Note: Descriptions are shown in the official language in which they were submitted.
CA 02569904 2006-11-30
DENDRIMER CROSS-LINKED COLLAGEN
Field of the Invention
[0001 ] T:he present invention relates to a novel method of crosslinking
collagen using
dendrimers, to the resulting dendrimer cross-linked collagen matrix and to
modification of the
dendrimer collagen matrix to incorporate biomolecules.
Background of the Invention
[0002]Collagen, the most abundant protein in the body, is the major
constituent of connective
tissues. As such, it has been widely applied in biomaterials applications as,
for example, a
wound dressing, a matrix for controlled release of active agents or a tissue-
engineering scaffold
[1-5]. Ccillagen as a biomaterial offers such advantages as biocompatibility,
low toxicity to most
tissues, and well documented structural, physical, chemical and immunological
properties. It can
be readily isolated and purified in large quantities and can be processed into
a variety of forms
[6]. Collagen scaffolds have been applied to the engineering of such tissues
as cartilage [7],
cornea [84 0] and dermal skin [11].
[0003]However, in its purified form, collagen forms a weakly crosslinked
thermo gel.
Therefore, for tissue engineering applications, covalent intermolecular
crosslinks between
collagen molecules in macromolecular fibrils using appropriate biocompatible
molecules is
essential for the development of stable materials with a high degree of
mechanical integrity.
While glutaraldehyde has been widely used as a collagen crosslinking agent
[12,13] and is
generally thought to result in one of the highest crosslink densities [ 14],
cytotoxicity, and a lack
of understanding of the mechanisms of the reaction make it desirable to find
alternative effective
crosslinking mechanisms [ 15,16].
[0004] Alternative procedures have been explored for physically crosslinking
collagen, including
dehydrothermal treatment, ultraviolet irradiation [17,18] as well as novel
chemical crosslinkers
including diisocyanates, acyl azide [19], and 1-ethyl-3-(3-dimethyl
aminopropyl) carbodiimide
(EDC) [20]. Most of these crosslinkers including glutaraldehyde, hexamethylene
diisocyanate
and acyl azide are "bridge-forming" meaning that the crosslinker acts as a
chemical "bridge"
between collagen molecules. However, with EDC, "zero-length" crosslinks are
formed, meaning
that the collagen molecules are linked directly. Since amines are the limiting
functional groups in
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collagen for crosslinking [20], the use of amine rich compounds in combination
with EDC has
been examined. Collagen gels crosslinked in the presence of diamines [21]
showed little
improvernent in mechanical properties and biological stability relative to EDC-
crosslinked
controls. This is possibly due to the relatively short length of the diamines
selected and
potentially a lack of adequate amounts of free amine groups in the diamines to
sufficiently
enhance the reaction. In comparison, longer and more amine rich lysine
containing peptides
have shown promising results as agents to facilitate collagen crosslinking
[22]. Others have used
multifunc:tional amines for crosslinking of polymers other than collagen [23].
[0005]The extracellular matrix (ECM) is the natural scaffold for the cells,
acting as a mechanical
support and creating a microenvironment to which the cells can respond.
Constructing a matrix
or scaffold which simulates the ECM environment is therefore desirable and a
widely used
strategy in tissue engineering. Such a scaffold has the potential to promote
cell growth and to
restore key functions to damaged tissues and organs. To mimic the high
proportion of collagen
present in most native tissues, collagen scaffolds are widely used in tissue
engineering.
[0006]However, the biological function of these tissues is in large part due
to the presence of
other extracellular components. For example, the extracellular matrix protein,
laminin, has been
previously used to promote neurite growth [24]. The YIGSR sequence of laminin
has been
incorporated into tissue engineering scaffold materials to promote peripheral
[25], and central
[26] nerve regeneration. In corneal applications, YIGSR grafted to a collagen-
acrylate copolymer
scaffold has been shown to promote human corneal epithelial stratification and
neurite ingrowth
[27].
[0007]The dynamic interactions of collagen scaffolds with the surrounding
biological
environment in vivo make it desirable to incorporate additional biological
functionality into a
crosslinked collagen matrix in the form of cell adhesion molecules like
peptides and growth
factors. However, most currently available crosslinking technologies, such as
those described
above, will not permit functionalization of the matrix without potentially
altering the biological
properties of the collagen itself. Thus, it would be desirable to develop
methodology which
results in anechanically strong collagen matrices and permits the
incorporation of biological
functionality into a collagen matrix without altering the properties of the
matrix.
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Summary of the Invention
[0008]A novel collagen matrix has now been developed in which collagen
solutions are cross-
linked with multifunctional dendrimers resulting in mechanically strong
collagen hydrogels with
high crosslinking densities. The dendrimer crosslinked collagen showed unique
thermal
characteristics, with high temperature transitions and multiple denaturation
temperature peaks in
contrast to other crosslinked collagens. The dendrimer collagen matrix is
particularly suitable for
use as a tissue engineering scaffold in vitro and in vivo and for the
incorporation and delivery of
biomolecules in vivo.
[0009]Thus, in one aspect of the present invention, a dendrimer crosslinked
collagen matrix is
provided.
[0010]In another aspect, a method of preparing dendrimer crosslinked collagen
is provided
comprising the steps of incubating a collagen solution with a dendrimer
solution in the presence
of an agent capable of facilitating the crosslinking for a period of time
suitable to achieve the
desired arnount of crosslinking.
[0011]In another aspect of the present invention, dendrimer crosslinked
collagen is provided for
use as a tissue engineering scaffold.
[0012]These and other aspects of the invention will become apparent from the
following detailed
description and figures in which:
Brief Description of the Figures
Figure 1 graphically illustrates the relative equilibrium water uptake of
various collagen
samples, including uncrosslinked, EDC crosslinked, glutaraldehyde crosslinked
and dendrimer
crosslinked collagen;
Figure 2 graphically illustrates the denaturation temperatures (Td) of
collagen samples measured
by DSC before and after crosslinking;
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Figure 3 graphically illustrates the relative degradation percentage of
collagen samples during
exposure to a collagenase solution (pH 7.4, 37 C, 24h);
Figure 4 graphically illustrates the number of activated carboxylic acid
groups in various
collagen samples;
Figure 5 is a schematic of a generation 2 polypropyleneimine dendrimer;
Figure 6 graphically illustrates visible light transmission through the
various collagen samples as
measured. spectrophotometrically;
Figure 7 i.llustrates TEM photos of various collagen samples (Magnification
20,000, bar = 200
nm) including uncrosslinked (A), EDC-crosslinked (B), glutaraldehyde-
crosslinked (C) and
dendrimer crosslinked (D) collagen samples.
Figure 8 graphically compares Young's modulus of various crosslinked collagen
samples (a); the
maximum load measured for various crosslinked collagen samples (b); and the
displacement at
maximum load of different collagen samples (c);
Figure 9 graphically compares the effect of collagen concentration on the
mechanical properties
of various collagen samples;
Figure 10 graphically illustrates the mechanical properties of dendrimer
crosslinked collagen gel
samples;
Figure 11 (a-d) are representative photomicrographs of human corneal
epithelial cells grown on
various collagen samples at 120 minutes;
Figure 12 (a-d) are representative photomicrographs of human corneal
epithelial cells on the
collagen gels after 4 days of culture;
Figure 13 graphically illustrates the cell quantification analysis results of
various collagen
samples at a) 120 minutes and b) 3 and 4 days;
Figure 14 is an H-NMR spectra of YIGSR (a), dendrimer G2 (b), and YIGSR-
modified
dendrimer (c);
Figure 15 is a MALDI-TOF spectra of a) a dendrimer and b) a YIGSR-modified
dendrimer;
Figure 16 is a comparison of mechanical properties (a, Young's modulus; b,
Maximum load) of
YIGSR-modified (6.4 g/mg collagen) and unmodified collagen samples;
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Figure 17 illustrates HCEC adhesion on YIGSR-modified collagen gels after 2
hours of culture;
Figure 18 illustrates HCEC proliferation on YIGSR-modified collagen gels after
2 days of
culture;
Figure 19 illustrates HCEC proliferation on YIGSR-modified collagen gels after
4 days of
culture;
Figure 20 illustrates HCEC proliferation on YIGSR-modified collagen gels
determined by
Cyquant assay;
Figure 21 graphically illustrates DRG neurite extension on YIGSR-modified
collagen gels
compared with an unmodified control; and
Figure 22 illustrates DRG nerve cell in-growth on an unmodified control (left)
and YIGSR-
modified (right, 6.4 g/mg collagen) collagen gels. Neurites extended longer on
YIGSR-modified
collagens.
