ORIGINAL ARTICLE

Study on the potential of RGD- and PHSRN-modified alginatesas artificial extracellular matrices for engineering bone

Ryusuke Nakaoka • Yoshiaki Hirano •

David J. Mooney • Toshie Tsuchiya •

Atsuko Matsuoka

Received: 15 November 2012 / Accepted: 4 March 2013

� The Japanese Society for Artificial Organs 2013

Abstract Alginate is a polysaccharide that can be

crosslinked by divalent cations, such as calcium ions, to

form a gel. Chemical modification is typically used to

improve its cell adhesive properties for tissue engineering

applications. In this study, alginates were modified with

peptides containing RGD (arginine–glycine–aspartic acid)

or PHSRN (proline–histidine–serine–arginine–asparagine)

sequences from fibronectin to study possible additive and

synergistic effects on adherent cells. Alginates modified

with each peptide were mixed at different ratios to form

gels containing various concentrations and spacing

between the RGD and PHSRN sequences. When normal

human osteoblasts (NHOsts) were cultured on or in the

gels, the ratio of RGD to PHSRN was found to influence

cell behaviors, especially differentiation. NHOsts cultured

on gels composed of RGD- and PHSRN-modified alginates

showed enhanced differentiation when the gels contained

[33 % RGD-alginate, suggesting the relative distribution

of the peptides and the presentation to cells are important

parameters in this regulation. NHOsts cultured in gels

containing both RGD- and PHSRN-alginates also demon-

strated a similar enhancement tendency of calcium depo-

sition that was dependent on the peptide ratio in the gel.

However, calcium deposition was greater when cells were

cultured in the gels, as compared to on the gels. These

results suggest that modifying this biomaterial to more

closely mimic the chemistry of natural cell adhesive

proteins, (e.g., fibronectin) may be useful in developing

scaffolds for bone tissue engineering and provide three-

dimensional cell culture systems which more closely

mimic the environment of the human body.

Keywords Bone tissue engineering � Polymeric scaffolds �Peptide modification � Cell differentiation � 3D Cell culture

Introduction

Scaffold materials are often critical for successful tissue

regeneration using tissue engineering techniques. The

scaffold materials should be designed as artificial extra-

cellular matrices that provide a space for transplanted cells

to attach, proliferate, and differentiate into a desired cell

phenotype in order to regenerate the target tissues. More-

over, the artificial extracellular matrices provide (1) a space

for cells to organize a three-dimensional (3D) structure, (2)

mechanical integrity, and (3) a hydrated space for the

diffusion of nutrients and metabolites to and from the cells

[1–7]. Hydrogels have been studied for their application in

R. Nakaoka (&) � T. Tsuchiya � A. Matsuoka (&)

Division of Medical Devices, National Institute of Health

Sciences, 1-18-1 Kamiyoga, Setagaya-ku,

Tokyo 158-8501, Japan

e-mail: nakaoka@nihs.go.jp

A. Matsuoka

e-mail: matsuoka@nihs.go.jp

Y. Hirano

Department of Chemistry and Material Engineering,

Faculty of Chemistry, Materials and Bioengineering,

Kansai University, 3-3-35 Yamate, Suita-shi,

Osaka 564-8680, Japan

D. J. Mooney

School of Engineering and Applied Sciences, Harvard

University, Pierce Hall 319, 29 Oxford Street, Cambridge,

MA 02138, USA

Present Address:T. Tsuchiya

Faculty of Medicine, Tokyo Medical and Dental University,

1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan

123

J Artif Organs

DOI 10.1007/s10047-013-0703-7

many fields, such as medicine, pharmacy, tissue engi-

neering, and basic sciences [1, 2, 4]. The space for cell

organization, mechanical strength, and diffusion rate of

various molecules through hydrogels can be modified in

various manners, including altering the crosslinking den-

sity and chemically modifying the molecules composing

the hydrogels. In the context of bone regeneration, gels

cannot provide sufficient strength for load-bearing situa-

tions. However, they can be used in non-loaded situations

(e.g., as a component of a composite material containing

both a mechanically strong matrix and a space-filling gel).

Alginates are naturally derived polysaccharides com-

posed of (1-4)-linked beta-D-mannuronic acid (M units)

and alpha-L-guluronic acid (G units) monomers [8]. It is

well known that divalent cations cooperatively bind

between sequential G units of adjacent alginate chains,

resulting in ionic crosslinking that causes hydrogel for-

mation from alginate aqueous solutions. This crosslinking

mechanism allows alginate solutions containing cells to be

gelled under gentle conditions that do not harm the cells.

