Surface modification of biomaterials for conjugation with human fetal osteoblasts

Article abstract of DOI:10.2533/chimia.2013.213

Surface functionalization of hydroxyapatite (HA) and ss-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible

Full text of DOI:10.2533/chimia.2013.213

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 213
doi:10.2533/chimia.2013.213
Chimia 67 (2013) 213–217 © Schweizerische Chemische Gesellschaft
Surface Modification of Biomaterials
for Conjugation with Human Fetal
Osteoblasts
Françoise Borcard§a, Phally Konga, Céline Journota, Davide Staedlera, Philip N. Sturzeneggerb,
Franziska Krauss Juilleratb, Urs T. Gonzenbachb, Lucienne Juillerat-Jeanneretc, and Sandrine
Gerber-Lemaire*a
§SCS-Metrohm Foundation Award for best oral presentation
Abstract: Surface functionalization of hydroxyapatite (HA) and β-tricalcium phosphate (TCP) bioceramics with
chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to
the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts
resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology
in the preparation of cell-engineered bone implants.
Keywords: Cell adhesions · Copper-free click reaction · Functionalized biomaterials · Human fetal osteoblasts
surgery with its increased risk of infection, Staudinger ligation with triarylphos-
pain, donor site morbidity, disease trans- phines and dipolar cycloadditions to al-
Introduction
mission and rejection (in the case of al- kynes, under mild reaction conditions.[8,9]
lografts), as well as monetary aspects call In this study, copper-free [3 + 2] dipolar
for the development of synthetic bone graft cycloaddition (click reaction)[10] was in-
materials which today are used in only vestigated to form a covalent linkage be-
15% of cases.[1] Recent advances in the un- tween modified FsOs and biomaterials.[11]
Over the last decade, bone-related
diseases such as osteogenesis imperfecta,
osteoarthritis, osteomyelitis, and osteo-
porosis, traumatic injuries and orthope-
dic surgeries have dramatically increased.
derstanding of the chemical and biological We disclose herein the synthesis of a chem-
The reasons for this drastic development
processes of bone tissue regeneration have ical ligand containing an activated alkyne
led to a new generation of synthetic bone for the copper-free click reaction and its
substitute materials that show good poten- ligation to azido-modified FsOs. The cy-
can be found in the ageing of the popula-
tion and the democratization of high-risk
sports.[1] The current gold standard to treat
tial for the treatment of large bone defects. totoxicity of this reaction for the cells was
One of the biggest challenges in bone also evaluated for several days. We also
tissueengineeringisthecompletecoloniza- determined whether the adhesion of FsOs
tion of the graft biomaterial by osteoblasts, to bioceramics of different compositions
especially in the inner area of implants. – hydroxyapatite (HA) and β-tricalcium
Due to the lower availability of nutrients phosphate (TCP) – can be improved by
and oxygen, the cells present in the inner chemical functionalization of the scaffold
large bone defects is still autologous iliac
crest graft.[2] Another option for patients
and surgeons is the use of allograft-based
bone tissue from other humans and in most
cases from cadavers. Many orthopedic al-
lograft procedures have been approved by
the FDA and applied for years in hospitals.