Detailed Description of the Invention
[00 1 3]Dendrimer crosslinked collagen is herein provided which is suitable
for the incorporation
of biomolecules and for the delivery of such biomolecules to a desired site in
vivo. The amine
groups on, the dendrimers, which permit collagen crosslinking, are also useful
for the
incorporation of biomolecules into the dendrimer collagen matrix without
significantly altering
the crosslinking density or the biological properties of the dendrimer
collagen matrices.
[0014]The term "dendrimer" is used herein to refer to a polymeric molecule
composed of a
repeating monomer (or dendrimer core). A dendrimer has a branching shape and
end groups that
are functional for cross-linking collagen. Depending on the dendrimer core
(central or core
monomer), the dendrimer may have 3, 4, 6, 8 or more branches and therefore is
multifunctional.
There are a large number of molecules which can be used as the core monomer
for a dendrimer.
For the purposes of the present invention, examples of suitable dendrimer
cores include an alkyl-
diamine such as ethyl-diamine and propyl-diamine; an alkyl dicarboxylic acid
such as malonic
acid, succinic acid and adipic acid; an aldehyde, and an isocyanate.
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[0015] The term "biomolecule" is used herein to refer to an entity that is
biologically active or
functional to provide a required linkage, stimulation or therapy. Thus, a
biomolecule may
comprise, but is not limited to, a peptide such as cell adhesion peptide
YIGSR, RGD, IKVAV
and RNIAEIIKDI; a glycoprotein such as laminin; a protein such as a growth
factor; a
polysaccharide such as heparin; and a naturally occurring or synthetic linker,
therapeutic, growth
stimulant or cell adhesion stimulant.
[0016]Th.e dendrimer crosslinked collagen of the present invention is prepared
by incubating a
suitable dendrimer with a collagen solution under conditions which result in
polymerization. In
order for the crosslinking to occur, a facilitating agent must be added to the
reaction mixture.
The facili.tating agent is any agent capable of causing the crosslinking
between collagen and the
dendrimer to occur. For example, where an amine-terminated dendrimer is used,
crosslinking is
facilitated by a carbodiimide, such as EDC or DDC (N,N'-
Dicyclohexylcarbodiimide)and
optimal conditions for this polymerization include, but are not necessarily
restricted to, a pH of
between 5.0 and 6.0, preferably a pH of 5.5, and incubation overnight at 37
C. Where a
carboxyl-terminated dendrimer is used, a carbodiimide or other facilitating
agent may be used
under polymerization conditions as described above. In any case, it is
desirable to degas the
collagen to maintain the mechanical properties of the resulting collagen
matrix. A stability
agent, such as N-hydroxysulfosuccinimide (NHS) or 1-Hydroxybenzotriazole
(HOBT), may also
be used in the crosslinking reaction. These agents include hydrophilic active
groups that react
rapidly with amines on target molecules and increase the stability of the
active intermediate
which ultimately reacts with the attacking amine. Although not necessary for
crosslinking to
occur, stability agents, such as NHS, significantly increase the yield of
crosslinked product.
[0017]The amount of dendrimer and collagen used to make the crosslinked
product is not
particularly restricted, and will depend on the nature of the dendrimer used
for cross-linking (the
greater the number of amine or carboxyl cross-linking groups on the dendrimer
i.e. the
generatiori of the dendrimer, the less the amount of dendrimer required). It
will also depend on
the desired cross-linked product. If a crosslinked product with free/available
cross-linking
groups is desired, then an amount of collagen and dendrimer is used in which
dendrimer
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functional groups are in excess to cross-linking groups of the collagen. The
greater the amount
of collagen used in relation to dendrimer, the greater the number of dendrimer
amine groups that
are utilized in the crosslinking reaction. Collagen:dendrimer ratios of
greater than 10:1 in terms
of dendrimer result in a significant excess of dendrimer functional groups.
Further increases of
dendrimer, thus, would not increase the level of crosslinking.
[0018]The use of multifunctional dendrimers, i.e. multi-branch dendrimers for
making a
crosslinked collagen matrix advantageously provides an increased number of
free amine groups
available for crosslinking with activated carboxylic acid groups of the
collagen relative to
carbodiimide and glutaraldehyde crosslinking counterparts. While not wishing
to be restricted to
any particular mode of action, it is believed that the dendrimers act as
"bridges" linking the
collagen molecules. Furthermore, in addition to introducing a large number of
amine groups,
dendrimers provide groups that are more accessible for crosslinking than those
in the collagen.
Therefore, the use of dendrimers for collagen crosslinking increases both the
extent of
crosslinking with collagen, and quality of the crosslinking (bridge linkage
versus "zero-length"
crosslinking).
[0019]The dendrimer crosslinked collagen product has features of high
mechanical strength and
high crosslinking densities in comparison to crosslinked collagen
counterparts. High
crosslinking densities are evidenced by its unique thermal characteristics of
high temperature
transitions and multiple denaturation temperatures which are not evident in
other crosslinked
collagens. High mechanical strength is evidenced by Young's modulus of at
least about 0.2
Mpa, and preferably, at least about 1.0 Mpa, as well as displacement at
maximum load of less
than aboul' 3.0 mm, and preferably, less than 2.0 mm.
[0020]The dendrimer collagen matrix may additionally incorporate a biomolecule
to enhance the
utility of the matrix. The biomolecule may be incorporated into the dendrimer
collagen matrix
by linkage to the dendrimer prior to crosslinking of the dendrimer to the
collagen. The linkage
of the biomolecule to the dendrimer may vary with the nature of the
biomolecule; however,
generally, this involves incubation of the biomolecule with the dendrimer
under conditions
suitable to catalyze linkage of the biomolecule to the functional groups on
the dendrimer also
utilized for collagen cross-linking. If the biomolecule contains the same
functional groups as the
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collagen crosslinking groups, it may be added into the reaction mixture during
the collagen
crosslinking reaction. The biomolecule may require modification to incorporate
a linker that will
allow ready linkage of the biomolecule to the dendrimer functional group.
Examples of suitable
linkers are known to those of skill in the art and include, for example,
carboxylic acids, amines,
hydroxyls or hydroxyl amines. In addition, additives which facilitate the
linkage of the
biomolecule to the dendrimer may be required, such as EDC to facilitate amine-
carboxylic acid
linkages, as one of skill in the art will appreciate. The amount of
biomolecule admixed with
dendrimer is such that the functional groups on the dendrimer are in excess of
the biomolecule so
that functional groups remain available on the dendrimer for subsequent or
simultaneous
collagen cross-linking.
[0021 ]Alternatively, the biomolecule may be linked to the dendrimer collagen
matrix following
the crosslinking reaction. This linkage is conducted by incubating the matrix
with the
biomolecule under conditions of temperature and pH which facilitate the
bonding of the
biomolecule to the matrix, and particularly, to available functional groups of
the dendrimer. The
incubation may be conducted in the presence of components required to
facilitate the
biomolecule-dendrimer linkage to occur as previously described. The amount of
biomolecule in
this case in not restricted as the dendrimer collagen matrix is formed and it
is no longer
necessary to maintain available dendrimer functional groups for crosslinking.
[0022]The dendrimer collagen matrix, optionally incorporating a selected
biomolecule, is useful
as a tissue engineering scaffold in vitro and for targeted sites in vivo, to
encourage the growth of
tissue for replacement or repair of damaged tissue, as well as for the
delivery of therapeutic
agents to a diseased region. The incorporation of biomolecules into the
present collagen matrix
further enhances their use as tissue engineering scaffolds.