However, since alginate hydrogels absorb very few pro-

teins, mammalian cells cannot adhere to the hydrogels. Cell

adhesion is a major requirement for the survival of most

mammalian cell types. Therefore, modification of alginate

molecules is necessary for cells to adhere and proliferate on

or in the hydrogels. Modification of alginate molecules by

covalently attaching cell adhesion ligands, specifically

arginine–glycine–aspartic acid (RGD) peptides, has been

performed to improve cell anchorage and interaction with

the modified alginate hydrogels [9–13]. Although the

resultant alginate hydrogels have been found to be useful in

tissue engineering studies, it is unclear whether their

capability to mediate cell phenotype can be further

enhanced by additional modification.

RGD is a well-known peptide sequence found in fibro-

nectin, a major protein discovered almost two and half

decades ago [14] that has a variety of functions, including

adhesion. RGD peptides have been used to improve the cell

adhesion activity of many biomaterials [9–13, 15–17].

However, another important sequence for cell adhesion in

fibronectin, the proline–histidine–serine–arginine–aspara-

gine sequence (PHSRN), has been reported to markedly

improve cell adhesion strength due to the synergistic

interactions of this peptide and the RGD sequence with

integrin receptors [18]. Therefore, it is possibly that the

modification of alginate with both RGD and PHSRN may

change the strength of cell adhesion to the gels, resulting in

significant effects on cell functions, such as proliferation

and differentiation. As a first test of this possibility, we

made gels from RGD- or PHSRN-modified alginates and

their mixtures and then cultured normal human osteoblasts

on or in the gels in order to study the effect of these pep-

tides on cell behavior, especially on cell differentiation.

Materials and methods

Materials

ProNova MVG, an alginate with a high G content

(Mw = 3 9 105), was purchased from ProNova Biopoly-

mers (Oslo, Norway). The two cell adhesive peptides,

Gly-Gly-Gly-Gly-Arg-Gly-Asp-Ser-Pro (RGD) and

Gly-Gly-Gly-Gly-Pro-His-Ser-Arg-Asn (PHSRN) were

purchased from Cosmo Bio Co. Ltd (Tokyo, Japan). All

other chemicals (JIS special grade) were purchased from

Sigma (St. Louis, MO) and used without any further

purification step.

Chemistry

Alginate was chemically modified according to the

method of Rowley et al. [9]. Briefly, sulfo-NHS, water-

soluble carbodiimide, and the peptide were added to

100 ml of 1 % alginate solution in 0.1 M MES buffer (pH

6.5, NaCl 0.3 M), followed by continuous stirring at room

temperature to perform the coupling reaction. After dial-

ysis against ddH2O using a dialysis tube (MWCO 3500;

Spectrum Laboratories, Rancho Dominguez, CA), the

solution was sterilized by filtration and lyophilized. The

actual modification of alginates was estimated from the

results of amino acid analysis, using an amino acid ana-

lyzer (LC-10A; Shimadzu Co., Kyoto, Japan). Before the

amino acid analysis, the peak areas of each amino acid

were determined by analyzing an amino acid mixture

standard solution (Type H; Wako Pure Chemical Indus-

tries Ltd., Osaka, Japan). The weighed peptide-modified

alginates were hydrolyzed and analyzed to determine the

amount of each amino acid in the peptides from their

peak area. Based on the molecular weights of the alginate

and peptides, the number of the peptide per one alginate

molecule can be calculated from the results of the anal-

ysis. To form gels, peptide-modified alginates were

weighed and dissolved in ddH2O containing 0.2 %

(NaPO3)6 to obtain a 2 % solution. To prepare the algi-

nate hydrogels, 0.2 ml of a water-based slurry of calcium

sulfate (0.21 g/ml) was mixed for every 5 ml of the 2 %

alginate solution, and the mixture was cast between par-

allel glass plates with 1-mm spacers. A mixture of RGD-

modified and PHSRN-modified alginate solutions was

used to form gels made from the two alginates (MIX gels)

in the calcium sulfate slurry. Hydrogel discs were pun-

ched out of the film with a hole punch (diameter 13 mm)

and kept in medium without fetal calf serum (FCS)

overnight at 4 �C before seeding cells were added. The

hydrogel discs utilized throughout this study were 13 mm

in diameter with a thickness of 1 mm.

