Activated dopamine derivatives as primers for adhesive-patch fixation of bone fractures

Article abstract of DOI:10.1039/c5ra23142f

For the stabilization of complex bone fractures, tissue adhesives are an attractive alternative to conventional implants, often consisting of metal plates and screws whose fixation may impose additional trauma on the already fractured bone. This study reports on the synthesis and evaluation of activated dopamine derivatives as primers for fiber-reinforced-adhesive patches in bone-fracture stabilization strategies. The performance of synthesized dopamine derivatives are evaluated with regard to the adhesive shear strength of formed bone patches, as well as cell viability and surface properties. Dopamine-derived primers with methacrylamide, allyl, and thiol functional groups were found to significantly increase the adhesive shear strength of adhesive patches. Furthermore, deprotonation of the primer solution was determined to be essential in order to achieve good adhesion. In conclusion, the primer solutions that were found to give the best adhesion were the once where dopa-thiol was used in combination with either dopa-methacrylamide or dopa-allyl, resulting in shear bond strengths of 0.29 MPa.

Full text of DOI:10.1039/c5ra23142f

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PAPER
Activated dopamine derivatives as primers for
adhesive-patch fixation of bone fractures†
Cite this: RSC Adv., 2016, 6, 26398
*
K. Olofsson, V. Granskog, Y. Cai, A. Hult and M. Malkoch
For the stabilization of complex bone fractures, tissue adhesives are an attractive alternative to conventional
implants, often consisting of metal plates and screws whose fixation may impose additional trauma on the
already fractured bone. This study reports on the synthesis and evaluation of activated dopamine derivatives
as primers for fiber-reinforced-adhesive patches in bone-fracture stabilization strategies. The performance
of synthesized dopamine derivatives are evaluated with regard to the adhesive shear strength of formed
bone patches, as well as cell viability and surface properties. Dopamine-derived primers with
methacrylamide, allyl, and thiol functional groups were found to significantly increase the adhesive shear
strength of adhesive patches. Furthermore, deprotonation of the primer solution was determined to be
essential in order to achieve good adhesion. In conclusion, the primer solutions that were found to give
the best adhesion were the once where dopa-thiol was used in combination with either dopa-
methacrylamide or dopa-allyl, resulting in shear bond strengths of 0.29 MPa.
Received 4th November 2015
Accepted 3rd March 2016
DOI: 10.1039/c5ra23142f
www.rsc.org/advances
continue to motivate research in this area. For more load
bearing applications, the adhesive can be improved by the
Introduction
formation of
a ber reinforced adhesive patch (FRAP),
Conventional methods of stabilizing complex bone fractures
rely on implants comprising metal-based plates that are applied
by open surgery and xated with pins and screws. While
conventional methods are effective, limitations concerning
accessibility, stress shielding and patient discomfort have to be
considered. In the development of new minimally-invasive
fracture-stabilization techniques, cross-linked bone adhesives
are promising candidates.1 Since adhesives do not require
drilling, fracture xation can be achieved via minimally invasive
surgery, enabling treatment of fractures where conventional
implants are unsuitable. With adhesives, it is possible to obtain
a more homogeneous weight-bearing load between bone frag-
ments than when pins and screws are utilized.1 Many materials
evaluated as potential bone adhesives have been originally
developed for other areas, such as dentistry and so
tissue adhesives, and were thus originally not designed as
adhesives for bone. Cyanoacrylates, urethanes, and alkylene
bis(oligolactoyl)-methacrylates, as well as a number of dental
cements and brin-based adhesives that are already in use as
sealants or adhesives for so tissues or in dentistry, are prom-
ising candidates that are still unsuitable as bone adhesives due
to problems with e.g. toxicity, biodegradability, insufficient
mechanical strength, or unsatisfactory stability in wet envi-
ronment.2–6 The foreseen advantages of using adhesives
comprising structural bers embedded in a cross-linked adhe-
sive matrix applied outside of a fracture.7,8 FRAP xation makes
it possible to tune the properties of the patch according to the
loading conditions for each specic fracture by manipulating
the number of ber layers or increasing the adhesion area to
allow for adjustments of the nal strength of the patch.9 The
FRAP-xation approach, with patching outside of a fracture, is
furthermore believed to interfere less with the natural bone
healing process, than if adhesive is applied directly in the
crevice of a fracture. To facilitate the surgical procedure, curing
upon external stimuli such as e.g. UV or high-energy-visible
(HEV) light is desirable. In the pursuit of a suitable matrix for
FRAP, the thiol–ene coupling (TEC) reaction between thiols and
unsaturated bonds is envisioned as a promising cross-linking
strategy. Aspects promoting the use of the TEC cross-linking
in surgical procedures are: insensitivity to oxygen inhibition,
selectivity, and possibilities of cross-linking using minimally
invasive optical bers.10
We have previously shown that the triazine-based-building
blocks;
tris[2-(3-mercaptopropionyloxy)ethyl]isocyanurate
(TAT) and 1,3,5-triallyl-1,3,5-triazinane-2,4,6-trione (TAA), with
thiol and allyl functional groups respectively, can be cross
linked into
a promising matrix for adhesion to bone
substrates.11 To further improve the adhesion of the cross
linked triazine-based matrix (TA-matrix), primers utilizing the
remarkable adhesive abilities of marine mussels were envi-
sioned. Marine mussels secrete byssal threads built up of so
called mussel foot proteins (mfps) that are able to bind strongly
KTH Royal Institute of Technology, Fibre and Polymer Technology, Teknikringen 56-58,
Stockholm, SE-10044, Sweden. E-mail: malkoch@kth.se
† Electronic supplementary information (ESI) available: Calculations of surface
free energy and supporting gures and tables. See DOI: 10.1039/c5ra23142f
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Scheme 1 Synthetic pathways to dopamine derivatives: dopamine 1, dopa-allyl 2, dopa-methacrylamide 3, dopa-thiol 4, dopa-alkyne 5, and
dopa-sp-allyl 6.
