A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived...
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A novel cyclic RGD-containing peptide polymer improvesserum-free adhesion of adipose tissue-derived mesenchymalstem cells to bone implant surfaces
Peter Tatrai • Bernadett Sagi • Anna Szigeti • Aron Szepesi • Ildiko Szabo •
Szilvia B}osze • Zoltan Kristof • Karoly Marko • Gergely Szakacs •
Istvan Urban • Gabor Mez}o • Ferenc Uher • Katalin Nemet
Received: 31 July 2012 / Accepted: 31 October 2012 / Published online: 8 November 2012
� Springer Science+Business Media New York 2012
Abstract Seeding of bone implants with mesenchymal
stem cells (MSCs) may promote osseointegration and bone
regeneration. However, implant material surfaces, such as
titanium or bovine bone mineral, fail to support rapid and
efficient attachment of MSCs, especially under serum-free
conditions that may be desirable when human applications
or tightly controlled experiments are envisioned. Here we
demonstrate that a branched poly[Lys(Seri-DL-Alam)]
polymer functionalized with cyclic arginyl-glycyl-aspar-
tate, when immobilized by simple adsorption to tissue
culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-
Oss� bovine bone substitute, significantly accelerates
serum-free adhesion and enhances seeding efficiency of
human adipose tissue-derived MSCs. Moreover, when
exposed to serum-containing osteogenic medium, MSCs
survived and differentiated on the peptide-coated scaffolds.
In summary, the presented novel polypeptide conjugate can
be conveniently used for coating various surfaces, and may
find applications whenever quick and efficient seeding of
MSCs is required to various scaffolds in the absence of
serum.
1 Introduction
Combining bone implant materials, such as titanium or
bone mineral, with mesenchymal stem cells (MSCs) to
promote osseointegration and stimulate bone regeneration
represents a promising novel direction in tissue engineer-
ing. MSCs seeded on titanium alloy plates were shown to
accelerate adhesion between metal and adjacent bone [1],
and superior bone formation on MSC-loaded titanium mesh
scaffolds has been reported in both orthotopic and ectopic
settings [2, 3]. Surface-modified titanium screws wrapped
in sheets of rabbit MSCs were surrounded by well-differ-
entiated bone tissue after 8 weeks of subcutaneous
implantation in SCID mice [4], indicating that MSCs may
facilitate integration of titanium implants into host bone.
Similarly, enhancement of bone regeneration and new bone
formation was demonstrated when inorganic bone matrix
substitutes, such as hydroxyapatite/tricalcium phosphate, or
the natural bovine bone mineral Bio-Oss�, were combined
with MSCs [5–7].
P. Tatrai � G. Szakacs
Research Center for Natural Sciences, Hungarian Academy
of Sciences, Budapest, Hungary
P. Tatrai � K. Nemet (&)
Department of Experimental Gene Therapy, National Blood
Transfusion Service, Dioszegi ut 64, Budapest 1113, Hungary
e-mail: [email protected];
[email protected]; [email protected]
B. Sagi � F. Uher
Stem Cell Laboratory, National Blood Transfusion Service,
Budapest, Hungary
A. Szigeti � A. Szepesi � K. Nemet
Creative Cell Ltd, Budapest, Hungary
I. Szabo � S. B}osze � G. Mez}oResearch Group of Peptide Chemistry, Hungarian Academy
of Sciences, Budapest, Hungary
Z. Kristof
Department of Plant Anatomy, Eotvos Lorand University,
Budapest, Hungary
K. Marko
Institute of Experimental Medicine, Hungarian Academy
of Sciences, Budapest, Hungary
I. Urban
Department of Restorative Dentistry, Loma Linda University,
Loma Linda, CA, USA
123
J Mater Sci: Mater Med (2013) 24:479–488
DOI 10.1007/s10856-012-4809-x
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MSCs applied for scaffold seeding can be derived from
various tissue sources. Adipose tissue may be a preferred
choice for multiple reasons. Fat is an abundant tissue with
high frequency of MSCs that readily differentiate toward
the osteogenic lineage [8]. In addition, the retrieval of fat,
either by lipoaspiration or surgical excision, is associated
with relatively low donor-site morbidity, and the presence
of both osteogenic and vasculogenic progenitors in the
freshly isolated stromal–vascular fraction (SVF) may be a
particular appeal for the engineering of vascularized bone
grafts [9]. Adipose tissue-derived MSCs (Ad-MSCs) can
proliferate and undergo osteoblastic differentiation on
titanium scaffolds as well as on mineral matrices [10, 11].