[0023]In one embodiment, polypropyleneimine octaamine dendrimers can be used
to generate
dendrimer crosslinked collagen with mechanical properties appropriate for its
use as a tissue
engineering scaffold. The dendrimer crosslinked collagen of the present
invention may be used
as a scaffold in vitro, to grow tissue for subsequent implant or
transplantation. Alternatively, the
dendrimer crosslinked collagen can be inserted at a desired site in vivo to
promote or regenerate
tissue growth.
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[0024]In another embodiment, dendrimer crosslinked collagen incorporating a
biomolecule can
be used as a tissue engineering scaffold in connection with various
cells/tissues. The
biomolecule, for example, a cell adhesion factor, can advantageously promote
or accelerate cell
adhesion and growth. As exemplified in the specific examples herein, dendrimer
crosslinked
collagen incorporating a cell adhesion biomolecule promoted cell adhesion and
proliferation of
corneal epitheal cells and neurite cells in comparison to other crosslinked
collagen samples.
[0025]Embodiments of the invention are described by reference to the following
specific
examples which are not to be construed as limiting.
Example 1 - Preparation of Dendrimer Cross-linked Collagen and Properties
thereof
Collagen crosslinking
[0026]Uncrosslinked collagen controls were prepared by neutralizing a 0.4%
type I collagen
solution from rat tail tendon (Becton Dickinson, Mississauga ON) with 0.1N
NaOH and
subsequerit incubation in a 37 C oven overnight to gel. For preparation of the
EDC-crosslinked
collagen, 5 ml of a 0.4% type I collagen solution was added to a pre-cooled
glass vial with 1 ml
1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide
(NHS) in
aqueous solution (2.3 mg EDC (Sigma Aldrich, Oakville ON), 1.4 mg NHS (Sigma
Aldrich,
Oakville ON)) and mixed thoroughly. The pH was adjusted to 5.5 with 0.1 N NaOH
and/or 0.1 N
HCl and the resultant solution was placed in a 37 C oven overnight to gel.
This pH has been
suggested as the optimum pH for activation of carboxylic acid groups with EDC
for subsequent
functionalization [15].
[0027]Glutaraldehyde crosslinked collagen was prepared as a control. After
neutralization with
0.1N NaOH, the collagen solution was mixed with an aqueous solution of
glutaraldehyde (Sigma
Aldrich, Oakville ON) to a final concentration of 0.9%. The solution was left
in a 37 C oven
overn.ight to gel and crosslink.
[0028]Diamine (ethylene diamine), triamine (tris (2-aminoethyl amine) and
dendrimer
crosslinked collagens were prepared using the following procedure. To 5 mL of
a 0.4% type I
collagen solution was added 1 mL of an aqueous solution containing 2.3mg EDC,
and the
multifunctional amine crosslinking agent, (Sigma Aldrich, Oakville ON) in an
amount dependant
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on the weight ratio of collagen to dendrimer, and 1.4 mg NHS. The pH of the
solution was
adjusted to 5.5 and the solution was placed in a 37 C oven overnight for
crosslinking and
gelation. Three generations of polypropyleneimine dendrimers, generation 1(Gl)
with 4 amine
terminal arms, generation 2 (G2) with 8 arms and generation 3 (G3), with 16
arms, were used in
this study. Different ratios of collagen to dendrimer were studied to examine
the effect of
dendrimer amount on crosslinking in the resultant gels.
[0029]Since the gels prepared from the dilute collagen solutions (0.4% w/v)
were weak and
difficult to handle and characterize, all of the gels were freeze-dried to
obtain sponges for further
characterization. The sponges were stored at 4 C until characterization.
Characterization of collagen samples
Water uptake
[0030]Collagen sponge samples (n>3, approximately 5 mg) were dried completely
overnight,
weighed and incubated in 3 mL of PBS (pH 7.2) at room temperature for 1 hour.
It was
determined that 1 hour was sufficient for these highly porous gels to reach
equilibrium. The wet
weight was then determined and the absolute water uptake calculated using the
equation:
W
Water Uptake (%) = W' W Wx 100%
d
where W, and Wd are the wet and dry weights as measured respectively. The
results were
normalized to the uncrosslinked collagen control, at 100%.
Differential Scanning Calorimetry (DSC)
[0031 ]The denaturation temperatures of the collagen samples were determined
using a TA DSC
instrument. Denaturation temperature has been previously suggested to provide
information
about the crosslinking density of collagen samples [14,28]. Collagen samples
(2 mg) were
immersed in 30 1 of demineralized water in aluminum hermetic pans for 2 hours
at room
temperature. Hermetic pans containing 30 1 demineralized water only were used
as the
reference. A heating rate of 5 C/min was applied in a temperature range from
15 to 100 C and
the endothermic peak(s) of the therrnogram was monitored and recorded. While
heating rate can
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affect the observed denaturation values, similar temperatures were obtained
with a heating rate of
2 C/minute.
Collagenase assay
[0032]A collagenase assay [12,22] was performed to further examine the
crosslinking in the
samples and provide information about their biological stability. Collagen
samples with a dry
weight of approximately 5 mg were incubated for 1 hour in 0.1M Tris-HCl
(pH7.4) containing
0.05M CaC12 at 37 C. Subsequently, 200 U of bacterial collagenase (Clostridium
histolyticum,
EC 3.4.24.3, Sigma Chemical Co.) in 1 mL of 0.1M Tris-HCl (pH7.4) was added.
After 24h at
37 C, the reaction was stopped by the addition of 0.25M EDTA and cooling the
mixture on ice.
The mixtures were then centrifuged and the supernatant analyzed for
hydroxyproline (Hyp) [13].
Briefly, aliquots of standard Hyp (2-20 g) prepared from a stock solution and
10 1 supematant
were mixed gently with sodium hydroxide (2N). The samples were hydrolyzed by
autoclaving at
120 C for 20min. Chloramine-T was subsequently added to the above solution,
mixed gently,
and the oxidation was allowed to proceed for 25 minutes at room temperature.
This was followed
by the addition of Ehrlich's aldehyde reagent (p-dimethylaminobenzaldehyde
dissolved in n-
propanol/perchloric acid 2:1 v/v) to each sample and the development of the
chromophore by
incubating the samples at 65 C for 20min. The absorbance of each sample was
read at 550 nm
using a spectrophotometer and compared to a standard calibration curve to
quantify the amount
of Hyp.
Measurement of activated carboxylic acid groups
[0033]As previously described [15], the total number of NHS-activated
carboxylic acid groups
prior to crosslinking and the amount available after the crosslinking reaction
were determined.
Briefly, the free amine groups of collagen samples were blocked using the
acylating agent, acetic
acid NHS ester (HAc-NHS). An aqueous solution containing HAc-NHS was added to
a 0.4%
collagen solution (NHS:NH2 = 5:1) and the reaction allowed to proceed for 5
hours at room
temperature (pH - 6.5 to 7.5). The collagen samples with blocked amine groups
were reacted
with EDC and NHS at pH 5.5 as described. The samples, including those with
blocked amine
groups, were then washed for 1 hour in 20 mL of 0.2 M NaH2PO4 buffer (pH 4.5)
to remove
unreacted NHS, and subsequently immersed in 1 mL of 0.1 M Na2HPO4 buffer (pH9.
1) for a
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period of 2 hours. The amount of NHS released was measured
spectrophotometrically at 260 nm
assuming E = 9700M-Icm"1.
RESULTS
Crosslinking of Low Concentration Collagen Gels
[0034]The general appearance of the various collagen gels prepared is
summarized in Table 1
below.
Table 1 Macroscopic Appearance and Relative Mechanical Properties of Gels
Collagen Sample Crosslinker pH Relative Gel Appearance
Mechanical
Strength
Uncrosslinked N/A 7.5 Fair Translucent
EI)C EDC+NHS 5.5 Poor Transparent
Diarnine EDC+NHS+ED 5.5 Poor Transparent
Triamine EDC+NHS+TA 5.5 Poor Transparent
Glutaraldehyde Glutaraldehyde 7.5 Poor Translucent
G1 Dendrimer EDC+NHS+dendrimer 5.5 Poor Transparent
G2,G3 Dendrimer EDC+NHS+dendrimer 5.5 Good Transparent
[0035]Results were generally quite consistent if bubble formation in the gels
was minimized.