to various substrates both in aqueous and dry environments.12 improve the adhesion between the triazine-based matrix
The adhesive properties of mfps is associated with the (TA-matrix) and bone. A library of dopamine derivatives was
sequences expressing the amino acid 3,4-dihydroxyphenyl- carefully synthesized to include two important features; (1)
L-alanine (DOPA).13–16 The ability to form strong hydrogen bonds unsaturated or thiol functionalities that enable the formation of
and the reported affinity toward hydroxyapatite, an abundant covalent bonds between primer and matrix and (2) DOPA
compound in bone, has in combination with the unique cohe- groups to enhance the adhesion to the bone through e.g.
sive strength of DOPA in aqueous environments resulted in hydrogen bonding. This was achieved by activating the primary
several reports with focus on DOPA-based compounds in the amine of dopamine via amidation reactions, enabling the
development of adhesives.14,17–24 Most of these reports have anchorage of allyl, methacrylamide, thiol, and alkyne-
focused on DOPA-based adhesives for so tissues and other functional groups, according to Scheme 1. The synthesis of
substrates, but little work has so far been done on evaluating primers was straight forward with acceptable yields aer puri-
the materials as bone adhesives.22,24 Herein, we present an cation and the structure was conrmed using NMR spectros-
alternative approach for the fabrication of E-glass-ber- copy. Evaluation of the performance of the derivatives as
reinforced adhesive thiol–ene patches for the xation of bone primers was conducted via formation of FRAPs for bone-
fractures, exploring the use of derivatives of dopamine as fracture stabilization. The primers were furthermore evaluated
adhesive primers.
with respect to cell viability and surface properties.
Cytotoxicity of dopamine derivatives
Results and discussion
In developing materials intended for us in the medical eld, it is
In previously published results we showed that the triazine- important to investigate the viability of cells in contact with the
based-building blocks; tris[2-(3-mercaptopropionyloxy)ethyl] material. Primer toxicity toward human dermal broblasts
isocyanurate (TAT) and 1,3,5-triallyl-1,3,5-triazinane-2,4,6- (hDF) was thus evaluated using alamarBlue® Assay, where
trione (TAA), with thiol and allyl functional groups respec- primer solutions with concentrations of 0.1–1000 mg mlꢀ1 were
tively, could successfully be cured into a matrix with promising added to hDF-cell culture. Cell viability, indicated by the
adhesion to bone substrates.11 In light of these results, activated metabolic activity from the alarmarBlue® Assay, decreased with
dopamine derivatives were envisioned as promising primers to increasing concentration of dopa-primer and dose limitations
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were calculated according to ISO 10993-5 standards, in which TA-matrix than the other primers. Combined with a lower
materials causing cell-viability reduction by less than 30% are hydrophilicity of the dopa-methacrylamide surface compared to
considered to be non-cytotoxic.25 Dose limitations of DOPA- the other primers, as measured by CA, it was expected that
primers were 2.5 mg mlꢀ1 (10.6 mM) for dopa-allyl, 30 mg mlꢀ1 dopa-methacrylamide would have a stronger interaction with
(136 mM) for dopa-methacrylamide, 45 mg mlꢀ1 (237 mM) for the TA-matrix than the other primers and thereby result in
dopamine, 90 mg mlꢀ1 (309 mM) for dopa-alkyne, 150 mg mlꢀ1 stronger adhesion.
(587 mM) for dopa-thiol, and 250 mg mlꢀ1 (423 mM) for dopa-sp-
allyl, and thus affected by small differences in the structures of
Fiber-reinforced-adhesive-patch (FRAP) xation of generic
the primers. These dose limitations are in the same range as
bone fractures
values reported by Ben-Shachar et al. in 2004, who reported that
A FRAP with a thiol–ene coupled matrix in combination with the
adhesive properties provided by a dopa-functional primer and
the load-bearing advantages of bers, was foreseen as a robust
strategy for bone-fracture xation. Other studies on dopa-
functional materials as bone adhesives have shown that these
materials have potential,22,24 why dopa-functional compounds
were foreseen as promising primers for the FRAP-xation
approach used herein. Differences in formulations,
substrates, test setup and other conditions between the studies,
however make comparisons of actual values from the different
studies inconclusive. The commercial dental primer thiol–ene
was therefore used herein for comparison.
extracellular dopamine could cause cell death at concentrations
above 200 mM.26 The in vitro elution test for the TA-matrix
showed no signs of toxicity, with cell-viability values of 111% ꢁ 9.