Success in both allografts and autografts
results from the physical and biological
similarity between the donor and host tis-
sue.[3] Nevertheless, the need for a second
area migrate to the surface. To overcome
with a specific ligand allowing the copper-
this problem we propose to establish a co- free click reaction with FsOs.[12]
valent linkage between implant materials
and human fetal osteoblasts (FsOs) allow-
ing a better colonization of the scaffold. Results
These cells have an interesting potential
for therapeutic use in bone tissue engineer- Synthesis of the Chemical Ligand
ing, due to their rapid growth rate, their for the Copper-free Click Reaction
ability to differentiate in vitro into mature
The synthesis of activated cyclooc-
osteoblasts and their histocompatibility.[4,5] tyne, DIBO, was performed according to
Duringthelastdecade,thedevelopment the published data.[13] After activation with
of bioorthogonal ligation has improved the 4-nitrophenyl chloroformate to give car-
possibility to study cellular processes.[6] bonate 2, the resulting intermediate was
Among the chemical motifs developed treated with derivative 3 containing a tet-
for these reactions, azide appears as raethyleneglycol chain required for higher
one of the most versatile bioorthogo- solubility in biological medium and a bio-
nal reporters due to its stability under tin label for the detection with a fluores-
physiological conditions and its small cent streptavidin to afford the model ligand
size that has generally only minimal ef- 4 used for further evaluation of the copper-
fects on the structure of the tagged free click reaction on azido-modified FsOs
molecules.[7] Azides can react through (Scheme 1).
*Correspondence: Dr. S. Gerber-Lemairea
Tel.: +41 21 693 93 72
E-mail: Sandrine.Gerber@epfl.ch
aInstitute of Chemical Sciences and Engineering
EPFL, CH-1015 Lausanne
bNonmetallic Inorganic Materials
ETHZ, CH-8093 Zürich
cUniversity Institute of Pathology
CHUV-UNIL, CH-1011 Lausanne

214 CHIMIA 2013, 67, Nr. 4
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Scheme 1. Synthesis
Synthesis of the Chemical Ligand
for the Copper-free Click Reaction
on Biomaterials
O
Cl
of the model linker for
the copper-free click
reaction on FsOs.
O
H N
N
H
2
O
O
3
O
3,4,5-Triacetoxy benzoic acid (5) was
S
O
3
O
H
first converted into the corresponding acyl
chloride by reaction with oxalylchloride in
the presence of DMF and then coupled to
N-Boc-1,6- hexanediamine to afford in-
termediate 6. After cleavage of the carba-
mate under acidic conditions, the resulting
amine 7 reacted with activated DIBO 2 in
thepresenceoftriethylamine. Deprotection
of the three hydroxyl groups under basic
conditions provided the desired compound
8 which was further used for the function-
alization of the biomaterial and the click
reaction with FsOs (Scheme 2).
NO2
H
HN
NH
NEt3,DMF,
DIPEA, PyBOP
H
O
8
8 %
O
N 2
O
1
2
CH2Cl2,20%
O
O
O
HN
H
O
NH
O
N
H
N
H
3
S
H
Scheme 2. Synthesis
of the ligand for the
copper-free click
reaction on bioma-
terials.
H
N
H
O
NH2
O
N
N
H
B
o
c
O
OH
6
6
1. (C
O
Cl) ,DMF in D
CM
4
N
H
Cl in dio
xane
2
Copper-free Click Reaction with
FsOs
3
0 %(3ste s)
p
NHBoc
2.
H N
AcO
OAc
Ac
O
OAc
2
Ac
O
OAc
O
Ac
O
Ac
O
5
Ac
NEt3,DCM
Compound 4 presenting activated cy-
clooctyne can react with azide moieties
without copper catalyst since the loss of
the ring strain (18 kcal/mol) and the ad-
ditional strain imposed by two aromatic
rings decrease the activation energy of the
click reaction. In order to express azido-
modified proteins, human fetal osteoblasts
were cultured in the presence of the non-
natural amino acid l-azidohomoalanine
(AHA) to insert an azide moiety in cel-
lular proteins, without cell alterations.[8]
Copper-free click reaction of compound
4 (Fig. 1A–D) with azido-modified FsOs
was performed at room temperature for
1 h in culture medium without FCS, con-
taining additional HEPES (20 µM) for
buffering. Cell nuclei were labeled with
DAPI and the cell functionalization was
monitored by fluorescence microscopy
using Cy3-streptavidin. In the absence of
preincubation of cells with AHA no label-
ing was detected. Ligand 4, containing an
activated alkyne moiety, allowed fast click
reactions with azido-modified FsOs at the
concentration of 20, 50 and 100 µM in the
incubation medium (Fig. 1).