The EDC., diamine and triamine crosslinked collagen gels had relatively poor
mechanical
properties compared to the other gels. The glutaraldehyde crosslinked gels
were also relatively
mechanically weak. In comparison, the G2 and G3 dendrimer crosslinked gels
exhibited
comparatively good mechanical strength. The collagen solution without the
addition of the EDC
and with the addition of dendrimers, did not gel at pH 5.5, the optimal
condition for the
carbodiimide crosslinking reaction. This result provides evidence for the
carbodiimide
crosslinking and for the necessity of the carbodiimide in the crosslinking of
collagens with
dendrimers. The gels were freeze dried for further characterization.
Water uptake
[0036]Water uptake results for the various collagen samples are illustrated in
Figure 1. The water
uptake of EDC-crosslinked collagen decreased very little relative to the
thermo-gelled sample
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(approximately 10%), possibly due to the "zero-length" crosslinking nature and
suggesting that
EDC crosslinking alone may not be suitable for generating highly crosslinked
samples.
Similarly, the combination of a di- or tri-amine and EDC had little impact on
the water uptake
relative to the control samples. However, as expected, the glutaraldehyde-
crosslinked collagen
samples had a much lower water uptake, with a decrease of approximately 50%
relative to the
thermo-gelled controls, indicating a higher degree of crosslinking in these
samples. Similarly,
the G2 and G3 -dendrimer crosslinked collagen samples showed similar decreases
in the water
uptake of'between 50% and 70%, inferring that significant crosslinking was
occurring with the
use of a combination of EDC and dendrimers. The GI dendrimer crosslinked
samples showed
similar results to the di- and tri-amine-crosslinked samples.
[0037]Water uptake results suggest that altering the ratio of G2 and G3
dendrimers to collagen
had a small but relatively insignificant effect on the crosslinking.
Furthermore, somewhat
surprisingly based on the results with the G1 dendrimers, the use of a G3
dendrimer with 16
functional amine groups did not decrease water uptake relative to the use of
G2 dendrimers with
only 8 furictional groups. It is likely that the additional functional groups
present on the G3
dendrimers cannot effectively participate in crosslinking due to steric
factors .
Differential Scanning Calorimetry (DSC)
[0038]Measurement of denaturation (shrinkage) temperatures (Td) of collagen
samples by DSC
is commonly used to evaluate the efficiency and extent of crosslinking
[14,28]. In general,
crosslinking of the collagen gels with various crosslinkers resulted in an
increase in the
denaturation temperature as shown in Figure 2. The EDC-crosslinked collagen
showed only a
slight increase in the denaturation temperature from 48 C in the uncrosslinked
sample to 55 C.
Denaturation temperatures of 54 C, 48 C and 45 C were noted for the ethylene
diamine,
triamine and G1 dendrimer crosslinked samples, respectively. Consistent with
the water uptake
and macroscopic uptake results, these results suggest that the level of
crosslinking in these
samples was not significant. Glutaraldehyde-crosslinking, which involves
reaction with the free
amine groups present in collagen, resulted in a further increase of the Td to
71 C. Higher Td
values of between 80 C and 90 C were noted following crosslinking with the G,
and G3
dendrimers at pH 5.5. This indicates that the use of dendrimers with higher
numbers of
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functional amine groups for crosslinking may result in a higher crosslinking
density than that
obtained using glutaraldehyde. While there was a trend toward higher
denaturation temperatures
with increased amounts of dendrimer with the G2 dendrimer-crosslinked samples,
differences
were not significant. A similar trend was not observed with the G3 dendrimer-
crosslinked
samples. Furthermore, there were no clear differences in the observed
denaturation temperatures
between the G2 and G3 dendrimer-crosslinked samples.
[0039]A unique characteristic feature of G2 and G3 dendrimer-crosslinked
collagens but not the
Gl dendrimer sample was noted during DSC peak assigmnent. These samples showed
multiple
peaks in the DSC scans as noted in Table 2 below.
Table 2: Presence of multiple denaturation peaks in dendrimer crosslinked
collagen
Sample Denaturation Temperature
(0C)
Uncrosslinked 47.9
EDC crosslinked 54.7
Glirtaraldehyde crosslinked 71.5
G2 crosslinked (20:1) 51.4 85.4
G2 crosslinked (10:1) 40.0 82.2 89.3
G2 crosslinked (5:1) 37.8 51.0 67.4 80.4 92.0
G3 crosslinked (20:1) 51.5 67.8 89.1
G3 crosslinked (10:1) 41.1 70.3 80.8
G3 crosslinked (5:1) 45.4 68.1 80.6 89.9
[0040]The presence of these multiple peaks could be the result of complexity
and heterogeneity
in the dendrimer-crosslinked samples due to the multifunctionality of the
dendrimers. These
peaks were denaturation peaks as confirmed by a second DSC; since the
denaturation of collagen
is an irreversible process, the denaturation peak(s) present in the first DSC
scan did not appear in
subsequent scans.
HAM-LAW\ 156340\1 14
CA 02569904 2006-11-30
Collagenase assay
[0041 ]The degradation and therefore biological stability of the collagen
samples was studied by
exposing materials to a collagenase solution. The degraded collagens were
quantified by analysis
of hydroxyproline release, a major component of collagen. The results are
shown in Figure 3 as
percentages of degraded collagen relative to uncrosslinked samples. Following
crosslinking, the
degradation of the samples decreased to various extents depending on the
nature of the
crosslinkers used. Approximately 60% of the EDC-crosslinked collagen was
degraded, possibly
due to the less efficient "zero-length" crosslinking that occurs with this
method. Consistent with
the results of others, glutaraldehyde showed significant ability to improve
the biostability of the
collagen samples. As little as 7% of the glutaraldehyde-crosslinked collagens
were degraded
under identical conditions. With the aid of G2 and G3 dendrimers, the
carbodiimide-crosslinking
could achieve comparative biostability to that noted with glutaraldehyde. The
results suggest
that collagen to dendrimer in a 10:1 ratio (wlw) resulted in the greatest
improvement in the
proteolytic resistance of the collagen in these samples. However, the
stability of all of the
dendrimer-crosslinked samples was similar.
Measurement of activated carboxylic acid groups
[0042]To provide further evidence of reaction and to determine the extent of
this reaction, the
number of activated carboxylic acid groups in carbodiimide-crosslinking
reactions was
monitoredl. The results are shown in Figure 4. The difference between the
number of crosslinked
samples and that of the amine-blocked collagens was the amount of activated
carboxylic acid
groups consumed during the crosslinking reactions. Analysis of an amine
blocked collagen
sample suggests that the total number of activated carboxylic acid groups
available for
crosslinking was 87 per 1000. This is slightly lower than the estimate for
total number of
carboxylic acid groups of 120 per 1000, suggesting that the EDC does not
activate 100% of the
available carboxylic acid groups in the collagen or that not all of the amide
groups were
hydrolyzed. The EDC crosslinked collagen had 71/1000 activated carboxylic acid
groups
suggesting that 16/1000 had been consumed by the crosslinking reaction. In the
G2 and G3
dendrimer crosslinked samples, the number of the consumed carboxylic groups
was between 40
and 69 pei- 1000, clearly demonstrating that the introduction of dendrimers
into the EDC
crosslinking reaction resulted in crosslinking via the carboxylic acid groups
and improved the
HAM_LAW\ 156340\ 1 15
CA 02569904 2006-11-30
extent of the reaction, likely due to large amount of free amine groups of
dendrimers. Again, no
clear trend was observed with changes in the weight ratios of collagen to
dendrimers and G2
versus G3.
Example 2 - Utility and BiocompatabiGty of Dendrimer Crosslinked Collagen
Collagen gel preparation
[0043]All the reagents used were purchased from Sigma Aldrich (Oakville ON)
except when
otherwise specified. Concentrated collagen suspensions, the generous gift of
Inamed
Corporation (USA), consisted of pepsin-digested bovine cornium purified
predominantly type I
collagen with less than 20% type III collagen. The 6% suspension was in
phosphate buffered
saline, pH 7.0-7.6. The suspensions were acidified with 1N HCI and diluted to
make clear
collagen solutions prior to further treatment.