Surface properties
The idea of a primer is to improve the interactions between
a substrate and an adhesive through the application of a thin
intermediate layer of primer. The low-molecular-weight primers
synthesized herein had slightly different molecular structures
and different functional end groups believed to be able to
interact covalently with the TA-matrix. For this to occur good
wetting is essential and an evaluation of the surface properties of
both primers and TA-matrix was therefore foreseen as a means
to predict the performance of the primers. To evaluate the
surface properties of primer surfaces, circular glass substrates
(15 mm in diameter) were spin-coated with primer solution
(5 mL, 84.5 mM) and analyzed using static contact-angle (CA)
measurements. CAs were measured for water, ethylene glycol,
and diiodomethane to enable calculations of surface free energy
according to the van Oss-Chaudhury-Good (OCG) theory.27 CAs
showed that all primers were signicantly more hydrophilic than
the cross-linked TA-matrix (Table 1). Whereas the TA-matrix had
a water CA of 86ꢂ, all primers, apart from dopa-methacrylamide
and dopamine, displayed a water CA around 20ꢂ. Dopa-
methacrylamide, however, displayed a water CA of 55ꢂ, much
more similar to that of the TA-matrix. Dopa-methacrylamide and
the TA-matrix also had signicantly more similar CAs for
ethylene glycol than any of the other substrates.
Due to large variations between bone samples from e.g.
different animal species, anatomical sites, and individuals, it is
difficult to get a representative model substrate in vitro. To
minimize inuence of external factors and focus this investi-
gation on the adhesion between the bone and the patch, bone
pieces with sizes of 50 ꢃ 15 ꢃ 3 mm were cut from bovine
marrow pipe and simple generic fractures were created using
a bow saw by dividing the bone pieces in two. The bone
substrates were furthermore sanded to minimize inuence of
differences in surface roughness and patches spanning across
the fracture were formed in layers on a 10 ꢃ 15 mm area on
each side of the fracture (Fig. 2a). 4 mL aqueous primer solution
(84.5 mM) was applied to moist bone, followed by TA-matrix
alternated with ve layers of E-glass bers. The matrix layers
were cured using HEV light with a total dose of 142 kJ cmꢀ2
Patches were prepared on both sides of each bone sample to
.
Additionally, surface free energy was determined for the
TA-matrix and for all primer systems according to the van Oss-
Chaudhury-Good theory27,28 (ESI†). It was found that the surface
free energy of dopa-methacrylamide was more similar to the
Table 1 Contact angles of the cured TA-matrix and FRAP-primer
surfaces measured for water, ethylene glycol, and diiodomethane
CA (water) CA (ethylene CA (diiodomethane)
Surface
[ꢂ]
glycol) [ꢂ]
[ꢂ]
TA-matrix
86.1 ꢁ 1.6 53.5 ꢁ 0.6
5.4 ꢁ 1.1 12.5 ꢁ 0.4
20.3 ꢁ 1.0 27.8 ꢁ 2.1
23.7 ꢁ 0.8 13.9 ꢁ 1.7
17.8 ꢁ 0.3 13.0 ꢁ 0.5
28.8 ꢁ 1.3
44.0 ꢁ 0.8
43.0 ꢁ 0.7
36.5 ꢁ 0.4
39.4 ꢁ 0.6
37.5 ꢁ 3.1
37.0 ꢁ 2.0
Dopamine
Dopa-sp-allyl
Dopa-alkyne
Dopa-allyl
Fig.
1 Dose limitations of dopamine derivatives (defined as the
Dopa-methacrylamide 55.4 ꢁ 2.1 43.5 ꢁ 1.5
concentration where the cell viability of human dermal fibroblasts is
70%).
Dopa-thiol
19.1 ꢁ 2.2 12.1 ꢁ 1.6
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Fig. 3 Shear bond strength of FRAPs determined by tensile tests
(dental-adhesive CLEARFIL™ SE BOND used as a comparison).
dopa-methacrylamide showed stronger shear bond strengths
than FRAPs with dopa-allyl. Polarity differences between the
amide bonds might also have an effect, as the polarity would
inuence the secondary forces between the primer molecules
and/or the bone tissue.
Inuence of addition of NaOH. In order to investigate the
impact of NaOH addition, the patches displaying the highest
Fig. 2 Patch fabrication on bone specimen: (a) schematic simplified adhesive strength (i.e. the patches with dopa-methacrylamide
patch build up (only two fiber layers are shown even though five layers
of fibers were used in the study), (b) a schematic representation of
a stabilized bone specimen, and (c) a photograph of a FRAP-stabilized
bone specimen ready for testing.
and dopa-thiol) were also prepared without addition of NaOH.
The effect of NaOH on primer structure was investigated using
NMR analysis of a solution of dopa-methacrylamide prepared in
50% D2O in MeOH with a 1 : 1 molar ratio addition of NaOH to
dopa-groups. The 13C-NMR spectra clearly indicated a change in
generate a double lap-shear mode at testing and stored in PBS
buffer for 24 h before tensile tests were performed (Fig. 2b).