6
7
1. 2,N 3
Et ,DMF
O
O
2:
7
O
O
H
N
O
O
2. LiOH, THF/H O
7 %(2steps2)
N
H
1
8
N 2
O
H
O
OH
OH
A
B
D
C
Evaluation of the Cytotoxicity of
the Copper-free Click Reaction on
FsOs
Fig. 1. Copper-free click reactions performed on FsOs. (A) To evaluate
the non-specific binding, FsOs without preincubation with AHA were
exposed to biotin-containing compound 4, then to fluorescent Cy3-
streptavidin to label biotin and DAPI to label cell nuclei. (B–D) FsOs were
The biocompatibility of this reaction
was evaluated by monitoring the cell vi-
ability using the WST-1 assay of func-
preincubated with AHA and subsequently exposed to compound 4 (20,
tionalized FsOs for up to 144 h after the
chemical reaction (Fig. 2). No cytotoxicity
on FsOs was observed for the copper-free
50, 100 µM) for 1 h at 25 °C using copper-free click reaction, then to
fluorescent streptavidin to label biotin and to DAPI to label cell nuclei.
click reaction. This technique can thus be
with ligands conjugated to biomaterials Then the azido-labeled FsOs (105 cells per
further applied to long-term cellularization
of implant biomaterials.
of different chemical compositions. For disc of biomaterial) were added to bioma-
that purpose, the ligand 8 combining a terial discs which were either unfunction-
pyrrogallol moiety for attachment to the alized or had been pre-functionalized with
inorganic matrix of the biomaterial[14] and compound 8. After the washing and fixa-
Click Reaction on HA and TCP
Biomaterials
After having proved that copper-free
click reaction can be used as an efficient
tool for the functionalization of FsOs sur-
face, we further investigated this reaction
DIBO was used. The hydroxyapatite (HA)
tion steps, the cells were stained with crys-
and β-tricalcium phosphate (TCP) bioma-
tal violet. Cell adhesion to the biomaterials
terials were functionalized by incubation was imaged with a stereomicroscope and
with a 0, 5 or 10 µM solution of 8 for 24 h. the number of adhered cells was quantified

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 215
acid AHA. A 50 to 100 µM concentration
3.0
2.5
2.0
1.5
1.0
0.5
0.0
of ligand 4 was optimal to achieve a high
rate of functionalization. Then the viability
of functionalized FsOs was evaluated for
up to 144 hours after the click reaction. No
decrease of the cell survival was observed
compared to the non-functionalized cells,
suggesting that this modification is not
toxic for FsOs.
C1
C2
4
A second chemical ligand 8 contain-
ing a pyrrogallol moiety for the binding
to the biomaterial discs[14] and a cyclic ac-
72
96
Incubation time after the click [h]
Fig. 2. FsOs were pre-incubated overnight with AHA (C2 or 4) or with
120
144
tivated alkyne for the covalent linkage to
FsOs was synthesized in reasonable yield.
The HA and TCP biomaterials were then
functionalized at different concentrations
with ligand 8 and AHA-labeled FsOs were
added. A significantly higher number of
adhered FsOs was observed on the func-
tionalized biomaterials and was strongly
correlated with the concentration of ligand
8 used for surface modification of the bio-
vehicle as control (C1), then the copper-free click reaction was performed
for 1 h at 25 °C (4) or cells were incubated in complete medium for 1 h at
25 °C as control (C1 and C2). Cells were further incubated for 72 h to 144
h, and then the WST-1 assay was performed. Results are the means +sd
of triplicate wells of a representative experiment out of three independent
experiments.
materials. Better results were obtained for
TCP bioceramic than HA bioceramic, the
number of adhered cells being higher with
this material. This observation was in ac-
A
B
***
cordance with previous results obtained for
***
other functionalizing ligands.[15]
80
70
60
50
40
30
20
10
0
In conclusion, surface functionaliza-
80
70
60
50
40
30
20
10
0
***
tion of HA and TCP biomaterials provides
an efficient tool to promote the adhesion of
bone precursor cells to the scaffolds using
***
the copper free click reaction. The biocom-
***
patibility of this chemical transformation
opens the way to further development of
functionalized bioceramics as cell-engi-
neered bone implant materials.