[0044]A thermally crosslinked collagen control was prepared by neutralizing
the collagen
solution with IN NaOH and subsequent incubation in a 37 C oven overnight in
order to allow for
gelation to occur. EDC-crosslinked collagen gels were prepared by mixing the
collagen solution
with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) / N-
hydroxysuccinimide (NHS)
aqueous solution (molar ratio of EDC: NHS: COOH = 5:5:1) in pre-cooled
syringes on ice. The
pH was subsequently adjusted to 5.5 with 1 N NaOH and/or 1 N HCl and the
resultant solution
was injected into glass moulds in a 37 C oven overnight to gel.
[0045]Gh>.taraldehyde-crosslinked collagen gels were prepared as positive
controls. After
neutralization with IN NaOH, the collagen solution was mixed with an aqueous
solution of
glutaraldehyde (1%) to a final glutaraldehyde concentration of 0.02%. The
solution was left in a
37 C oven overnight for gelation and crosslinking.
[0046]Dendrimer-crosslinked collagens were prepared using the following
procedure. The
collagen solution was mixed (-10 minutes) with an aqueous solution containing
EDC, dendrimer
and NHS :in pre-cooled syringes on ice. The pH of the solution was adjusted to
5.5, the optimal
reaction condition for carbodiimide crosslinking [20] and the solution was
injected into glass
moulds and reacted in a 37 C oven overnight. The EDC and NHS ratios remained
constant
based on above. However, different ratios of collagen to dendrimers were
studied to examine the
HAM LAW\ 156340\I 16
CA 02569904 2006-11-30
effect of dendrimer amount on crosslinking in the resultant gels. The chemical
structure of a
generation 2 polypropyleneimine octamine dendrimers used for crosslinking is
shown in Fig. 5.
[0047]The final collagen concentration ranged from 2% to 5% based on different
dilution
factors. In all cases, due to the high viscosity of the collagen solutions
used for gel preparation,
it was desirable to avoid the introduction of air into the mixture as this
altered the appearance
and mechanical properties of the gels formed. Once formed, the resultant gels
were removed
from the moulds, immersed in glycine solution (0.5% in PBS) at room
temperature to neutralize
any residual activated carboxylic acid groups and to extract the N-
hydroxysuccinimide reaction
product, or in the case of the glutaraldehyde-crosslinked gels, to neutralize
any residual
glutaraldehyde. The final gels were rinsed three times with PBS over a 12 hour
period. Prepared
gels were stored hydrated in a 4 C refrigerator until use.
Characterization of collagen samples
Transparency measurements
[0048]The collagen samples were examined for transparency by scanning within
the visible
range of wavelengths (390nrn - 780nm) with Beckman DU-640 spectrophotometer.
Transmission Electron Microscopy (TEM)
[0049]Samples were fixed for 2 hours with 2% glutaraldehyde in 0.1M sodium
cacodylate buffer
(pH7.4), rinsed twice in buffer, post-fixed for 1 hour in a 0.1 M sodium
cacodylate buffer
containing 1% osmium tetroxide, and finally rinsed twice with buffer. Then
samples were
gradually dehydrated by ethanol (50%, 70%, 95%, 100%) for at least 1 hour at
each
concentration. The samples were then infiltrated with Spurr's resin through a
resin:ethanol series
of 1:2, 1:1, 2:1, 100% Spurr's with continuous mixing on a rotator throughout
the infiltration
process. Once in 100% Spurr's resin, the samples were then cut into blocks of
a width of lmm,
placed into flat embedding moulds and polymerized at 60 C overnight. The
embedded samples
were sectioned with a diamond knife on a Leica Ultracut UCT microtome, post-
stained with
uranyl acetate and then viewed in a JEOL JEM 1200 EX transmission electron
microscope
operating at 80kV.
HAM_LAVJ~ 156340\1 17
CA 02569904 2006-11-30
Glucose permeation
[0050]Glucose permeability of the dendrimer-crosslinked collagen gel samples
was determined
using a custom-made device previously described [29]. Other samples were
mechanically not
strong enough to be placed into the apparatus without leaking. The glucose
concentrations of
solutions in the each of the chambers were periodically measured based on the
enzymatic
conversion of glucose to glucose-6-phosphate followed by production of
dinucleotide and
quantified UV absorption. The permeability coefficient of glucose in PBS (pH
7.4) was
calculated from the rate of glucose concentration change with time.
Mechanical properties
[0051 ]In order to prepare collagen gel samples for Instron testing, a custom
designed mould was
prepared. A polymer mesh was incorporated in the gel sample in the area where
the gels would
be gripped in the test in order to make the handling and gripping of the
samples in the testing
machine easier as well as to provide an accurate measure of the strength of
the gel unaffected by
the grips. The area between the grips was free of mesh so that only the gel
was tested.
Appropriate gel forming solutions were poured into the mould, and the mould
was placed under
two flat glass plates in order to make the samples. A weight was placed on top
of the glass plate
to ensure ;solution contact with the mould and the plates. The mould was then
placed overnight
in a 37 C oven under humidified conditions. The gel was removed from the mould
and rinsed
with Milli-Q water at least three times over 10 hours to remove unreacted
crosslinking reagents.
The gels vvere then blotted dry gently with filter paper and mounted on the
grips of an Instron
Series IX Automated Materials Testing System. A crosshead speed of 5 mm/min
and full-scale
load range; of 500 N were used for the test which was conducted at 23 C and a
humidity of 50%.
Young's modulus, maximum load and displacement at maximum load were recorded
as
indications of the mechanical properties of the various collagen samples.
Suture strength
[0052]Suture strength of collagen gels was also determined since this is
anticipated as the
location of failure at implantation. A method similar to that recommended for
vascular
prostheses and triflate heart valves (ANSUAAMI) was used as suggested
previously [30].
HAM LAw\ 156340\1 18
CA 02569904 2006-11-30
Briefly, fully hydrated gels were suspended between two diametrically
positioned nylon 10/0
sutures (33 m diameter), selected based on their used in ocular surgeries,
penetrating through the
gels at 2 inm from the edge. The free ends of each suture were clamped in the
grips of the Instron
and the samples were drawn to break at a crosshead speed of 5 mm/min. The
suture itself was
found to llave a breaking load of -56 g, which as well above the failure point
of the tested gels.
The maximum load at breaking was recorded as a very practical indication of
gel performance
during surgical suturing.
In vitro cell culture studies
[0053]For cell culture, 0.5 cm disks of the gels were exposed to keratinocyte
serum-free medium
(KSFM, Invitrogen Life Technologies, Burlington ON) containing antibiotics
(penicillin/streptomycin 1:100, gentamycin 1:1000). Immortalized human comeal
epithelial
cells [8] were used to evaluate corneal epithelial compatibility of the
various collagen surfaces.
The cells were seeded on the gels at a density of 104 cells per well. The
cells were incubated for
approximately 15 minutes to allow the cells to adhere to the surfaces before
keratinocyte serum-
free medium containing epidermal growth factor (5 ng/mL) was added. Medium was
replaced
every two days and the surfaces were examined daily by light microscopy. To
quantify cell
adhesion and growth, a CYQUANT assay (Molecular Probes, Invitrogen Life
Technologies,
Burlington ON) was performed at specified times.
RESULTS
Collagen gel preparation
[0054]While all of the collagen solutions became gels under the specified
reaction conditions, a
scaffold for a tissue engineered cornea must be transparent and strong enough
to withstand
suturing. Unlike the other crosslinking methods, which resulted in gels with
varying degrees of
transparency, the dendrimer crosslinked collagen samples were all transparent
and, relative to the
other samples, easy to manipulate.