Inuence of dopa primers on adhesive shear strength of
FRAPs. The adhesive shear strength of the patches was evaluated
using lap-shear tests. Additionally, NaOH was added as an
additive to the primer solution to deprotonate the acidic phenols
and thereby increase the adhesive properties of the patches
through hydrogen bonding. The shear bond strengths calculated
from the lap-shear tests (Fig. 3), conrmed that activated
derivatives of dopamine could successfully be used as primers to
improve the shear bond strength of FRAPs. The shear-bond
strengths for the individual primer solutions with NaOH
added (Fig. 3 grey, no pattern) show that the primers performed
differently depending on their structure. Primers with alkyne,
allyl, methacrylamide, and thiol, all enhanced the adhesive
strength of formed FRAPs, compared to FRAPs prepared without
primer. Dopa-thiol and dopa-methacrylamide (0.18 and 0.17
MPa, respectively), gave the strongest patches, closely followed
by dopa-allyl (0.14 MPa). Compared to patches without primer,
patches with dopa-thiol and dopa-methacrylamide displayed
approximately two times as high bond strengths. The differences
between dopa-allyl and dopa-methacrylamide could possibly be
explained by differences in reactivity towards TAT in the TA-
matrix. Although dopa-allyl should have a higher reactivity
during TEC, dopa-methacrylamide should be more prone to
homo-polymerization,29 which might explain why FRAPs with
the structure of the catechol, as the peaks for the carbon atoms
attached to the hydroxyl groups shied from 143.8 and 145.3
ppm to 151.2 and 153.6 ppm (ESI, Fig. S3†). The other aromatic
carbons also shied slightly, while the rest of the peaks were
unaffected by the NaOH addition. The 13C-NMR spectra did not
imply oxidation from catechol to quinone, as that would have
resulted in peaks around 180 ppm30 and it was concluded that
the catechol group was deprotonated by the addition of NaOH.
Interestingly, the addition of NaOH to primer solutions did
indeed increase the adhesive shear strength of the patches 6.3
and 5.1 times, using primer solutions of dopa-methacrylamide
and dopa-thiol, respectively (Fig. 3).
Dopa-sp-allyl 6 was initially synthesized to evaluate the
impact of adding a spacer between the dopa-group and the allyl
group. Dopa-sp-allyl was envisioned to have better adhesion
than the shorter dopa-allyl primer, due to the increased
mobility brought by the tri(ethylene oxide) spacer. Additionally,
dopa-alkyne 5 was envisioned to improve adhesion due to the
possibilities of binding to more than one TAT molecule in the
matrix via thiol–yne chemistry (TYC). Unfortunately, these two
primers did not perform as good as anticipated (Fig. 3). In fact,
dopa-sp-allyl showed the lowest adhesion of all tested dopa-
mine derivatives, prompting further investigation. NMR spectra
of the dopa-alkyne primer before and aer addition of NaOH
(ESI, Fig. S4†), revealed that the increase in pH caused hydrolytic
cleavage of the ester bonds in the structure. The same sensitivity
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towards hydrolysis is expected for dopa-sp-allyl due to the pres- with a spectral window of 20 ppm, an acquisition time of 4 s,
ence of similar ester bonds. Degradation of the primers in this and a relaxation delay of 1 s. 13C-NMR spectra were acquired at
way aer addition of NaOH would explain why dopa-sp-allyl and 101 MHz with a spectral window of 240 ppm, an acquisition
dopa-alkyne did not increase the adhesion of FRAPs as expected, time of 0.7 s, and a relaxation delay of 2 s.
as the primers could no longer interact with both bone and
Static contact-angle measurements were performed on
matrix as envisioned. The signicant differences in the adhesion a CAM200 contact-angle meter (KSV Instruments, Helsinki,
of patches where the degraded primers were used compared to Finland), using the sessile-drop technique and an automatic
patches where primers with higher hydrolytic stability were used, dispenser. Contact angles were measured for water, ethylene
furthermore support the theory that the added functional groups glycol, and methyl diiodate at 23 ꢂC and at a relative humidity of
promoted interactions towards the matrix.
50%, using a drop volume of 5 ml for water and ethylene glycol,
Inuence of allyl-to-thiol ratio. Earlier published results, and a drop volume of 3 ml for methyl diiodate (the drop volume
showed that off-stoichiometry between allyl and thiol groups in had to be decreased to 3 ml because of the low surface tension of
the TA-matrix can inuence the properties of the cured matrix.31 the liquid). OneAttention soware, using Laplace tting, was
The efficiency of the HEV-induced crosslinking of an equimolar used for calculating the contact angles at three different posi-
mixture of TAA and TAT was monitored by FT-Raman analysis. tions for each substrate. Surfaces for contact-angle measure-
The disappearance of the thiol and allyl peaks, at 2574 and 1645 ments were prepared via spin coating of the primer solutions on
cmꢀ1 respectively, showed that the matrix could be efficiently glass substrates.