To generate clinically useful Ad-MSC-seeded implants,
it is paramount to achieve efficient attachment and pro-
longed survival of the cells on the implant surface. Cell
adhesion and survival can be promoted by physical and
chemical surface treatments, including biomimetic coating.
Arginyl-glycyl-aspartate (RGD) peptides have been
broadly applied for the biomimetic functionalization of
various coating polymers [12]. RGD peptides have been
shown to improve adhesion and osteoblastic differentiation
of human MSCs on both titanium and bone mineral scaf-
folds [13–16]. Major advantages of synthetic peptides over
natural pro-adhesive coatings such as fibronectin include
resistance to proteolytic degradation, designable orienta-
tion of bioactive motifs, and cost-efficient production [12].
Ad-MSCs may be seeded on the implant intraopera-
tively, or may be retrieved in a preliminary session and
cultured ex vivo prior to the implantation. While intraop-
erative seeding of scaffolds with freshly isolated SVF is an
intriguing approach, many studies suggest that ex vivo
culturing and osteogenic induction of Ad-MSCs prior to
implantation is indispensable for a clinically relevant
enhancement of bone healing [9]. The ex vivo expansion of
Ad-MSCs requires medium supplemented with fetal calf
serum, but the presence of animal serum is a major obstacle
for human clinical use. Thus, it is essential to facilitate
ex vivo attachment and survival of Ad-MSCs under serum-
free conditions. Also, serum-free conditions may be
desirable to rule out the confounding effects of undefined
factors influencing the behavior of Ad-MSCs.
Previously, we have shown that AK-cyclo[RGDfC], a
synthetic branched chain polypeptide (poly[Lys(DL-Alam)],
where m = 2.5–5) conjugated with the cyclic RGD peptide
c[RGDfC] promotes serum-free adhesion and survival of a
variety of cells on tissue culture (TC) plastic and glass [17].
In these earlier experiments, however, neither Ad-MSCs
nor clinically relevant implant surfaces were investigated. In
the present study, a recently developed and improved variant
of the formerly studied polymer, SAK-cyclo[RGDfC] was
investigated with regard to its effect on the serum-free
adhesion and survival of human Ad-MSCs on clinically
relevant scaffolds. The surfaces to be modified included TC
plastic, Ti6Al4V, and bovine bone mineral. Moreover, to
simulate post-implantation conditions and to test whether the
treated surfaces can support prolonged cell attachment and
bone formation, osteoblastic differentiation was induced
with serum-containing osteogenic medium.
2 Materials and methods
2.1 Isolation and modification of Ad-MSC
Work with human Ad-MSC was performed with permis-
sion from the Ethical Committee of the Hungarian Medical
Research Council (ETT). Lipoaspirate from a 30-year-old
healthy donor was digested with 0.1 % w/v collagenase
(Sigma) and adherent cells were plated in MSC expansion
medium (DMEM/F12 1:1 with 10 % v/v FBS, 5 ng/ml
FGF-2, 2 mM L-glutamine, and 50 lg/ml gentamicin; all
reagents from Invitrogen/Life Technologies, Carlsbad, CA,
USA). At passage three, Ad-MSC was transduced with a
lentivirus containing an EF1 promoter-driven eGFP
expression cassette. Following transduction, GFP-positive
cells were sorted on a FACSAria I flow cytometer (BD
Biosciences, Franklin Lake, NJ, USA). Cells between
passages 5 and 7 were used in the experiments.
2.2 Plasticware and scaffolds
All plasticware (TC and suspension multiwell dishes) were
from Greiner Bio-One (Mosonmagyarovar, Hungary).
Titanium disks of 5 mm diameter and 1.44 mm thickness
were laser-cut from sheets of implant-grade titanium alloy
(Ti6Al4V), and acid-etched in 1:3 diluted mixture of
concentrated HF and HNO3. Geistlich Bio-Oss� (GBO)
bovine bone mineral granules (granule size: 0.25–1 mm)
were purchased from Geistlich Biomaterials (Wolhusen,
Switzerland).