***
5µM
10µM
5µM
10µM
Fig. 3. The quantification of the number of cells adhered to the discs of bioceramics was per-
formed by analysis of stereomicroscopy pictures. A: HA and B: TCP. The number of cells bound
to the bioceramic discs was compared between unfunctionalized scaffolds (0 µM) and scaffolds
functionalized with compound 8 (5 µM or 10 µM) using a Student’s t-test : *** : p < 0.001. The
correlation coefficient (R2) was calculated between the number of adhered cells on scaffolds func-
tionalized with 5 µM and 10 µM of compound 8. R2 = 1.00. Results are the mean ±sd of triplicates
of three independent experiments.
Experimental Section
Chemical Syntheses
Commercial reagents (Fluka, Aldrich,
VWR, Switzerland) were used without fur-
ther purification. Anhydrous solvents were
obtainedbyfiltration(PureSolvMDSeries,
Innovative Technology). TLCs for reac-
tion monitoring were performed on Merck
silica gel 60 F254 plates, and spots were
revealed with UV light and reaction with
KMnO4 or ninhydrine. IR spectra were
recorded on a Perkin-Elmer-1420 spec-
by the IMAGE J software for each mate- Discussion
rial (Fig. 3). Quantification of the number
of bound cells to the discs of biomateri-
The colonization of the inner surface of
als showed that the functionalization of bonesubstitutematerialsby osteoblastshas
the discs with compound 8 significantly (p to be reached to ensure homogeneous bone
<0.001) increased the number of adhered reconstruction and success of the implant.
cells, with a very strong dependency (10 In this study, copper-free click reaction on
µM vs 5 µM: p <0.001, correlation = 1) human fetal osteoblasts was first evaluated
on the concentration of compound 8 used to determine if a chemical compound can
for the functionalization of the two types be bound to the cell surface without any
of biomaterials. No cells were bound on cytotoxicity. After the synthesis of a model
the unfunctionalized discs of bioceramics. ligand 4 containing an activated alkyne, a
1
trometer. H-NMR spectra were recorded
on a Bruker ARX-400 spectrometer (400
MHz) using CDCl3 or MeOD as solvent
and calibrated using the solvent’s residual
signal at 7.27 or 3.31 ppm as an internal
reference. 13C-NMR spectra were recorded
on a Bruker ARX-400 spectrometer (100.6
MHz) using CDCl3 or MeOD as solvent
TCP biomaterial showed a higher number
of bound cells compared to HA.
polyethylene chain and a biotin moiety, the
copper-free click reaction was performed
with this compound on modified FsOs and
monitored by fluorescence microscopy us-
ing a fluorescent streptavidin. Cell surface
modification was only observed on FsOs
pre-incubated with the non-natural amino
and calibrated using the solvent’s residual
signal at 77.0 or 49.0 ppm as an internal
reference. Chemical shifts are expressed
in parts per million (ppm) and coupling
constants (J) in Hertz. Mass spectra were
obtained on a Nermag R-10-10C spec-
trometer with chemical ionization (NH3)

216 CHIMIA 2013, 67, Nr. 4
Laureates: awards and Honors, sCs FaLL Meeting 2012
and mode m/z (amu) [% relative base peak (ESI): (m/z): calcd for C35H44N4O7S+H: product was purified by HPLC ((XTerra
(100%)]. Semi-preparative HPLC was 665.3009, found: 665.3010. [α]D25 = 0.1 (c Prep RP C18, (25×150 mm Waters)) to
performed on a Waters Autopurification = 0.1, DCM).