cured using HEV-light irradiation (ESI, Fig. S5†). Adding a layer
Curing was performed using a lamp intended for light-cured
of dopa-thiol underneath the matrix, however, resulted in dental materials, Bluephase® 20i, predominately radiating HEV
reduced curing efficiency of the matrix (ESI, Fig. S6†), where light with wavelengths of 410 nm and 470 nm. Two different
small peaks corresponding to both allyl and thiol groups were irradiation programs were used: the TURBO program (2000 mW
visible even aer curing. This could thus inuence the adhesive cmꢀ2) and the HIGH POWER program (1200 mW cmꢀ2).
bond strength of the patches and an ene-to-thiol ratio of 1.1
All mechanical tests were performed on an Instron 5566
should thus be preferred. The best results were in fact achieved static-testing machine with a 10 kN load cell and a cross-head
when dopa-thiol was used in combination with either dopa-allyl speed of 5 mm minꢀ1, using a preload of 1 N and a pre-load
or dopa-methacrylamide, both combinations showing shear speed of 2 mm minꢀ1. All measurements were conducted at
bond strengths of 0.29 MPa, more than 1.6 times higher than 23 ꢂC and at a relative humidity of 50%. Samples were accli-
for the individual primer solutions (Fig. 3). With ene and thiol matized to the testing temperature prior to measurements and
combined, both compounds in the primer solution had a dopa kept in PBS buffer as long as possible before being attached to
group able to interact with the bone and a functional group able the machine. Data was collected using Bluehill soware.
to interact with the matrix, while maintaining an ene-to-thiol
ratio of 1 : 1 in the entire primer-matrix system.
Synthesis of dopamine derivatives
The combination of dopa-thiol with either dopa-
methacrylamide or dopa-allyl interestingly gave adhesive The synthesis paths for all primers used in this study are dis-
strengths similar to that of the commercially available dental played in Scheme 1.
primer: CLEARFIL™ SE BOND. Statistical evaluation (ESI, Table
Synthesis of N-(3,4-dihydroxyphenethyl)pent-4-enamide –
S3†) even showed that there was no signicant difference dopa-allyl (2). Dopamine hydrochloride 1 (8.00 g, 42.1 mmol)
between the dopa-thiol/dopa-allyl combination and CLEARFIL™ was dissolved in a solution of dimethyl sulfoxide (DMSO) (40.0
SE BOND. It should however be noted that, when CLEARFIL™ ml) and triethylamine (TEA) (7.07 ml, 50.7 mmol). 4-Pentenoic
SE BOND is used in dentistry, another sample-preparation anhydride (6.94 ml, 38.0 mmol) was slowly added, aer which
procedure is used, which may inuence the results.
the reaction was allowed to proceed under agitation overnight.
Diethyl ether (1000 ml) was added and the mixture was extrac-
ted four times with 100 NaHSO4(aq) (10 w%). The organic phase
was dried over MgSO4 and subsequently ltered and the solvent
removed under reduced pressure. Further purication was
carried out by ash chromatography using ethyl acetate and
heptane to give 2 as a white solid (4.84 g, 54% yield).
Experimental
Materials
All chemicals were purchased from Sigma Aldrich and used as
received, unless otherwise specied. E-glass fabric (25 g mꢀ2
,
1H NMR (400 MHz, CD3OD) d 6.67 (d, 1H, Ar), 6.62 (d, 1H,
Ar), 6.51 (dd, 1H, Ar), 5.76 (m, 1H, –CH]), 4.98 (dd, 2H, ]CH2),
3.31 (t, 2H, –CH2–), 2.61 (t, 2H, Ar-CH2–), 2.29 (m, 2H, –NHCO–
CH2–), 2.21 (m, 2H, –CH2–CH]); 13C NMR (101 MHz, CD3OD)
d 175.3, 146.2, 144.7, 138.2, 132.0, 121.0, 116.8, 116.3, 115.8,
42.2, 36.4, 35.9, 31.0 (M ¼ 235.28 g molꢀ1).
nish FE 800, plain) was purchased from R&G Faserverbund-
werkstoffe GmbH and used as received. CLEARFIL™ SE Bond,
a commercial dental primer was purchased from Kuraray
America, Inc. All cell-culture reagents were purchased from
Thermo Scientic™ HyClone™.
Synthesis of N-(3,4-dihydroxyphenylethyl)-methacrylamide –
dopa-methacrylamide (3). Dopamine hydrochloride 1 (16.6 g,
Instrumentation
1H and 13C-NMR analysis were performed on a Bruker Avance 87.3 mmol), and TEA (14.8 ml, 106 mmol) were dissolved in
1
NMR instrument. H-NMR spectra were acquired at 400 MHz DMSO (80.0 ml), aer which methacrylic anhydride (11.8 ml,
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79.4 mmol) was slowly added (Scheme 1). The reaction mixture
was thereaer le under agitation for 90 min until all anhy-
dride had reacted, as analyzed with 13C-NMR. Diethyl ether
(1750 ml) was added and the mixture was extracted four times
with 200 ml NaHSO4(aq) (10 w%). The organic phase was dried
over MgSO4 and subsequently ltered aer which a small
spoon of methyl hydroquinone was added as inhibitor before
evaporation of the solvent. Further purication of the organic
phase was carried out by ash chromatography using ethyl
acetate and heptane. In addition, product from the water
phase was allowed to crystallize to give a total of 9.8 g of 3 as
a white solid (56% yield).