2.3 Synthesis and characterization of the biomimetic
coatings
Biomimetic polypeptide conjugates were synthesized by
covalently binding cyclo[Arg-Gly-Asp-D-Phe-Cys] (cyclo
[RGDfC]) peptide to a branched poly[Lys(Seri-DL-Alam)]
backbone henceforth referred to as SAK. A schematic
representation of the conjugate is shown in Fig. 1. The
preparation of the SAK polypeptide, as well as the chlo-
roacetylation of the N-terminus of the branches, was per-
formed as previously reported [18, 19]. The average
polymerization rate of the applied branched chain poly-
peptide (poly[Lys(Ser0.9-DL-Ala2.7)]) was around 200. The
chloroacetyl (ClAc) substitution of the side chains was
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30 % by elementary analysis. The cyclo[RGDfC] and
cyclo[RGDfV] peptides were synthesized as previously
described [17]. The chemoselective ligation of cysteine-
containing cyclic RGD peptides was performed under
slightly alkaline conditions (0.1 M Tris buffer, pH 8.0) at
RT for one day. Unreacted ClAc-goups were blocked with
cysteine afterwards. Peptides were separated from the
polymer conjugate by dialysis (cut-off 10,000) followed by
freeze-drying. The substitution of the branches with the
cyclic peptide ranged between 10 and 30 % in the various
lots. In preliminary experiments, no significant influence of
the substitution rate was observed on the biological activity
of the bioconjugates.
2.4 Surface treatment
Surfaces were coated by physical adsorption of SAK-
cyclo[RGDfC] or one of the following controls: (a) the
SAK backbone without bioactive peptide; (b) unconjugated
cyclo[RGDfV] peptide [20]; (c) human plasma fibronectin
(FN) (Millipore, Billerica, MA, USA). Synthetic peptides
were dissolved in deionized water; FN was reconstituted in
PBS. Unless specified otherwise, 10 lg/ml solutions were
applied for 2 h at 37 �C. Peptide-coated surfaces were
allowed to air dry and stored at 4 �C until use; FN-coated
surfaces were used immediately.
2.5 Cell seeding
Ad-MSCs were harvested, and suspended in serum-free
adhesion medium (StemPro� MSC SFM from Invitrogen
with 2 mM L-glutamine and 25 lg/ml gentamicin). For the
generation of the dose–response curve, a 96-well TC plate
was pretreated with the dilution series (0.04–10.0 lg/ml) of
SAK, cyclo[RGDfV], SAK-cyclo[RGDfC], and FN solu-
tions, then 5 9 103 cells/well were allowed to attach to the
plate for 4 h and incubated for another 24 h in serum-free
medium before quantification. For the time-lapse experi-
ment, cells were seeded at a density of 5,200/cm2 on a
24-well TC plate. In adhesion experiments with scaffolds,
5 9 103 cells in 100 ll were dispensed on 5–6 GBO
granules, and 4 9 104 cells in 100 ll were pipetted on the
TiAlV disks. Cells were allowed to attach for 45 min, then
supernatants with unattached cells were aspirated, scaffolds
were rinsed thoroughly with PBS by pipetting, and either
used immediately or incubated further in fresh medium.
2.6 Time-lapse microscopy
Cells were seeded in adhesion medium supplemented with
10 mM HEPES on a 24-well plate, kept in a 37 �C
chamber without CO2, and photographed at *5-min
intervals for 3.5 h. At each time point, three non-overlap-
ping fields of view (FOVs) were recorded per well.
Adhered cells were counted by visual estimation, based on
the observed difference between floating cells and those in
the initial phase of adhesion. Early visual signs of adhesion
included loss of spherical shape and smoothening of con-
trast between the darker center of the cell and the lighter
halo at the cell periphery. The three FOVs were summed,
and percent attached cell values were plotted against time
on a cumulative Kaplan–Meier curve.
2.7 Assessment of cell quantity following adhesion
The quantity of GFP-expressing Ad-MSCs attached to
titanium was assessed by placing the titanium disks upside
down into a transparent 96-well plate and measuring GFP
intensity on a Perkin-Elmer (Waltham, MA, USA) Victor
X3 plate reader. On GBO granules, cells were allowed to
grow for additional 24 h after attachment in adhesion
medium, and then quantified by resazurin reduction assay.
Resazurin (Sigma-Aldrich, St. Louis, MO, USA) was
added to the medium at a final concentration of 0.1 mg/ml.