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123

Cell studies

Normal human osteoblasts (NHOsts) were purchased from

Lonza Walkersville Inc., (Walkersville, MD) and main-

tained in alpha-MEM containing 10 % FCS.

The NHOsts were seeded on or in the alginate hydrogel

disks for estimating the effects of coupled peptides on their

behavior. To encapsulate cells in the alginate gels, we

suspended the NHOsts in medium containing 2 % alginate,

then mixed the mixture with the calcium sulfate slurry as

described in the previous section to form the gels. The

number of NHOsts seeded on top of the hydrogel discs or

in wells was 2.5 9 104, and the approximate cell number

encapsulated in each gel disc was calculated to be

2.4 9 104. Changes in the differentiation level of NHOsts

during the culture were determined from their alkaline

phosphatase (ALP) activity and osteocalcin production.

The NHOsts were cultured with osteogenic supplements,

10 mM beta-glycero phosphate, ascorbic acid (50 lg/ml),

and 10-8 M dexamethasone. After the NHOsts had been

cultured on alginate gels for 2 weeks, their ALP activities

were estimated using the original procedure of Ohyama

et al. [19]. Briefly, the NHOsts were first washed in

phosphate buffered saline (PBS), followed by the addition

of 1 ml of 0.1 M glycine buffer (pH 10.5) containing

10 mM MgCl2, 0.1 mM ZnCl2, and 8 mM p-nitrophenyl-

phosphate sodium salt. The cells were then incubated at

room temperature for 7 min, following which the absor-

bance of the added buffer was detected at at 405 nm using

a lQuant spectrophotometer (Bio-tek Instruments, Inc.,

Winooski, VT) to evaluate the alkaline phosphate activity

of the test cells. To estimate osteocalcin production from

the NHOsts, the culture medium was replaced with med-

ium without FCS 24 h before the medium was collected for

analysis. The amount of osteocalcin in each culture med-

ium was estimated using the Gla-osteocalcin ELISA kit

(Takara Bio Inc., Shiga, Japan). After measuring the ALP

activities of the NHOsts and collecting the supernatants for

an evaluation of osteocalcin production, we transferred the

gels to a plastic tube filled with 0.2 % Nonidet-P40 aque-

ous solution; this was followed by sonication to obtain cell

lysates. The amount of DNA in each lysate was measured

utilizing the PicoGreen dsDNA quantification kit (Invitro-

gen, Carlsbad, CA). Based on the DNA amount, the cell

number ratio on the respective alginate gel against that on a

normal culture dish was calculated and utilized for the

standardization of other data obtained.

The number of NHOsts encapsulated in the gels was

estimated utilizing the Tetracolor One assay (Seikagaku

Co., Tokyo, Japan), which incorporates an oxidation–

reduction indicator based on the detection of metabolic

activity. After adding 1 ml of the culture medium con-

taining 5 % of Tetracolor One to the medium, the

absorbance of the medium at 450 nm was estimated by the

lQuant after a 2-h incubation. After the assay, the ALP

measurements of the NHOsts were also performed by

incubating the gels in 1 ml of glycine buffer, followed by

the same procedure as described above. After the ALP

measurements, the buffer was discarded and the medium

with osteogenic supplements was added for further culti-

vation of the encapsulated cells. These measurements were

performed once a week during the 4-week culture period.

After the Tetracolor One assay and the ALP measurement

at the 4-week time point during the culture period of the

gels, the encapsulated NHOsts were collected by centri-

fugation after the gel had been dissolved in EDTA citrate

buffer (pH 6.8). The DNA content in the lysates prepared

from the collected NHOsts by sonication in the Nonidet

solution was determined by a PicoGreen kit and used to

estimate the DNA contents from the results from Tetra-

color One assay. The estimated DNA contents were uti-

lized for calculating the cell number ratio and normalizing

the ALP data to the DNA content in each culture. The

calcium deposited during osteogenesis of the NHOsts was

qualitatively evaluated by alizarin red staining. The amount

of osteocalcin secreted into the culture medium of the

encapsulated NHOsts was measured by the Gla-osteocalcin

ELISA kit.

Statistics

Each experiment was performed three times to verify its

reproducibility, and representative data are shown as fig-

ures. Data were expressed as the mean value ± the stan-

dard deviation of the obtained data. The Tukey criterion

was used to control for multiple comparisons after per-

forming analysis of variance and to compute the least

significant difference between means.