Transparency Measurements
[0055]Fig. 6 summarizes light transmission through the sa.mples in the visible
light range
(390nm - 780nm), measured as an indication of gel transparency. The EDC and
dendrimer-
HAM_LAw\ 156340\1 19
CA 02569904 2006-11-30
crosslinked collagen samples had very high levels of light transmittance
through the entire range
of wavelengths. Light transmission though the glutaraldehyde-crosslinked
samples was
somewhat lower while a significantly (p<0.05) lower level of light
transmission was observed
with the uncrosslinked thermal gels. This is likely due to fibril formation
which is characteristic
of this gelling process.
Transmission Electron Microscopy (TEM)
[0056]TEM was used to examine gel morphology via the formation of collagen
fibers/fibrils
during gel preparation as well as the relationship between fibril formation
and the crosslinkers.
As shown in Figure 7, no fibrils were observed in the dendrimer-crosslinked
collagen samples at
a magnification of 20 k. Therefore, these gels will have a high level of
transparency.
Conversel y, in the thermally gelled collagen samples (Figure 7-a) , numerous
collagen fibers
with a size in the order of 100 nm were noted. Unlike the highly ordered
fibril alignment that is
present in natural corneal stroma, these fibers were randomly aligned. In the
EDC-crosslinked
samples, there were few fibrils (Figure 7-b) although some fine fibrils with
sizes on the order of
10-100 nm were observed in the glutaraldehyde-crosslinked samples (Figure 7-
c). Uneven
distribution of the fibrils was also observed in these samples, possibly due
to the imperfect
mixing of the solutions when the samples were prepared.
Glucose permeability
[0057]Since the avascular cornea has a high nutrient permeability, glucose
permeability is an
important characteristic in corneal tissue engineering scaffolds. The glucose
permeability of the
comeal stroma has been estimated to be approximately 0.7x 10-6cm2/s [30]. 3%
dendrimer-
crosslinked collagens had similar or higher glucose permeability at 0.8-1.1 x
10-6cm2/s. By
decreasing the collagen concentration to 2%, the permeability can be increased
to 2.2x 10-6cm2/s.
Mechanical Properties
[0058]Mechanical properties of collagen gel samples, including Young's
modulus, maximum
load and displacernent at maximum load were measured using an Instron Series
IX Automated
Materials 'Testing System. The results for these tests for the various
crosslinkers are summarized
in Figure 8. It was not possible to obtain data for the thermally gelled
collagen samples as they
HAM_LAW\ 156340\I 20
CA 02569904 2006-11-30
were extremely weak and did not withstand clamping. Clearly, the dendrimer-
crosslinked
collagens had the highest Young's modulus at 1.4 0.1 MPa and strength at
maximum load at
1.2 0.17 N. These properties in the other samples were at least an order of
magnitude lower. As
expected, the displacement data showed the opposite trend with the dendrimer-
crosslinked
collagens having the smallest displacement compared with other samples.
[0059]The effect of collagen concentration in the dendrimer-crosslinking
reaction of the
mechanical properties of the resultant gels was also examined. The results are
summarized in
Figure 9. While the trend suggests an increase in Young's modulus and maximum
load with
increasing collagen concentration and a decrease in displacement (p<0.05), the
differences
between the 2% and 3% collagen were not significant. While higher
concentrations generally
resulted in improved mechanical properties, the high viscosity of these
samples resulted in
mixing difficulties and therefore higher variances in these results. For this
reason, all remaining
samples were prepared using 3% for the ease of sample handling and
consistency.
[0060]The effect of dendrimer amounts on mechanical properties of dendrimer-
crosslinked
collagen samples was also examined. As seen in Figure 10, different collagen
to dendrimer
weight ratios (40:1, 20:1, 10:1 and 5:1) were used to prepare the samples for
the test. Consistent
with DSC measurements of denaturation temperature from a previous study [31 ],
increasing the
amount of dendrimer in the reaction mixture to amounts greater than
stoichiometric had no
significant effect on the mechanical properties of the gel (p>0.05).
[0061 ]The effect of another important factor - reaction pH - on the gel
properties was also
examined and found to not significantly affect Young's modulus although
slightly higher values
were found with increasing pH (results not shown). However, at pH values above
6.0, the
formation of fibrils deteriorated the optical properties of the gels, making
them unsuitable for
corneal scaffolds. At pH values lower than 5, gelation did not occur.
Suture Strength
[0062]Suture strength of the dendrimer-crosslinked collagen gels was also
measured as a
practical indication of gel performance during surgical suturing. Maximum load
of the sutured
dendrimer-crosslinked collagen gels was 5.50 0.92 g compared with the strength
of nylon 10/0
HAM_LAW\ 156340\1 21
CA 02569904 2006-11-30
sutures of -56 g. It was also much lower than that of natural cornea, which
has a higher strength
than that of sutures and which therefore did not break [27,32]. However, it
was much higher
than the strength of the EDC- and glutaraldehyde-crosslinked samples, which
were difficult to
suture and could not be placed in the apparatus.
In Vtro Corneal Epithelial Cell Culture
[0063]Representative photomicrographs of human corneal epithelial cells on the
various surfaces
at 120 minutes and on day 4 of culture are shown in Figures 11 and 12,
respectively.
Surprisingly, there are clearly distinct differences in the number of cells
present, with the
crosslinked gels presumably showing better adhesion than the physically
crosslinked thermal
gels initially. To better quantify these differences, a Cyquant assay,
measuring cell adhesion was
performed at short times of 120 minutes (Figure 13a) and at longer times (3
and 4 days post
seeding) (Figure 13b) to assess cell proliferation. Similar levels of initial
adhesion were
observed on the EDC- and dendrimer-crosslinked gels and not statistically
different (p>0.05).
The adhesion of the cells on the glutaraldehyde-modified surfaces was only
slightly lower and
also not statistically different from the dendrimer and EDC-crosslinked gels.
However, that
observed on the uncrosslinked thermal gels was much lower (p<0.05). Generally
all of the
surfaces supported the proliferation of corneal epithelial cells, with similar
levels of adhesion at
3 and 4 days post plating. However, it is of interest to note that the
glutaraldehyde-crosslinked
gels consistently showed decreased cell numbers at 4 days relative to three
days, potentially
indicative of the release of cytotoxic glutaraldehyde byproducts.
CONCLUSIONS
[0064]Polypropyleneimine octaamine dendrimers were studied as a means of
generating highly
crosslinked collagen by amplifying the reaction between collagen molecules
using the water-
soluble carbodiimide, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide
hydrochloride (EDC).
Compared with EDC only and glutaraldehyde-crosslinked collagens, dendrimer-
crosslinked
collagen gels had the best optical and mechanical properties. The Young's
modulus of the gels
was a factor of more than 10 greater with dendrimer-crosslinking compared to
EDC-
crosslinking. In vitro cell adhesion and growth studies with human corneal
epithelial cells show
that dendrimer-crosslinking does not adversely affect the biological
compatibility of the collagen
and suggest that dendrimer-crosslinking may actually result in improved
biological interactions.
HAM_LAW\ 156340\1 22
CA 02569904 2006-11-30
Thus, dendrimers have been successfully applied to collagen crosslinking to
produce transparent,
mechanically stronger and more biocompatible collagen gels having utility as
tissue engineering
scaffolds.
Example 3- Preparation of Collagen Matrix including Biological Ligands
Covalent attachment of YIGSR to dendrimers
[0065]YIGSR was added to aqueous dendrimer solutions containing EDC and NHS
and reacted
overnight at room temperature with stirring. The molar ratio of YIGSR to
dendrimer was 1:1,
meaning that the number of NH2 groups for covalent attachment of the peptide
was in significant
excess and residual amine groups could be used for collagen crosslinking. A
ratio of 5:5:1
EDC:NHS:COOH of YIGSR was used. The YIGSR-modified dendrimer product was
purified by
dialysis with Spectro/Por membrane (MWCO 500) in water for 2 days. The
purified product was
freeze dried for characterization or further reaction.