Following 1 h of incubation at 37 �C, 5 % CO2, superna-
tants were transferred to an optical 96-well plate (Greiner
Bio-One), and fluorescence was read at Ex 540 nm/Em
579 nm in a Victor X3 plate reader. The same resazurin
reduction method was applied to quantify cells in the dose–
response experiment.
2.8 Fluorescent labeling of microfilaments
For the visualization of microfilamental organization,
Ad-MSCs seeded on SAK-cyclo[RGDfC]-treated versus
uncoated or control-coated 96-well TC plastic plates were
fixed with 4 % w/v PBS-buffered paraformaldehyde (PFA)
after 30 min or 4 h of adhesion, incubated for 30 min at
37 �C with 0.1 lg/ml Texas Red isothiocyanate (TRITC)-
conjugated phalloidin (Sigma-Aldrich), and nuclei were
stained with DAPI.
Fig. 1 Schematic representation of the SAK-cyclo[RGDfC] conjugate
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2.9 Osteogenic differentiation
Seeded scaffolds were first incubated in expansion medium
for 2 days before replacement of expansion medium with
osteogenic medium (StemPro Osteogenesis Differentiation
Kit, Life Technologies). On GBO, osteogenic differentia-
tion was detected by alkaline phosphatase (ALP) cyto-
chemistry after 10 days. Samples were fixed in 4 % w/v
PBS-buffered PFA, then washed with PBS and incubated
for 5 min at room temperature (RT) with 5-bromo-
4-chloro-3-indolyl phosphate (BCIP), 0.02 % w/v, and
nitro blue tetrazolium (NBT), 0.03 % w/v, dissolved in
ALP buffer (100 mM TRIS, 100 mM NaCl, 5 mM MgCl2,
0.05 % v/v Tween-20, pH 9.5). On the Ti6Al4V disks,
calcification was evaluated by Alizarin red staining after
4 weeks of differentiation. Cell-scaffold constructs were
fixed in 4 % w/v PBS-buffered PFA, washed, and incu-
bated for 1 h at RT with 2 % w/v Alizarin red (Sigma),
pH 4.2.
2.10 Statistical analysis
Curves were fitted to dose–response data with GraphPad
Prism v4.00 software. Corresponding data points of the
curves were compared pairwise by Student’s t test. Similarly,
Student’s t test was used for the comparison of different
groups in the serum-free adhesion experiments. In each case,
P \ 0.05 was considered as statistically significant.
3 Results
3.1 Establishment of optimal coating concentration
and seeding time
To establish the optimal concentration of SAK-cyclo
[RGDfC] for coating, a dose–response curve between 0.04
and 10 lg/ml was generated (Fig. 2). Out of 5 9 103 cells
seeded, (3.2 ± 0.8) 9 103 were detected on the untreated
plastic after 24 h (horizontal baseline with dotted confidence
interval in Fig. 2). SAK-cyclo[RGDfC], similarly to FN,
exerted a concentration-dependent positive effect on the
number of adhered cells. Data were best approximated with a
hyperbolic saturation model, and curves were fitted accord-
ingly. Half-maximal adhesion-enhancing effect of SAK-
cyclo[RGDfC] was observed at 0.1 lg/ml (FN: 0.17 lg/ml),
and the curve was practically in saturation at 10 lg/ml. At
this concentration, SAK-cyclo[RGDfC], when compared
with bare TC plastic, increased the number of adhered cells
by 69 ± 13 % (FN: 65 ± 12 %). The curves of SAK-
cyclo[RGDfC] and FN were not significantly different,
whereas the peptide coating differed from untreated plastic
starting from concentrations as low as 0.08 lg/ml. In the
following experiments, all coatings were applied at the sat-
urating concentration of 10 lg/ml. Remarkably, unconju-
gated cyclo[RGDfV] failed to improve adhesive properties
of the plastic, and the SAK backbone alone exerted a robust
cell-repellent effect with a log-linear dose response
(R2 = 0.86).
For the determination of optimal seeding time, the kinetic
profile of Ad-MSC adhesion to conjugate-coated versus
untreated TC plastic was characterized by time-lapse
microscopy. Phase contrast micrographs were taken at 5-min
intervals; in Fig. 3a, snapshots recorded at 2 min, 30 min,
59 min, and 3.5 h post seeding are shown. Micrographs were
evaluated visually, and the percent of attached cells was
plotted against time (Fig. 3b). The curves showed rapid
saturation in the case of SAK-cyclo[RGDfC] and FN.