ZQ System equipped with a 2767 Sample
Manager, a 2525 Binary Gradient Module 5-[(6-Aminohexyl)carbamoyl]ben-
and a 2996 Photodiode Array Detector, zene-1,2,3-triyl Triacetate (7)
give 8 (10 mg, 38%). IR (film): 3275,
2935, 1695, 2595, 1515, 1450, 1330, 1200,
1135, 1035, 865, 755, 720, 645, 585 cm–1.
1H NMR (400 MHz, MeOD): δ = 7.38 (d,
coupled toWaters Micromass ZQ analyzer.
The HPLC purifications were performed
Few drops of DMF were added to 1H, aromatic, CH), 7.37–7.29 (m, 7H,
3,4,5-triacetoxy benzoic acid (1 g, 3.4 aromatics, (CH)7), 6.82 (s, 2H, (CH)2),
2
on XTerra Prep RP C18 (19 × 150 mm)
mmol, 1 equiv.) in dry DCM (6 mL). The 5.42 (m, 1H, C(1’)H), 3.22 (dd, 1H, J =
3
2
columns, using reverse-phase conditions (2 reaction was cooled to 0 °C and oxalyl- 15.0 J = 2.0 Hz, CH2), 3.11 (t, 2H, J =
to 100% acetonitrile with 0.1% TFA over
chloride (0.31 mL, 3.7 mmol, 1.5 equiv.) 2.0 Hz, CH ), 2.82 (dd, 1H, 2J = 15.0 3J =
20 min). MS: Nermag R-10-10C, chemical was added drop wise. After 15 min the 3.8 Hz, CH22), 1.60–1.29 (m, 10H, 5 CH2).
ionization (NH3) mode m/z (amu) [% rela- reaction mixture was allowed to reach rt 13C NMR (100 MHz, MeOD): δ = 169.12,
tive base peak (100%)].
and stirred for 1 h. The solution was con- 156.64, 152.34, 151.03, 136.52, 129.57,
centrated under reduced pressure and the 127.92, 127.81, 126.89, 126.84), 125.74,
residue was suspended in dry DCM (4.5 125.48, 124.99, 123.47, 112.39, 109.57,
11,12-Didehydro-5,6-
dihydrodibenzo[a,e][8]annulen-5-yl mL). A solution containing N-Boc-1,6- 76.40, 45.74, 40.28, 39.36, 29.40, 29.10,
4-nitrophenyl Carbonate (2)
hexadiamine (0.72 g, 3.4 mmol, 1 equiv.) 26.25, 26.06. MS (ESI): HRMS (ESI):
4-Nitrophenyl chloroformate (0.9 g, and DIPEA (1.73 mL, 10.1 mmol, 3 equiv.) (m/z): calcd for C H30N2O +H: 515.2182,
4.6 mmol, 2 equiv.) and pyridine (0.9 mL, in DCM (4.5 mL) was added drop wise at 0 found: 515.2180. 3[0α]D = 60.1 (c = 0.004,
25
11.4, 5 equiv.) were added to a solution of °C. After 15 min the reaction mixture was CH2Cl2).
11,12-didehydro-5,6-dihydrodibenzo[a,e] allowed to reach room temperature and
[8]annulen-5-ol (1, 0.5 g, 3.6 mmol, 1 stirred for 1 h. The solution was concentrat-
equiv.) in DCM (68 mL) and the reaction ed under reduced pressure and the residue Bioceramics
mixture was stirred for 8 h at 25 °C. The so- was purified by flash column chromatogra- Discs (d = 2.5 cm, h = 0.5 cm) of dense-
lution was washed with brine (2 × 23 mL). phy (AcOEt/EP 1:1). The resulting product ly sintered HA and TCP biomaterials were
Coating of HA and TCP
The organic phase was dried with MgSO
was dissolved in 4 M HCl in dioxane and incubated in a solution of 8 at increasing
and concentrated under reduced pressure4. stirred for 1 h at room temperature. HCl concentrations (0, 5 and 10 µM) for 24 h at
The crude product was then purified by
was coevaporated three times with diethyl room temperature in the dark under slight
flash column chromatography (PE/DCM ether to afford a white paste (400 mg, 30% stirring. The discs were washed three times
2:1) to give 2 as a white solid (0.8 g, 88%). for 2 steps). IR (film): 3265, 2935, 2860, with water and dried under vacuum for
The analytical data were in accordance
2225,1770, 1635, 1585, 1550, 1490, 1435, 24 h.