Characterization of YIGSR-modified dendrimers
[0066]The purified YIGSR-modified dendrimer (YIGSR-m-dendrimer) was
reconstituted into
deuterated water for H-NMR analysis. Spectra for the dendrimer, YIGSR and
YIGSR-m-
dendrimei- were recorded and the peaks compared. MALDI-TOF (Matrix-Assist
Laser
Desorption Ionization time-of-flight) mass spectrometry was also used to
characterize the
dendrimer, YIGSR and YIGSR-m-dendrimer. The Micromass TofSpec 2E MALDI-TOF
mass
spectrometer was operated in reflectron mode using alpha-cyano-4-
hydroxycinnamic acid as the
matrix. In reflectron mode, an electrostatic mirror bounces the ions back and
focuses them at a
second detector allowing for better resolution and mass accuracy.
Collagen gel preparation
[0067]All the reagents used were purchased from Sigma Aldrich (Oakville ON)
except when
otherwise specified. Concentrated collagen suspensions (6%), the generous gift
of Inamed
Corporation (USA), contained pepsin digested bovine cornium purified type I
collagen
predominantly with less than 20% type III collagen. The 6% suspension was in
phosphate
buffered saline, pH 7.0-7.6. All of the suspensions were acidified with 1N HCl
and diluted to
make clear collagen solutions prior to further treatment.
HAM_LAVJ\ 156340\1 23
CA 02569904 2006-11-30
[0068]Dendrimer-crosslinked collagens were prepared by mixing the collagen
solution with an
aqueous solution containing EDC, generation 2 polypropyleneimine octaamine
dendrimer (Fig.
5), and NHS (molar ratio of EDC:NHS:COOH=5:5:1) in pre-cooled syringes on ice.
The pH of
the solution was adjusted to 5.5, the optimal reaction condition for
carbodiimide-crosslinking
[20] and the solution was injected into glass moulds in a 37 C oven overnight
for crosslinking
and gelation. 3% collagen gels and a collagen to dendrimer weight ratio of
10:1 were used
throughout this study based on previous results [23]. In all cases, due to the
high viscosity of the
collagen solutions used for gel preparation, the introduction of air into the
mixture was avoided
as this altered the appearance and mechanical properties of the gels formed.
This was achieved
by carefully removing air bubbles from the collagen suspensions before they
became viscous
solutions. YIGSR bulk-modified collagen gels were prepared following the same
procedure
using a combination of dendrimers with chemically attached YIGSR and
unmodified dendrimers
as crosslinkers. A series of YIGSR-modified collagen gels with different
amounts of YIGSR
were prepared by using various YIGSR-m-dendrimer percentages (100%, 10%, 1%)
in the
crosslinking solution.
[0069]Once crosslinked, the gels were removed from the moulds and immersed in
glycine
solution (0.5% in PBS) at room temperature to react with any residual
activated carboxylic acid
groups and to extract out the N-hydroxysuccinimide reaction product. The final
gels were rinsed
with PBS at least three times over a period of 12 hours to remove any residual
reaction products.
The gels were stored in 4 C refrigerator. Gels for in vitro cell culture
studies were prepared under
sterile cor-ditions in a class II biosafety cabinet. All the reagents were
either autoclaved or
sterilized by filtering with 0.2gm filters.
Quantification of YIGSR in collagen gels
[0070]In order to directly quantify the YIGSR content in the modified collagen
gels, YIGSR was
radiolabeled with 1251 using lodogen method [33]. Briefly, 125I was added to a
YIGSR in a
precoated lodogen vial. After stirring at room temperature for 20 minutes, the
labeled YIGSR
was purified by dialysis against water using Spectro/Por dialysis membranes
(MWCO 500). The
radioactivity of the dialysate was monitored until no further free iodide was
detected. YIGSR
solution containing 10% 125I labeled was used to attach to dendrimer and then
collagen gel
HAM LAW\ 156340\1 24
CA 02569904 2006-11-30
preparation. These collagen gels were counted in a gamma counter to determine
the amount of
YIGSR in the gels.
YIGSR surface modification of the collagen gels
[0071 ]Dendrimer only crosslinked collagen gels were immersed in an aqueous
solution
containing EDC, NHS and the YIGSR. Surfaces with varying peptide coverage were
prepared by
applying different amounts of peptide. The molar ratio of EDC:NHS:YIGSR was
5:5:1. The pH
of the reaction solution was maintained at 5.5 and the reaction was carried
out at room
temperature overnight with slight agitation. The modified surfaces were
thoroughly rinsed with
Milli-Q water to remove unreacted peptides and excess EDC and NHS. Surface
density of
YIGSR was determined using 125 1 radiolabeled peptide.
Surface characterization of modified gels
[0072]X-ray photoelectron spectroscopy (XPS) analysis was performed with a
Leybold MAX
200 XPS System (Cologne, Germany), using a non-monochromatised Mg Ka X-ray
source
operating at 15 kV and 20 mA. The spot size used was 2x4 mm. The energy range
was calibrated
by placing the Au 4f peak at 84 eV or the main C 1 s peak at 284.5 eV. Survey
scans were
performed f r o m 0 to 1000 eV, and low resolution and high resolution C 1 s
spectra were obtained
at 90 and 20 takeoff angles of the collagen gels before and after YIGSR
modification.
Bulk characterization of dendrimer modified collagen gels
[0073] Mechanical properties of collagen gels were examined to test the
effects of YIGSR
modification. In order to prepare collagen gel samples for Instron testing, a
custom designed
mould was prepared. A polymer mesh was incorporated in the gel sample in the
area where the
gels would be gripped in the test in order to make the handling and gripping
of the samples in the
testing machine easier as well as to provide an accurate measure of the
strength of the gel that
was unaffected by the grips. The area between the grips was free of mesh so
that only the gel was
tested. Gel forming solution was poured into the mould, and the mould was
placed under two flat
glass plate:s in order to make the samples. A weight was placed on top of the
glass plate to ensure
solution contact with the mould and the plates; otherwise the gel preparation
procedure was as
with samples for other tests. Prior to testing, the gels were blotted dry
gently with filter paper
and mounted on the grips of an Instron Series IX Automated Materials Testing
System. A
HAM-LAW\ 156340\1 25
CA 02569904 2006-11-30
crosshead speed of 5 mm/min and full-scale load range of 500 N were used for
the test which
was conducted at 23 C and a humidity of 50%. Young's modulus, maximum load and
displacenient at maximum load were recorded as indications of the mechanical
properties of the
various collagen samples.
In vitro corneal epithelial cell culture characterization
[0074]The response of human corneal epithelial cells to the modified surfaces
was examined to
assess whether there were differences that resulted from the YIGSR
modification. For cell
culture, 0.5 cm disks of the sterile gels were pretreated with keratinocyte
serum-free medium
(KSFM, Invitrogen Life Technologies, Burlington ON) containing antibiotics
(penicillin/streptomycin 1:100, gentamycin 1:1000). Immortalized human corneal
epithelial
cells [Griffith et al., 1999], were seeded on the gels at a density of 104
cells per well. The cells,
in a small volume of medium (100-200 l), were incubated on the surfaces for
approximately 15
minutes. This permitted the cells to adhere to the surfaces and ensured that
the cells were not
washed off the surface of the disks. Following this, epidermal growth factor-
containing
keratinocyte serum-free medium was added to cover the surfaces. Medium was
replaced every
two days and the surfaces were examined and photographed daily. To quantify
cell adhesion and
proliferation, a CYQUANT (Molecular Probes, Invitrogen Life Technolgies,
Burlington ON)
assay was performed at specified times.
In vitro early nerve in-growth
[0075]Early nerve in-growth studies were performed using Dorsal Root Ganglia
(DRG) from
chicken einbryo. Collagen gel samples with varying amounts of incorporated
YIGSR were
sterilized by incubating in 1% chloroform in PBS for 4 days at 4 C and
subsequently washed in
PBS followed by PBS containing antibiotics. Low concentration collagen gels
were prepared
from diluted collagen solutions for initial adhesion of the DRG. The dorsal
root ganglia were
isolated from chick embryos and separated from fibroblasts as previously
described. Selected
DRG's were then dipped into low concentration collagen gels on ice and placed
on sample
surfaces. Cells were cultured in keratinocyte serum free medium (KSFM) medium
(Invitrogen
Life Technologies, Burlington ON) supplemented with dexamethasone, dibutryl
cyclic adenosine
monophosphate (dB cAMP; Sigma), dimethylsulfoxide (DMSO; BDH chemicals). Media
was
changed every other day. DRG's were allowed to extend for 5 days.
