A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived...

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


Z. Kristof

Department of Plant Anatomy, Eotvos Lorand University,

Budapest, Hungary

K. Marko

Institute of Experimental Medicine, Hungarian Academy


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

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

480 J Mater Sci: Mater Med (2013) 24:479–488

123

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

J Mater Sci: Mater Med (2013) 24:479–488 481

123

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


123

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


123

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)


123

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


123

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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A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived...

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