1370, 1315, 1180, 1120, 1050, 1010, 975,
890, 870, 820, 760, 730, 700 590 cm–1. Cell Source and Culture Conditions
1H NMR (400 MHz, MeOD): δ = 7.64 (s,
Human fetal osteoblasts were derived
with the reported characterization.[13]
Preparation of Ligand 4
11,12-Didehydro-5,6-dihydrodi- 1H, CH), 3.39 (t, 2H, CH ), 2.92 (t, 1H, by the explant technique from the femo-
benzo[a,e][8]annulen-5-yl 4-nitrophenyl CH2), 2.30 (s, 9H, 3 CH3), 21.66 (m, 4H, 2 ral bone of a 12-week old fetus according
carbonate (2, 0.67 g, 1.7 mmol, 1 equiv.) CH2), 1.44 (m, 4H, 2 CH2). 13C NMR (101 to a protocol accepted by the Lausanne
was added to a solution of N-(2-{2-[2- MHz, MeOD): δ = 169.50, 168.35, 133.72, Hospital and University Ethics Committee
(2- aminoethoxy)ethoxy]ethoxy}ethyl)- 121.00, 40.81, 40.63, 30.1, 28.44, 27.32, and with the mother’s oral and written ap-
5-[(4R)-2-oxohexahydro-1H-thieno[3,4- 26.96, 20.38, 19.96. MS (ESI): HRMS proval (protocol No 51/01, 2008). These
d]imidazol-4-yl]pentanamide (3, 0.72 g, (ESI): (m/z): calcd for C19H26N2O7+H: cells were used at passage 6. Cells were
1.7 mmol, 1 equiv.) and triethylamine (0.7 395.1818, found: 395.1823.
mL, 5.2 mmol, 3 equiv.) in DMF (194 mL),
routinely grown in DMEM medium con-
taining 4.5 g/l glucose, 10% fetal calf
serum (FCS) and antibiotics (all from
under an argon atmosphere. The reaction
11,12-Didehydro-5,6-dihydrodi-
mixture was stirred overnight at rt, concen- benzo[a,e][8]annulen-5-yl(6- Invitrogen, Basel, Switzerland).
trated under reduced pressure and the resi- {[(3,4,5-trihydroxy phenyl)carbonyl]
due was purified by silica gel flash chroma- amino}hexyl) Carbamate (8)
Click Reaction on FsOs
tography (DCM/MeOH 98:2) to afford 4 as
5-[(6-aminohexyl)carbamoyl]ben-
l-Azidohomoalanine (AHA) was pur-
a green solid (225 mg, 20 %). IR (film): zene-1,2,3-triyl triacetate (7, 75 mg, 0.2 chased from Invitrogen, bovine serum
3330, 2920, 2865, 1780, 1705, 1520, 1450, mmol, 1 equiv.) was dissolved in DMF (18 albumin (BSA), from Sigma-Aldrich,
1330, 1205, 1075, 955, 925, 860, 815, 760, mL) and carbonic acid, 5,6-dihydro-11,12- Buchs, Switzerland. FsOs (80–90% con-
730, 685, 645, 565, 545, 520, 515 cm–1. didehydro-dibenzo[a, e]cycloocten-5-yl fluent) were washed with warm (37 °C)
1H-NMR(400 MHz, CDCl3): δ = 7.52 (d, ester, 4-nitrophenyl ester (2, 80 mg, 0.2 PBS and incubated in methionine-free
1H, aromatic, 2J = 7.6 Hz, CH (aromatic)), mmol, 1 equiv.), followed by triethylamine DMEM culture medium containing 4.5 g/L
7.41–7.26 (m, 7H, 7 CH (aromatics)), 5.50 (0.08 mL, 0.6 mmol, 3 equiv.) were added. glucose, antibiotics (P/S) and 3% FCS for
(s, 1H, CH), 4.47 (m, 1H, CH), 4.29 (m, The reaction mixture was stirred overnight
30–60 min to deplete methionine reserves.
