systematic screening protocol with the centrifugal
adhesion assay was adapted to determine the biological
functionality of the peptide inserts on the viral particles. The
virus coating density has been reduced significantly and only a
minimal amount of the virus (10 .mu.g per well) was needed whereas
our previous studies with mesenchymal stem cells required milligram
quantities per coverslip. The virus-based substrates can be
screened by a simple and quantifiable process to aid many of the
future projects for determining receptor specificity, ligand
concentration effects, and competitive assays.
[0345] The results indicate that native TMV capsids provide little
to no cell binding specificity under the reported experimental
conditions. Cells attached to the substrates coated with virus
mutants containing RGD peptide sequences near the carboxy terminus
of the viral coat protein. However of the 9 different mutants
designed, only two mutants exhibited systemic infections
(Supplementary Table 1). The BHK cells seeded on TMV-RGD1 and RGD7
had similar attachment profiles, but promoted stronger attachment
for CHO cells. There are several differences between RGD1 and RGD7
insertions. RGD1 is based on a 7-mer insert that extends from the
carboxy terminus of the coat protein, whereas RGD7 insert a 12-mer
sequence that sits between residues serine 155 and glycine 156. The
difference in attachment strengths could be attributed to the
different flanking sequences, which may provide different tertiary
conformation of RGD peptide for improved binding for RGD1. There is
also one additional residue for the RGD1 mutant (-S155GPATG-RGD-
(SEQ ID NO: 53)) whereas RGD7 amino acid sequence has the binding
sequence (-S155AVTG-RGD- (SEQ ID NO: 54)). A single residue
extension and the different flanking sequence (a bend introduced by
proline in RGD1 whereas RGD7 has mostly small side chain amino
acids) could provide greater surface exposure and receptor binding
potential. Both possibilities are both logical explanations for the
adhesion strength differences. The two cell lines tested showed
weaker attachment to the surfaces coated with the TMV mutant that
contains the fibronectin synergy site, PHSRN (SEQ ID NO: 55) than
the TMV-RGD mutants. The collagen derived peptide inserts for
TMV-DGEA was a minor improvement in comparison to the native TMV
particles and TMV-P15 provided the strongest attachment for both
cell lines among all of the mutants screened by the centrifugal
assay. Additional mutations will be needed to determine ideal
sequences for promoting stronger cell attachments.
[0346] The TMV mutants reported here incorporate longer flanking
sequences than previously reported RGD peptide inserts displayed on
viral particles. It is currently thought that the integrin
specificity could rise from the difference between the flanking
sequences surrounding the RGD motif, but also from the structural
orientation of the ligand and the presence of other synergy site,
such as the PHSRN (SEQ ID NO: 55) on fibronectin. The first
isolated RGD mutant possesses the typical GRGDSPG (SEQ ID NO: 1)
insert with only four additional amino acids to flank the binding
site. The other RGD mutant contains a much longer 12 amino acid
insert, based on the sequence of fibronectin with four amino acids
flanking the amino and five amino acids flanking the carboxy end.
Both of these RGD inserts are not displayed as a loop, but as a
linear fragment displayed at the carboxy terminus of the TMV coat
protein. Alternative mutation sites as well as circular
permutations of the coat protein are potential routes to enclose
these binding sites in a loop. The sequence, PHSRN (SEQ ID NO: 55),
has been reported as the peptide fragment derived from the synergy
site of fibronectin that promote the interaction between native
fibronectin with its receptor, integrin .alpha.5.beta.1. Several
reports have shown that RGDS (SEQ ID NO: 56) peptides often bind
non-preferentially to several integrin receptors, making it
difficult to control its effects on stem cells and tissue
engineering scaffolds. In the crystal reconstructed image of
fibronectin, these two sequences are also slightly at an off-angle,
which might suggest that the positioning of the two regions could
affect binding to the integrin receptor. Based on the crystal
structure of TMV, we find that each carboxy region of the coat
protein are separated by 2-3 nm, which is similar to the distance
between the RGDS (SEQ ID NO: 56) and PHSRN (SEQ ID NO: 55)
sequences along the native fibronectin. Whether the TMV mutant or
other plant viruses can mimic this complex structural feature
remains to be seen.
[0347] Despite the growing scientific interest in adapting the
virus particles as nano-agents in biomedical applications, a key
issue remains. How will these fare when introduced to the body?
Manchester, Stuhlmann and colleagues have demonstrated that
fluorescently labelled CPMV particles can circulate in animal hosts
as potent vasculature imaging agents. Appending short polyethylene
chains to shield the virus particles have also been suggested by
researchers to avoid the host immune response. However, TMV is a
powerful antigenic carrier that has been used to display high copy
numbers of an epitope and activate cellular immune system to
protect vaccinated animal hosts against lethal challenge of
pathogens. Translating a virus-based vaccine agent as an
implantable tissue scaffold will require a balance between
recruiting stem cells to the scaffolds while repelling the
macrophages and other immune cells. One possibility is by culturing
the tissues ex-vivo with the virus scaffolds in serum-free,
chemically defined cultures then transplanting the engineered
tissue to the injury site. Due to its proteinaceous nature,
majority of the TMV particles are likely degraded over time by the
proteinases secreted by the cells. Our current studies have
modelled the viruses in three-dimensional scaffolds, and initial
studies suggest that such immunogenic responses can be avoided by
proper timing of the ex vivo cultures and tissue implantation. As
long as the virus provides an "electrical" jolt to jump start the
stem cell differentiation process, followed by degradation of the
virus particles and replacement by natural ECM proteins, the
virus-based biomaterial may yet be a feasible concept for tissue
engineering.