Median time to complete adhesion was 15 min for SAK-
cyclo[RGDfC] and 10 min for FN (difference not significant
between the conjugate and FN). On SAK-cyclo[RGDfC],
82 % of cells showed clear signs of initial adhesion after
30 min (FN: 71 %), and 93 % were neatly flattened at 3.5 h
(FN: 87 %). Based on these results, a seeding time of 45 min
was chosen for the adhesion experiments. While unconju-
gated cyclo[RGDfV] exerted a moderate pro-adhesive effect
(71 % of cells attached at 3.5 h), kinetics of adhesion was
markedly slower (median time 140 min). Within the time
frame investigated, very few or no cells attached to uncoated
plastic and SAK (7.0 and 0.0 % after 3.5 h).
3.2 The effect of SAK-cyclo[RGDfC]
on microfilamental organization of Ad-MSC
To assess actin stress fiber formation of Ad-MSC on coated
versus uncoated TC plastic, cells were allowed to attach for
Fig. 2 Dependence of Ad-MSC adhesion on the concentration of
coating solutions. Equal numbers of cells were seeded in tissue
culture multiwells coated with serial dilutions of the SAK-
cyclo[RGDfC] conjugate and controls. Cell numbers were determined
by resazurin assay
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30 min or 4 h and stained with fluorescently labeled
phalloidin (Fig. 4). In contrast with very limited spreading
on uncoated plastic and virtually no cellular interaction
with either SAK or unconjugated cyclo[RGDfV] after
30 min, advanced spreading with discernible cortical actin
layer and initial stress fiber formation was detected on both
conjugate-treated and FN-treated plastic. After 4 h, stress
fibers were robustly developed on SAK-cyclo[RGDfC] and
FN, whereas the few adhered cells on bare plastic and
cyclo[RGDfV] displayed poor cytoskeletal organization.
After rinsing away unattached cells, wells treated with
SAK were found empty at this time point.
3.3 Serum-free adhesion of Ad-MSC
to SAK-cyclo[RGDfC]-coated GBO granules
and Ti6Al4V disks
The peptide conjugate was tested on two types of clinically
relevant materials: GBO and Ti6Al4V. Scaffolds seeded for
45 min were photographed and subjected to quantitative
Fig. 3 Time course of
Ad-MSC adhesion to
SAK-cyclo[RGDfC]-coated vs.
untreated or control-coated TC
plastic. a Rapid flattening of
cells was observed on the
peptide conjugate coating and
FN. Note aggregation of
Ad-MSCs on cell-repellent SAK
(arrow). b Percentage of
adhered cells on various
coatings plotted against time
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measurement directly after adhesion (Ti6Al4V) or follow-
ing an additional 24-h incubation period (GBO) (Fig. 5a, b).
Very few poorly spread cells were seen on untreated, SAK-
and cyclo[RGDfV]-treated GBO. Rather than adhering to
the surface, Ad-MSCs tended to form large clumps.
Markedly higher cell densities were achieved on SAK-
cyclo[RGDfC]- and FN-treated scaffolds, with the majority
of cells assuming well-spread, spindle-like fibroblastic
morphology. Compared to FN-treated GBO, 146 ± 11 %
cells attached to SAK-cyclo[RGDfC], while only 23–37 %
adhered to the control surfaces (Student’s t test, P \ 0.001).
The cell-repellent effect of the SAK backbone was not
evident here. In accordance with the above, all eGFP-
expressing AdMSCs were easily removed by rinsing from
the uncoated and control-treated Ti6Al4V disks, whereas
the cells formed a firmly adherent, continuous layer which
resisted to rinsing on SAK-cyclo[RGDfC]- and FN-coated
titanium. Negligible GFP signal was detected on the
untreated and control-treated disks, in contrast with the
145 ± 20 % signal intensity of the conjugate-treated disk
as compared to its FN-coated counterpart (conjugate-treated
vs. untreated, P \ 0.001; conjugate-treated vs. FN,
P = 0.007).