Synthesis of (1-(13-oxo-3,6,9-trioxa-12-azaheptadec-16-en-1-
yl)-1H-1,2,3-triazol-4-yl)methyl-4-((3,4-dihydroxylphenethyl)
amino)-4-oxobutanoate – dopa-sp-allyl (6). Dopa-sp-allyl 6 was
synthesized via copper(I)-catalyzed azide–alkyne cycloaddition
(CuAAC). Dopa-alkyne 5 (0.537 g, 1.84 mmol) and N-(2-(2-(2-(2-
azidoethoxy)ethoxy)ethoxy)ethyl)-pent-4-enamide (0.504 g, 1.68
mmol) were dissolved in 5.00 ml THF aer which sodium
ascorbate (66.0 mg, 0.335 mmol) and CuIISO4 (42.0 mg, 0.168
mmol) dissolved in 2.00 ml of DI water were added. The reaction
was heated to 40 ꢂC and stirred overnight. An additional amount
of dopa-alkyne (98.0 mg, 0.336 mmol) was added to ensure full
conversion of azide groups and the reaction was followed using
13C NMR. Aer complete conversion had been achieved, the
solvent was removed under reduced pressure, and 100 ml DCM
was added. The mixture was extracted four times with 10 ml
H2O, aer which the combined water phase was extracted with
an additional 50.0 ml DCM. The DCM phases were then
combined and dried over MgSO4, ltered, whereaer the solvent
was removed under reduced pressure. Further purication was
carried out using ash chromatography in ethyl acetate and
methanol to yield 0.222 g of the pure product (22.4% yield).
1H NMR (400 MHz, CDCl3) d 7.82 (s, 1H, triazole), 6.79 (d, 1H,
Ar), 6.70 (d, 1H, Ar), 6.53 (dd, 1H, Ar), 5.78 (m, 1H, –CH ]), 5.17 (s,
2H, –COO–CH2–triazole–), 5.05–4.96 (dd, 2H, ]CH2), 4.50 (t, 2H,
–triazole–CH2–CH2–O–), 3.84 (t, 2H, –triazole–CH2–CH2–O–), 3.60
(s, 8H, –O–CH2–CH2–O–), 3.53 (t, 2H, –O–CH2–CH2–NHCO–), 3.41
(m, 4H, Ar-CH2–CH2–NHCO– and –O–CH2–CH2–NHCO–), 2.63 (t,
4H, Ar-CH2– and –NHCO–CH2–CH2–COO–), 2.40 (t, 2H, –NHCO–
CH2–CH2–COO–), 2.35 (t, 2H, –CH2–CH2–CH]), 2.25 (m, 2H,
–CH2–CH2–CH]); 13C NMR (101 MHz, CDCl3) d 173.2, 172.8,
171.9, 144.6, 143.4, 142.5, 137.2, 131.0, 125.5, 120.6, 116.0, 115.8,
115.6, 70.6, 70.5, 70.3, 70.0, 69.4, 57.7, 50.5, 41.1, 35.9, 34.8, 31.0,
29.8, 29.7 (MW ¼ 591.66 g molꢀ1).
1H NMR (CD3OD, 400 MHz) d 6.68 (d, 1H, Ar), 6.65 (d, 1H, Ar),
6.52 (dd, 1H, Ar), 5.64 (s, H, ]CHH), 5.34 (s, H, ]CHH), 3.38 (t,
2H, –CH2–NH–), 2.64 (t, 2H, Ar-CH2–), 1.91 (s, 3H, –CH3); 13C
NMR (CD3OD, 101 MHz) d 172.1, 147.1, 145.6, 142.3, 133.0, 121.9,
121.1, 117.8, 117.2, 43.5, 36.8, 19.6 (MW ¼ 221.26 g molꢀ1).
Synthesis of N-(3,4-dihydroxyphenetyl)-4-mercapto-butanamide
– dopa-thiol (4). Dopamine hydrochloride 1 (25.0 g, 132
mmol), NaHCO3 (23.3 g, 277 mmol), and g-thiobutyrolactone
(12.5 ml, 144 mmol) were dissolved in 250 ml deionized
water. The reaction mixture was heated to 95 ꢂC and was
allowed to reux for 2 h before being cooled down to room
temperature. 250 ml brine was added and the product was
extracted two times with 625 ml THF. The organic phase was
dried over MgSO4, ltered, and the solvent evaporated under
reduced pressure. Further purication was carried out by
ash chromatography using ethyl acetate and heptane, which
gave 28.3 g of the product as a slightly yellow solid (84%
yield).
1H NMR (400 MHz, CD3OD) d 6.66 (d, 1H, Ar), 6.61 (d, 1H, Ar),
6.50 (dd, 1H, Ar), 2.61 (t, 2H, Ar-CH2–), 2.44 (t, 2H, –CH2–SH),
2.25 (t, 2H, –NHCO–CH2–), 1.82 (p, 2H, –CH2–CH2–SH); 13C
NMR (101 MHz, CD3OD) d 175.44, 146.40, 144.92, 132.11,
121.18, 116.98, 116.46, 42.33, 36.05, 35.69, 31.49, 24.59 (MW ¼
255.33 g molꢀ1).