[0348] These and other modifications and variations to the present
invention may be practiced by those of ordinary skill in the art,
without departing from the spirit and scope of the present
invention, which is more particularly set forth in the appended
claims. In addition, it should be understood the aspects of the
various embodiments may be interchanged both in whole or in part.
Furthermore, those of ordinary skill in the art will appreciate
that the foregoing description is by way of example only, and is
not intended to limit the invention so further described in the
appended claims.
Sequence CWU 1
1
5617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Gly Arg Gly Asp Ser Pro Gly 1 5 212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Ala
Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser 1 5 10 312PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Glu
Asp Arg Val Pro His Ser Arg Asn Ser Ile Thr 1 5 10 44PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Asp
Gly Glu Ala 1 515PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly
Gln Arg Gly Val Val 1 5 10 15 611PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Gly Ala Gly Ser Gly Arg
Gly Asp Ser Gly Ala 1 5 10 710PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Thr Gly Arg Gly Asp Ser Pro
Ala Ser Ser 1 5 10 810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Gly Glu Pro Arg Gly Asp Thr
Tyr Arg Ala 1 5 10 910PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Ala Gly Ser Gly Arg Gly Asp
Ser Gly Ala 1 5 10 1011PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Gly Glu Pro Arg Gly Asp Thr
Tyr Arg Ala Tyr 1 5 10 1111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Glu Asn Gly Pro Arg Gly Asp
Asn Tyr Arg Ala 1 5 10 1216PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Ala Gly Ser Gly Glu Pro Arg
Gly Asp Thr Tyr Arg Ala Ser Gly Ala 1 5 10 15 136PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Pro
His Ser Arg Asn Gly 1 5 1410PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 14Glu Asp Arg Val Pro His Ser
Arg Asn Ser 1 5 10 154PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15Asp Ser Pro Gly 1
168PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Thr Gly Arg Gly Asp Ser Pro Gly 1 5
176PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Ser Ser Gly Pro Ala Thr 1 5 1810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Asp
Ser Pro Ala Ser Ser Gly Pro Ala Thr 1 5 10 1913PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Arg
Pro His Ser Arg Asn Ser Ile Thr Gly Pro Ala Thr 1 5 10
2015PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Asp Val Arg Pro His Ser Arg Asn Ser Ile Thr Gly
Pro Ala Thr 1 5 10 15 216PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 21Glu Ala Gly Pro Ala Thr 1 5
2216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val
Gly Pro Ala Thr 1 5 10 15 2351DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 23tctggtcctg caactggaag
aggagactct ccaggatgag gtagtcaaga t 512451DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
24atcttgacta cctcatcctg gagagtctcc tcttccagtt gcaggaccag a
512545DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25agcggcagag gcgacagcgg cgccggtcct gcaacttgag
gtagt 452645DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 26gctgtcgcct ctgccgctgc cggcaccaga
ggtccaaacc aaacc 452745DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 27agaggcgaca gccccgccag
cagctgaggt agtcaagatg cataa 452842DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 28ggcggggctg tcgcctctgc
cggtagaggt ccaaaccaaa cc 422951DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 29ggcgagccca gaggcgacac
ctacagagcc tgaggtagtc aagatgcata a 513048DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30ggctctgtag gtgtcgcctc tgggctcgcc agaggtccaa accaaacc
483145DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 31agcggcagag gcgacagcgg cgccggtcct gcaacttgag
gtagt 453245DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 32gctgtcgcct ctgccgctgc cggcagaggt
ccaaaccaaa ccaga 453348DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 33ccgagaggag atacatacag
agcatacggt cctgcaactt gaggtagt 483448DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
34tctgtatgta tctcctctcg gctctccaga ggtccaaacc aaaccaga
483548DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35ggaagaggag attcaccggc atcatcaggt cctgcaactt
gaggtagt 483648DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 36cggtgaatct cctcttcctg tcactgcaga
ggtccaaacc aaaccaga 483748DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 37aatggaccga gaggagataa
ttacagagca ggtcctgcaa cttgaggt 483848DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38tctgtaatta tctcctctcg gtccattctc agaggtccaa accaaacc
483945DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 39cgcggtgata cgtaccgtgc gagcggcgcc ggtcctgcaa
cttga 454048DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 40gtacgtatca ccgcgtggct ctccgctgcc
ggcagaggtc caaaccaa 484148DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 41tctggtcctg caactccaca
ctctagaaat ggatgaggta gtcaagat 484248DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42atcttgacta cctcatccat ttctagagtg tggagttgca ggaccaga
484345DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 43agagtgcccc acagcagaaa cagctgaggt agtcaagatg
cataa 454442DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 44tctgctgtgg ggcactctgt cctcagaggt
ccaaaccaaa cc 424548DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 45gtgccgcact caaggaattc aataacgggt
cctgcaactt gaggtagt 484648DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 46attccttgag tgcggcactc
tatcctcaga ggtccaaacc aaaccaga 484739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
47tggacctctg acggcgaggc cggtcctgca acttgaggt 394839DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
48agcaggaccg gcctcgccgt cagaggtcca aaccaaacc 394945DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
49agcggcagag gcgacagcgg cgccggtcct gcaacttgag gtagt
455045DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 50gctgtcgcct ctgccgctgc cggcagaggt ccaaaccaaa
ccaga 455123DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 51cgtgcctgcg gatgtatatg aac
235223DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 52cgttatcgta cgcaccacgt gtg 23539PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 53Ser
Gly Pro Ala Thr Gly Arg Gly Asp1 5 548PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Ser
Ala Val Thr Gly Arg Gly Asp1 5 555PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 55Pro His Ser Arg Asn 1 5
564PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Arg Gly Asp Ser 1
* * * * *