3.4 Bone differentiation of Ad-MSC on SAK-
cyclo[RGDfC]-coated GBO granules and Ti6Al4V
disks
Induction of ALP in Ad-MSCs growing on SAK-
cyclo[RGDfC]-coated GBO was assessed cytochemically
after 10 days of osteogenic differentiation (Fig. 6, upper
row). Dark staining on the surface of the granules indicated
ALP activity of the cells. By this time point, the few cells
that initially adhered to uncoated GBO were also flattened
and showed osteogenic induction; however, the extent of
ALP staining on SAK-cyclo[RGDfC]-coated GBO still
reflected the early advantage in adhesion. On the Ti6Al4V
disks, calcium deposition was evaluated by Alizarin red
staining after 4 weeks of differentiation (Fig. 6, lower
row). On uncoated Ti6Al4V, no cells were able to per-
manently attach and survive, and therefore no calcium was
accumulated. In contrast, the continuous layer of Ad-MSCs
formed on SAK-cyclo[RGDfC]-coated titanium during
45 min of seeding could survive and differentiate over
4 weeks, producing massive calcium deposits by the end of
the experiment.
4 Discussion
Although titanium and bone mineral as skeletal biomate-
rials exhibit excellent biocompatibility, the efforts to
strengthen their interactions with skeletal progenitor cells
are justifiable. Even in the presence of serum proteins and
after 8 h of adhesion, only little more than 20 % of all
seeded human MSCs were observed to attach to pristine
titanium [21]. GBO, a bone substitute widely applied in
oral reconstruction, was also found to poorly support the
adhesion and viability of MSCs in vitro, despite the pres-
ence of serum factors [22, 23]. Our results, in accordance
with previous reports [24], indicate that the in vitro per-
formance of these biomaterials is further deteriorated
by serum deprivation. This is a point of importance
since animal serum-free conditions are required when
Fig. 4 Microfilamental organization of Ad-MSCs seeded on SAK-
cyclo[RGDfC]-coated vs. untreated or control-coated TC plastic.
Actin cytoskeleton was labeled with TRITC-conjugated phalloidin
(grayscale) and nuclei were stained with DAPI (blue) after 30 min or
4 h of adhesion. Original magnification: 6009 (Color figure online)
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cell-seeded constructs are maintained in vitro with the aim
of human implantation. Moreover, avoidance of sera may
be desirable in any experimental setup where their many
undefined bioactive factors are thought to hinder evaluation
of results.
Therefore, the goal of our study was to devise a surface
derivatization method based on a recently developed cyclic
RGD-containing branched polypeptide conjugate to assist
attachment and survival of human Ad-MSCs on titanium
and bovine bone mineral in a serum-free environment.
Fig. 5 Adhesion of Ad-MSC to SAK-cyclo[RGDfC]-coated vs.
uncoated or control-coated GBO and Ti6Al4V. eGFP-expressing
Ad-MSCs were seeded on the scaffolds, photographed (a), and
quantified by resazurin reduction assay (GBO) or GFP fluorescence
measurement (Ti6Al4V) (b). Asterisks above the brackets show
statistically significant differences. **, P = 0.007; ***, P \ 0.001
Fig. 6 Osteogenic
differentiation of Ad-MSCs on
SAK-cyclo[RGDfC]-coated vs.
uncoated GBO and Ti6Al4V.
ALP activity was detected on
GBO by cytochemistry after
10 days (dark grey staining);
calcium deposition was
demonstrated on Ti6Al4V by
Alizarin red staining after
4 weeks. As controls,
FN-coated seeded scaffolds
with undifferentiated (undiff.)
cells, as well as uncoated seeded
scaffolds subjected to
osteogenic differentiation
protocol (osteo) are shown
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In contrast with polylysine, this type of branched chain
polypeptides is not toxic. A principal advantage of the
proposed method is that the labor-intensive chemical steps
of preparation are separated from the facile and quick step
of surface treatment which occurs by simple physical
adsorption. Unlike some conventional protocols employed
for the immobilization of cyclic RGD peptides on titanium
and bone mineral, such as silanization and bifunctional
crosslinking [13, 25], this method requires no special
equipment or chemical competence from researchers and
appliers of the functionalized surfaces.