1H, CH), 3.66–3.62 (m, 8H, 4 CH ), 3.60– at room temperature. DMF was removed Then they were incubated for 4 h in the
3.53 (m, 4H, 2 CH2), 3.47–3.36 2(m, 4H, under reduced pressure and the product presence of AHA (50 µM) in fresh com-
2 CH2), 3.19–3.09 (m, 2H, 2 CH), 2.96– was purified by flash column chromatog- plete culture medium. The culture medium
2.86 (m, 2H, 2 CH), 2.71 (d, 1H, 2J = 12.6 raphy (AcOEt/EP 1:1 then 2:1) (150 mg, was removed and the cell layer was washed
2
Hz, CH), 2.19 (t, 2H, J = 7.7 Hz, CH2), 46%). The resulting intermediate (33 mg,
twice with PBS.
Stock solution of compound 4 (2.5
2H, CH2). 13C-NMR (101 MHz, CDCl3): at 0 °C and a 1 M solution of LiOH (0.15 mM in PBS:DMF (7:3)) was prepared. For
δ = 172.8, 130.0, 128.1, 127.1, 126.1, mL, 0.15 mmol, 3 equiv.) was added. The microscopy experiments, the click reac-
123.8, 70.4, 70.1, 61.9, 60.3, 55.4, 46.2, reaction was monitored by MS until total tion was performed in a solution contain-
40.5, 39.2, 29.7, 28.0, 25.6, 25.4. HRMS conversion of the starting material. The ing compound 4 (20–100 µM) in DMEM
1.70–1.60 (m, 4H, 2 CH2), 1.45–1.40 (m, 0.05 mmol, 1 equiv.) was dissolved in THF

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 217
in a multiwell-plate reader (iEMS Reader 3 pictures for each treatment were taken
MF, Labsystems, Bioconcept) against and the cell number was evaluated using
containing additional HEPES (20 µM).
Incubation was performed for 1 h at 25 °C.
a blank containing culture medium and the IMAGE J software.
The reaction mixture was removed and
WST-1.
two washings with PBS at 37 °C allowed
Received: February 25, 2013
the complete removal of the reagents. The
Click Reaction on the Biomaterial
cells were then fixed using a 4% formal-
dehyde in PBS for 10 min at 25 °C. After
two washings with warm PBS, the cells
were incubated with PBS containing 1%
BSA for 1 h at 25 °C and the labeling
was performed. Streptavidin-Cy3 (Sigma-
Aldrich) (1:100) and DAPI (4',6-diamidi-
no-2-phenylindole, Roche Diagnostics) (1
µg/mL in PBS) were then added and incu-
bated with the cells for 30 min in the dark.
After two washings with PBS during 5 min
in the dark, the cells were fixed using a 4%
formaldehyde solution in PBS at 4 °C.
[1] M. Bohner, Mater Today 2010, 13, 24.
[2] A. Mahendra, A. Maclean, Injury, Int. Care
Petri dishes (9 cm, Costar) were coat-
ed with 1000 µg/mL Rat Tail collagen I
(Invitrogen) according to the supplier’s
Injured 2007, 38, 7.
[3] J. R. Porter, T.