Results

An amino acid analysis was performed to estimate the

number of peptides coupled to alginate molecules. The

number of RGD and PHSRN peptides per alginate mole-

cule used in this study was found to be 1.4 and 2.8,

respectively. These modified alginates were used

throughout this study.

NHOsts were first seeded on top of gel discs or wells.

Microscopic study after 1 day of culture revealed that few

cells adhered to an alginate gel without peptide modifica-

tion, whereas cell adhesion was slightly improved on

alginate gels modified with peptides (data are not shown).

After a 2-week incubation, the NHOsts on a normal culture

dish became confluent and differentiated, but no cells were

observed on the alginate gel without peptide modification,

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indicating that NHOsts could neither attach nor proliferate

on the gel without peptide modification (Fig. 1). Therefore,

the results of ALP activity and osteocalcin production from

NHOsts cultured on the unmodified alginate gel are not

shown in this study. When the NHOsts were seeded onto

RGD-modified alginate gel discs, they formed several

aggregates with a dark shadow after a 2-week incubation;

this shadow may be ascribed to a calcium deposition on the

aggregates. In contrast, few NHOsts were observed on

PHSRN-modified alginate gel discs, and those cells present

in this condition tended to form small aggregates without

the dark shadow after 2-week incubation. Culturing cells

on gels containing a mixture of PHSRN alginate and RGD-

modified alginate (MIX gels) led to an increase in the

number of NHOsts, and these NHOsts formed aggregates.

However, even though more cells were present on the MIX

gels than on the PHSRN gels, there were still large surface

regions on the MIX gels without adherent cells. Both the

size and the number of the cell aggregates changed with

changes in the ratio of RGD in the MIX alginate. The level

of the dark density of the shadows on the aggregates was

also affected by the RGD ratio.

Figure 2 shows the effects of the RGD ratio in MIX gels

prepared from RGD- and PHSRN-modified alginates. The

graphs are based on representative data from two inde-

pendent experiments after normalization against data from

a normal culture dish. The RGD ratio in the MIX gels

affected the cell number and the gels with 67 % RGD

showed the maximum cell number after a 2-week incuba-

tion (Fig. 2a). No cells were detected when NHOsts were

cultured on unmodified alginate gels after a 2-week incu-

bation as described above. The differentiation level of

NHOsts cultured on various peptide-modified alginate gels

was estimated using alkaline phosphatase (ALP) activity,

and the amounts of osteocalcin secretion into the medium

after a 2-week incubation. ALP activity of the NHOsts, and

the amounts of osteocalcin secreted from the NHOsts were

normalized by the cell number. When the NHOsts were

cultured on the gel, ALP activity after a 2-week incubation

was around 50 % of the value for NHOsts on a 24-well

tissue culture plate, irrespective of the RGD ratio in the gel

(Fig. 2b). In contrast, osteocalcin production was influ-

enced by the RGD ratio in the MIX alginates (Fig. 2c).

Similar to the cell number, osteocalcin production in the

medium was highest after a 2-week incubation when

NHOsts were cultured on the MIX gel with 67 % RGD.

Calcium deposition was too low to be estimated, possibly

due to the low cell number on the gels.

Studies with cells encapsulated within the various gels

were then performed. Since the cell number encapsulated

in each gel disc was calculated to be 2.4 9 104, the same

number of cells was seeded on a 24-well culture plate as a

control. Microscopic observation of NHOsts in alginate

gels after their encapsulation revealed that almost cells had

a round shape, irrespective of gel type. Even encapsulated

in a MIX gel with a 67 % RGD ratio, only a few spreading

cells were found by microscopic observation (data not

shown). Alizarin red staining indicated that (1) the white

deposits observed inside the gels contained calcium and (2)

NHOsts cultured in MIX-alginate gels deposited more

calcium than did NHOsts in unmodified- and PHSRN-

alginate gels. This latter finding was especially pronounced

in MIX gels with a 50 and 67 % RGD ratio (Fig. 3).