Synthesis
of
tris[2-(3-mercaptopropionyl-oxy)ethyl]
isocyanurate (TAT). TAT was synthesized by mixing 1,3,5-tris(2-
hydroxyethyl)isocyanurate (25.0 g, 95.7 mmol), 3-mercaptopro-
pionic acid (91.0 g, 0.857 mol) and p-toluene sulfonic acid mon-
ohydrate (11.0 g, 57.8 mmol) in 800 ml toluene. The mixture was
heated to 125 ꢂC and the reaction was run for 2 h. The reaction
was monitored by NMR. The reaction solution was cooled down
to room temperature and washed with H2O, followed by washing
with NaHCO3(aq) (10 w%) 3 times. The organic phase was
collected and toluene was removed under reduced pressure.
Remaining material was dissolved in DCM and washed with
NaHCO3(aq) (10 w%) 4 times. The organic phase was collected and
dried over MgSO4, followed by evaporation of solvents. 44 g of the
product was obtained as a clear oil (88% yield).
Synthesis
of
prop-2-yn-1-yl-4-((3,4-dihydroxyphenethyl)
amino)-4-oxobutanoate – dopa-alkyne (5). Dopa-alkyne 5 was
synthesized according to a previously published procedure32 as
follows: dopamine hydrochloride 1 (5.00 g, 26.4 mmol) was
dissolved in a solution of DMSO (25.0 ml) and TEA (4.41 ml, 31.6
mmol), aer which 4-oxo-4-(prop-2-yn-1-yloxy)butanoic anhy-
dride (synthesized according to a previously published proce-
dure33) (6.98 g, 23.7 mmol) was slowly added (Scheme 1). Aer 1
h, diethyl ether (1000 ml) was added and the mixture was
extracted four times with 100 ml NaHSO4(aq) (10 w%). The
organic phase was dried over MgSO4 and subsequently ltered
and the solvent evaporated under reduced pressure. Further
purication was carried out by ash chromatography with ethyl
acetate and heptane to give 2.61 g of 4 as a white solid (38%
yield).
1H NMR (400 MHz, CDCl3) d 4.37–4.34 (t, 6H, –CH2–CH2–O–),
4.20–4.17 (t, 6H, –CH2–CH2–N–), 2.76–2.71 (t, 6H, –CH2–CH2–
SH), 2.63–2.60 (t, 6H, –CH2–CH2–COO–), 1.66–1.62 (t, 3H, –SH).
13C NMR (101 MHz, CDCl3) d 171.62, 149.05, 61.36, 42.16, 38.39,
19.62 (MW ¼ 527.64 g molꢀ1).
1H NMR (400 MHz, CD3OD/D2O) d 6.83 (d, 1H, Ar), 6.77 (d,
1H, Ar), 6.66–6.63 (dd, 1H, Ar), 4.71 (d, 2H, –CH2–C^), 3.38 (t,
2H, –CH2–NHCO–), 2.97 (t, 1H, ^CH), 2.67 (m, 4H, –NHCO–
CH2–CH2–COO– overlap), 2.51 (t, 2H, Ar-CH2); 13C NMR (101
MHz, CD3OD) d 174.1, 173.5, 146.4, 144.9, 132.2, 121.2, 117.0,
Cytotoxicity study
A
cytotoxicity study was performed on solutions of all
116.5, 78.9, 76.3, 42.5, 36.1, 31.4, 30.3 (MW ¼ 291.30 g molꢀ1). dopamine-derived primers as well as on eluents from the cured
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adhesive-matrix. Human dermal broblasts (hDF) were bone with sealing tape. Wet bone pieces were wiped with
cultured in complete growth medium (CGM), containing Dul- surgical pads before 4 ml of primer solution were applied on each
becco's Modied Eagle's Medium (DME/F12) comprising 10% moist bone piece and then allowed to dry until the bone surface
fetal bovine serum (FBS), penicillin (100 U mlꢀ1) and strepto- regained its moist state. Matrix resin and reinforcing biaxial E-
mycin (100 mg mlꢀ1). Cells were incubated at 37 ꢂC in glass ber sheets were then consecutively applied to build up
a humidied atmosphere with 5% CO2. CGM renewal was a patch, consisting of ve ber sheets imbedded in the matrix.
carried out once every three days. Cells were detached at 80% The rst matrix layer was irradiated with HEV-light for 15 s (2 kW
conuence with trypsin/EDTA and seeded in 48-well tissue- cmꢀ2), and subsequent layers were irradiated for 5 s each (2 kW
culture plates with 50 000 hDFs and 500 ml CGM in each well.