T. Ruckh, K. C. Popat,
protocol and human fetal osteoblasts were
then grown in the dishes for 24 h until
Biotechnol. Prog. 2009, 25, 1539.
[4] J. Hohlfeld, A. D. de Buys Roessingh, N. Hirt-
Burri, P. Chaubert, S. Gerber, C. Scaletta, P.
the cells are 80–90% confluent. The cells
were washed with PBS and incubated in
methionine-free DMEM culture medium
(Invitrogen) for 60 min to deplete methio-
nine reserves. Then the cells were incubat-
ed overnight in the presence of AHA (50
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[5] M. O. Montjovent, C. Bocelli-Tyndall,
C.
Scaletta, A. Scherberich, S. Mark, I. Martin, L.
A. Applegate, D. P. Pioletti, Tissue Engin. 2009,
15A, 1523.
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µM final concentration) in fresh complete
2005, 1, 13.
methionine-free culture medium. The cul-
[7] M. F. Debets, C. W. J. van der Doelen, F. P. J.
Two additional washings with PBS were
T. Rutjes, F. L. van Delft, ChemBioChem 2010,
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ture medium was removed and the cell lay-
performed. The chamber was removed
ers were washed twice with PBS. Collagen
coating was degraded by adding 0.1 mg/
mL collagenase (Sigma-Aldrich) in PBS
from the slide glass by using the chamber
removal device and the evaluation of the
click reaction was performed by fluores-
cence microscopy and slides photographed
under a fluorescence microscope (Zeiss
Axioplan 2).
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at 37 °C for 45 min. The osteoblasts were
2182.
then counted. Three mL of the cell suspen-
[11] F. Borcard, A. Godinat, D. Staedler, H. Comas
sion (105 cells/mL) were added on unfunc-
Blanco, A.-L. Dumont, C. Chapuis-Bernasconi,
C. Scaletta, L. A. Applegate, F. Krauss Juillerat,
U. T. Gonzenbach, S. Gerber-Lemaire, L.
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tionalized HA and TCP biomaterials or on
HA and TCP biomaterials functionalized
with ligand 8, in a 6-well plate (Costar)
and incubated for 1 h at room temperature
Cell Viability Assay
Cell cultures were treated with com-
pound 4 (100 µM) in complete medium
and additional HEPES (20 µM) for 1 h at
[12] F. Krauss Juillerat, F. Borcard, D. Staedler,
under slight agitation. Then the discs were
C. Scaletta, L. A. Applegate, H. Comas, L. J.
washed once with PBS, fixed in cold meth-
room temperature. Then cell survival was
Gauckler, S. Gerber-Lemaire, L. Juillerat-
Jeanneret, U. T. Gonzenbach, Bioconjugate
Chem. 2012, 23, 2278.
anol (–20 °C) for 5 min and incubated in
5% BSA for 1 h at room temperature. Cells
evaluated after 72 h to 144 h using the
WST-1 assay. The culture medium was
[13] N. E. Mbua, J. Guo, M. A. Wolfert, R. Steet,
G.-J. Boons, ChembioChem 2011, 12, 1912.
[14] H. Comas, V. Laporte, F. Borcard, P. Mieville,
were stained for 5 min at room temperature
with a solution containing 0.05% crystal
removed, cells were rinsed with DMEM
without phenol red (Invitrogen) and in-
violet (Sigma-Aldrich) and 1.5% glacial
F. Krauss Juillerat, M. Caporini, U. T.
Gonzenbach, L. Juillerat-Jeanneret, S. Gerber-
cubated with the WST-1 reagent (Roche
acetic acid in water. Cells were imaged by
stereomicroscopy (MZ16 FA Leica/DFC
480 Leica, 0.63 X and 1.6 X, zoom 72 X),
Diagnostics), diluted 1:10 in DMEM with-
out phenol red for 3 h. Optical density of
the supernatants was measured at 450 nm
Lemaire, Appl. Mater. Interface 2012, 4, 573.
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7988.