The ability of RGD peptides to promote in vitro adhesion,
survival, and bone differentiation of osteoblastic cells on
titanium has been confirmed in a number of studies. Both
linear and cyclic RGD peptides increased attachment of
human bone marrow-derived osteoprogenitors to Ti6Al4V
disks [13], and poly(ethylene glycol)-(PEG-) immobilized
RGD stimulated proliferation and calcification of murine
MC3T3-E1 osteoblasts on polished pure titanium [26]. More
controversy exists with regard to the utility of RGD func-
tionalization of bone mineral substitutes such as hydroxy-
apatite (HA). While chemically crosslinked linear and cyclic
RGD motifs facilitated attachment of human osteoprogeni-
tors to HA cylinders [27], and polyglutamate-tethered RGD
enhanced adhesion of human bone marrow MSCs to sintered
HA disks [14], some authors claim that RGD functionali-
zation of HA may be detrimental to osteoblastic attachment
when natural adhesion factors are also present. They suggest
a mechanism whereby synthetic RGD motifs compete for
matrix receptors with their natural counterparts but are
unable to stimulate them with comparable efficiency [28].
In the serum-free system described herein, however,
such competition (at least at the initial stages of adhesion)
could not evolve, and the cyclo[RGDfC]-containing poly-
peptide conjugates exerted a striking effect on the speed and
efficiency of Ad-MSC adhesion. On TC plastic, as well as
on titanium and bovine bone mineral, the conjugates pro-
moted adhesion, and therefore improved the efficiency of
seeding. Enhanced cell affinity of the treated surfaces was
also substantiated by more expanded cellular morphology
and higher cytoskeletal organization of attached cells.
Moreover, the surfaces coated with SAK-cyclo[RGDfC]
supported in vitro bone differentiation induced by serum-
supplemented osteogenic medium.
In all aspects, the beneficial properties of the conjugates
were comparable in extent to those of fibronectin, and were
not reproduced by surface treatment with either the
SAK backbone or the unconjugated cyclo[RGDfV] peptide
alone. In fact, the oligo-(serine-DL-alanine)-derivatized
poly-L-lysine backbone SAK appeared to repel rather than
attract Ad-MSCs. On one hand, this observation was wel-
come, since the clear concentration dependence of the
repellent effect, indicating progressive masking of available
plastic surface with increasing SAK coat concentrations,
demonstrated the robust physisorptive ability of the backbone.
On the other hand, it was slightly unexpected, because poly-L-
lysine treatment of Ti6Al4V disks had been reported to
enhance adhesion and osteoblastic differentiation of human
bone marrow MSCs [29]. Nevertheless, SAK as a carrier did
not abolish the positive effect of cyclo[RGDfC] in the con-
jugates; on the contrary, it was indispensable for the proper
immobilization and positioning of the cyclo[RGDfC] motifs.
The dual behavior of SAK is not unparalleled, as it was pre-
viously shown that RGD peptides may exert their facilitative
effect even against a cell-repellent background. Bacteria-
repellent hyaluronic acid-chitosan surface treatment inhibited
murine osteoblast adhesion, but this negative effect was
reversed by the addition of RGD [15]. Similarly, the cell-
repellent side effect of the antibacterial poly-L-lysine-graft-
poly(ethylene glycol) coating was more than counterbalanced
by the addition of a long linear RGD peptide [30].
While the principal aim of this work was to improve
serum-free seeding of the scaffolds, there is considerable
indication that cyclo-RGD motifs may confer additional
benefits beyond an initial enhancement of cell adhesion.
Despite ambiguous in vitro results with titanium scaffolds
[31] and some reports with negligible or no effect of RGD
[32, 33], several in vivo studies demonstrated synergism
between immobilized RGD and other surface treatments
such as roughening or coating with organic, mineral, and
composite layers [34–39]. As mentioned above, data con-
cerning RGD-functionalized HA are also ambiguous, since
the same authors first reported promising in vitro results but
later assumed a more skeptical approach when faced with
lack of success in vivo [28]. However, these scientists’
model system differs significantly from the present studies
in a number of aspects, including the biomaterial used (HA
vs. GBO), and the application of added cells (empty vs.
seeded scaffolds). In our view, the reassuring in vitro results
presented herein give sufficient rationale for investigating
the in vivo performance of SAK-cyclo[RGDfC]-coated
titanium and GBO in animal models.
Acknowledgments The authors would like to thank all colleagues
at the Tissue Regeneration Department of the Twente University for
the kind support, as well as Eva Juhasz and Balazs Heged}us for the
help with time-lapse microscopy. This work was financially supported
by the grants BIO_SURF from the National Office for Research and
Technology (NKTH) and TAMOP-4.2.1-IKUT from the National
Development Agency (NFU).
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