cmꢀ2). Aer all ve layers of bers and matrix had been applied;
Solutions of either dopamine, dopa-allyl, dopa- the patch was irradiated for an additional 2 ꢃ 30 s (1.2 kW
methacrylamide, dopa-thiol, dopa-alkyne, or dopa-sp-allyl cmꢀ2), resulting in an average total dose of 142 kJ cmꢀ2. Patches
were added to each well to achieve a concentration series of were prepared on both sides of each bone sample to generate
0.1–1000 mg mlꢀ1 for each primer. An elution test for the TA- a double lap-shear mode at testing, as can be seen in Fig. 1b.
matrix was performed according to ISO 10993-5. A cured
All specimens were submerged in PBS for 24 h aer the
sample of the TA-matrix was rinsed in 70% ethanol for sterili- FRAPs had been applied, aer which lap-shear tests were per-
zation and then washed three times with sterile PBS. The TA- formed to determine the adhesive shear strength of the patches.
matrix was then submerged in CGM in a tissue-culture plate Strings were used to attach the specimens to the machine in
ꢂ
for 24 h at 37 C and 5% CO2, with a weight to volume ratio of order to compensate for irregularities of the bone pieces and to
0.2 g mlꢀ1. 500 ml of extract medium was then added to each enable uniaxial deformation. Triplicates for each patch formu-
well in a 48-well tissue-culture plate and 50 000 hDFs were lation were used and the maximum bond shear strength of the
seeded to each well with extract medium.
FRAPs was calculated using eqn (1).
All cells were cultured for 24 h and the viability was deter-
mined using alamarBlue® Assay (Life Technology) according to
the instruction from the manufacturer. The metabolic activity
of cells, indicated by the production of resorun from non-
uorescent resazurin, was measured with a plate reader
(Tecan Innite® M200 Pro) with excitation wavelength at 560
nm and emission wavelength at 590 nm. hDFs cultured in CGM
were used as negative control to dene the viability of 1 and
CGM without addition of cells served as blank control to dene
a viability of 0. Each data point was made in four replicas.
s ¼ F/A
(1)
where s is the shear strength [Pa], F is the load at break [N], and
A is the total adhesive area [m2]. The total adhesive area was
measured aer the mechanical testing using a digital caliper.
Conclusions
Dopamine derivatives were herein synthesized and evaluated as
primers for adhesive stabilization of bone fractures to improve
the interaction between bone and matrix. Results showed that
the use of dopamine-derived primers improved the adhesion
between the bone and the patch and also that the addition of
NaOH to the primer solution was essential to achieve a good
adhesion. It was furthermore concluded that similarities in
contact angles and surface free energy could add some under-
standing into why there was a stronger interaction between
dopa-methacrylamide and the matrix compared to the other
primers. Results moreover showed that it was benecial to use
a combination of dopa primers with both thiol and allyl or
methacrylamide end groups. Aer combining the results from
tensile tests, CA measurements, and cell-viability tests, the
mixture with dopa-thiol and dopa-methacrylamide is consid-
ered to be the most promising primer solution of the ones
tested herein for this application.
Procedure for the preparation of ber reinforced adhesive
patches (FRAPs)
Sample preparation. Bone pieces with sizes of 50 ꢃ 15 ꢃ 1
mm were cut from bovine bone of marrow pipe using an electric
tile saw and a bow saw. The pieces were polished using a detail
sander to get at pieces. 3 mm holes were drilled at both distal
ends of the bone pieces to facilitate attachment to the Instron
5566 during mechanical testing. A fracture was simulated by
sawing the bone pieces straight off into two halves, aer which
the pieces were polished with P80 sandpaper followed by P320
sandpaper. All bone pieces were kept wet in PBS buffer (pH 7.4,
phosphate buffer 0.01 M, NaCl 0.154 M) until FRAPs were
prepared.
Primer-solution and matrix-resin preparation. 3.38 ꢃ 10ꢀ5
mol primer was dissolved in 400 ml of deionized water to
a concentration of 84.5 mM. In primer solutions where NaOH
was used, NaOH (2.7 mg, 6.76 ꢃ 10ꢀ5 mol) was added to the
primer solution.
The matrix resin was prepared with equimolar amounts of
TAT and 1,3,5-triallyl-1,3,5-triazinane-2,4,6-trione (TAA). 0.1 w%
of the photo initiator camphor quinone was additionally added
to the resin to enable HEV-curing.
In conclusion, the use of primers derived from dopamine,
made it possible to enhance the adhesion of FRAPs and resulted
in FRAPs with shear strength values up to around 0.3 MPa,
comparable with the commercially used dental primer
CLEARFIL™ SE BOND.
Acknowledgements
FRAP-formation and testing. An area of 10 ꢃ 15 mm was Wilhelm Beckers Jubileumsfond is acknowledged for nancial
dened on all bone pieces by covering the other parts of the support. Additionally, Professor Mats Johansson at the
26404 | RSC Adv., 2016, 6, 26398–26405
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department of Fiber- and Polymer Technology at KTH Royal 17 W. M. Chirdon, W. J. O'Brien and R. E. Robertson, J. Biomed.
Institute of Technology in Stockholm, Sweden, is acknowledged
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19 H. Lee, N. F. Scherer and P. B. Messersmith, Proc. Natl. Acad.
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RSC Adv., 2016, 6, 26398–26405 | 26405