HAM-LAW\ 156340\t 26
CA 02569904 2006-11-30
[0076]After 5 days of culture, samples were fixed with 4% paraformaldehyde
(PFA, Sigma
Aldrich, C>akville ON) in PBS. Fluorescent immuno-staining for neurofilament-
200 was
performedl by using mouse monoclonal anti-neurofilament-200 (Sigma Aldrich,
Oakville ON) as
the primary antibody and fluorescently-labelled goat anti-mouse (Amersham
Biosciences) as the
secondary antibody. Fluorescent microscopy images were taken of the gels at a
magnification of
times and a montage was created to show the extension. The numbers of nerves
extending
150 m, 300 m, 450 m and 600 m were counted as a measure of neurite extension.
Results
Collagen gel preparation
[0077]All the dendrimer crosslinked collagen gels before and after YIGSR
peptide modification
were transparent and strong enough to manipulate. The gels were stable when
stored in
PBS/water at 4 C for at least 8 months.
Covalent attachment of peptides to dendrimers and characterization
[0078]YUJSR and negative control YISGR were attached to dendrimers using the
same reaction
as was used for dendrimer-mediated collagen crosslinking. The carboxylic acid
groups in the
peptides were activated by EDC and NHS to form reactive NHS esters, which
reacted with
amine groups in dendrimers to form chemical bonds.
[0079]The reaction between the dendrimers and the peptides was confirmed by H-
NMR and
MALDI TOF. H-NMR spectra of dendrimer, YIGSR and YIGSR-modified dendrimer are
shown
in Figure 14. Characteristic peaks from dendrimers (2.29-2.47ppm) and YIGSR
(6.62-6.9 1 ppm)
were fourid in the purified YIGSR modified dendrimer spectra, which indicated
the successful
attachmeiit of YIGSR to dendrimers. The estimated molar ratio of
YIGSR:dendrimer was found
to be 1:5.4. Therefore, compared with the initial molar ratio of
YIGSR:dendrimer (1:1), it is
estimated that only 18.5% of the initial YIGSR present was attached to the
dendrimers after
reaction and purification. Assuming all the YIGSR-modified dendrimers are
involved in the
crosslinking reaction of collagens, the maximum YIGSR content in the collagen
gels would be
expected to be 1.6X 10-2 mg/mg collagen.
HAM LAW\ 156340\1 27
CA 02569904 2006-11-30
[0080]MALDI mass spectra of dendrimer, YIGSR and YIGSR-modified dendrimer
further
confirmed the formation of YIGSR-modified dendrimer (Figure 15). Peaks for the
dendrimer
(773.7) and YIGSR (595.3) as well as for the YIGSR-modified dendrimer (1354.1)
were found
in the spectra as expected. Additional peaks present (1186, 1086, 1029, 955)
are thought to result
from deposition of the chemically-attached YIGSR due to its thermally labile
nature [34].
Incorporation of peptides in collagen gels and characterization of peptide
modified gels
[0081 ] 125I labeled YIGSR were used to quantify the amount of YIGSR
incorporated within the
collagen gels. It was found that 3.1-3.4X 10-2mg of YIGSR/mg collagen could be
incorporated,
suggesting that 24 to 26% of the initial YIGSR present was attached to
collagen. This result is
consistent with the estimate from the H-NMR analysis of the peptide-modified
dendrimers.
Gel Characterization
[0082]Surfaces of collagen gels before and after YIGSR modification were
examined by XPS.
Not unexpectedly, there were no significant differences (data not shown),
which demonstrates
that the reaction with the peptide-modified dendrimers did not adversely
affect the surface
properties of the gels. Denaturation temperatures of the collagen gels were
determined by DSC
to determine whether changes in the crosslinking density occurred with YIGSR
modification.
Multiple denaturation temperature peaks were found in YIGSR-modified collagen
samples
similar to the previously examined dendrimer-crosslinked collagens [23]. As
shown in Table 3,
slightly lower denaturation temperatures were found in YIGSR-modified collagen
samples,
indicating; the possible interference of YIGSR with the dendrimer-mediated
crosslinking reaction
resulting in a lower crosslinking density. Possibly due to this, slightly
poorer mechanical
properties in terms of modulus were observed in the YIGSR-modified collagens
as shown in
Figure 16a. However, they had a similar maximum load to the unmodified
dendrimer-crosslinked
collagens (Figure 16b).
Table 3
Collagens Denaturation temp. peakl( C) Peak2 ( C) Peak3 ( C)
YIGSR-modified 56 59.8 78.2
HAM_LAW~ 156340\1 28
CA 02569904 2006-11-30
Control 40 82 89
Surface modification of collagen gels with YIGSR
[0083]The surface coverage of YIGSR on the collagen surfaces was in the range
of 88.9-95.6
gg/cmz. While this accounts for only 5-6% of the maximal YIGSR coverage
calculated
theoretically from the availability of amine groups, it is much higher than
the densities 2.5x 10-5
g/cm2 reported in other studies previously [35].
In vitro corneal epithelial cell culture
YIGSR-bulk modified collagens
[0084]Representative photographs of human comeal epithelial cells (HCEC) on
YIGSR bulk
modified/unmodified collagen gel surfaces at 120 minutes are shown in Figure
17. It was found
that the ce:lls adhered to all of the collagen surfaces within 30 to 60
minutes. Furthermore,
morphology changes were observed in all cases. In comparison, the cells did
not adhere to the
control tissue culture plates and remained round and non-adherent after 2
hours of culture. The
presence of YIGSR resulted in the formation of clusters and visibly greater
levels of cell
attachment. Over longer periods of time, there was a trend showing that the
cells proliferated
faster on collagen gels with higher YIGSR content as shown in Figure 18 and
19. This trend was
confirmed by Cyquant assay as shown in Figure 20.
YIGSR-surface modified collagens
[0085]Similar to the YIGSR-bulk modified collagens, the cells adhered to all
collagen surfaces
within 60 minutes and changed morphology. However, as shown in Figure 19, over
a period of
1 week of culture, there was no significant improvement in the adhesion and
growth of the cells
on these surfaces relative to the unmodified collagen gels.
Dorsal Root Ganglia Neurite Extension
[0086]Neurite extension from DRG cells was found to be significantly enhanced
by the presence
of the YIGSR in the collagen gels. The length of the neurites and number of
neurites,
summarized in Figure 21, was significantly (p<0.05) enhanced by the presence
of the cell
HAM_LAW\ 156340\l 29
CA 02569904 2006-11-30
adhesion peptide (see Figure 22). Surprisingly, there was little or no effect
of peptide
concentration in the gel, although it is possible that the surface density of
the peptide on these
surfaces was relatively similar as this was a bulk modification. Visually, it
is clear that the nerve
density on these materials was also enhanced by the presence of the peptide.
Conclusions
[0087]The YIGSR peptide sequence of laminin was either chemically incorporated
into the bulk
structure of collagen gels or attached onto collagen gel surfaces by way of
dendrimers. The
structure of YIGSR-modified dendrimer was confirmed by H-NMR, MALDI mass
spectra and
the amourit of YIGSR incorporated in collagen was determined by 125 1
radiolabelling. The
incorporation reaction was carried out under mild aqueous conditions at room
temperature and
the amourrt of peptide incorporated can be tuned by varying reaction
conditions such as the
percentage of peptide modified dendrimers in crosslinker solutions for the
collagens. The
crosslinking density of the collagen gels was slightly affected by the
incorporated YIGSR,
resulting in small decreases of the modulus of the gels. However, the overall
mechanical
properties of the gels was not significantly altered. The incorporated YIGSR
peptide promoted
the growth of the corneal epithelial cells on collagen gel surfaces in both
terms of adhesion and
proliferation. As well, neurite extension and nerve cell density was enhanced
on these materials
relative ta unmodified or control peptide modified controls, although no
effect of peptide
concentration was observed.
HAM_LAW\ 156340\1 30
CA 02569904 2006-11-30
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