The numbers of NHOsts encapsulated in the various

alginate gels, expressed as normalized to the cell number,

following culture on a tissue culture plate were determined

after 4 weeks of culture. Twenty percent of the encapsu-

lated cells survived after 1 week, and the numbers were

largely unchanged at 2 weeks of culture (Fig. 4a). The cell

number decreased by 3 weeks, but the cell number in

Fig. 1 Light micrographs of

normal human osteoblasts

(NHOsts) cultured for 2 weeks

on various kinds of alginate

gels. RGD Arginine–glycine–

aspartic acid peptide, PHSRNproline–histidine–serine–

arginine–asparagine sequence

peptide, MIX alginate mixture

of RGD-modified and PHSRN-

modified alginate solutions

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123

MIX-alginate gels and RGD-modified alginate gels

increased slightly after 4 weeks, with the exception of the

MIX gel with 50 % RGD-modified alginate. The maximum

cell number ratio was observed when the cells were cul-

tured in MIX gels with 67 % RGD-modified alginate,

which is similar to the finding in the twodimensional cul-

ture (Fig. 2a), although statistically significant differences

were not observed among the cell number ratios in the

various alginate gels tested. The cell number ratio within

gels after a 4-week culture was similar to that found for

cells cultured on top of gels (2D-culture experiments, as

shown in Fig. 2a). The maximum ALP activity was

observed for encapsulated cells after a 3-week culture,

while the ALP activity of NHOsts cultured on the tissue

culture plates peaked at the 2-week culture time point and

decreased to a constant value (about 70 % activity of its

maximum value) thereafter (Fig. 4b). Osteocalcin in the

culture medium from NHOsts encapsulated in the alginate

gels could not be detected even after 4 weeks of culture.

Discussion

Many mammalian cells have been reported to poorly

adhere to alginate gels [9]. This has led to efforts to

improve alginate’s cell adhesive property in order to use it

as a scaffold for tissue engineering. Modification with

RGD peptides has been useful to improve the cell adhesive

property of many biomaterials [9–13, 15–17], and it has

previously been applied to alginate. As previously repor-

ted, although few cells adhere to native alginate, RGD

modification has been found to improve the attachment of

cells [9–13].

In addition to the RGD sequence, the PHSRN sequence

has also been reported to play an important role in the cell

adhesion properties of fibronectin, as it works synergisti-

cally with RGD to increase the binding strength to integrin

molecules [18, 20]. Since interactions between fibronectin

and integrins have been reported to be required for osteo-

blast differentiation [21], we hypothesized that co-presen-

tation of RGD and PHSRN in the same material may

increase cell adhesion and enhance the cell response to the

material. Many researchers have reported the effects of

material modification by RGD and PHSRN peptides on

various cell behaviors, such as cell adhesion [22–24], for-

eign giant cell formation [25], and rat calvaria osteoblast

differentiation [26]. The results of our study also suggest

that a combination of RGD and PHSRN synergistically

affect normal human osteoblast behaviors. As shown in

Fig. 2, the number of cells on the alginate gels after a

Fig. 2 The cell number ratio,

calculated from the DNA

amounts in cell lysates relative

to those extracted from NHOsts

cultured on a 24-well tissue

culture plate (a), alkaline

phosphatase (ALP) activity

normalized by the DNA amount

(b), and total amount of

osteocalcin secreted into

medium in a 24-well plate (c),

after a 2-week incubation of

NHOsts on various kinds of

MIX alginate gels composed of

RGD- and PHSRN-modified

alginates. Closed circles in

b and c indicate values obtained

from NHOsts cultured on tissue

culture plates. All data are

expressed as the mean value ±

standard deviation (SD)

(n = 3–4)

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2-week incubation was affected by the RGD/PHSRN ratio.

In addition, although the ALP activity of NHOsts adhering

to various alginate gels was about one-half that of cells

adherent to tissue culture plates, irrespective of gel com-

position, osteocalcin production from whole NHOsts was

enhanced when they were cultured on the MIX gels with a

67 % RGD ratio. Considering the cell number, it is to be

expected that NHOsts on the MIX gel with a 67 % RGD

ratio showed higher osteocalcin production than any other

gels tested. Consequently, the osteocalcin amounts were

normalized by the cell number ratio, and the results are

shown in Fig. 5. As expected, NHOsts on the MIX gels

with a RGD ratio of [33 % showed almost a similar

enhancement in osteocalcin production, i.e., tenfold higher

than that of control NHOsts and about 1.5- to twofold

higher than that from NHOsts on RGD-modified gels.

However, only NHOsts on the MIX gel with the 67 %

RGD ratio showed a statistical enhancement in osteocalcin

production. These findings suggest that RGD peptides

mainly play an important role in NHOst proliferation and

differentiation on the gels but that they require a synergistic

interaction with PHSRN peptides to enhance the prolifer-

ation and differentiation. These findings also suggest that

the probability of NHOsts recognizing both one RGD and

one PHSRN, similar to the recognition of a fibronectin

molecule by their integrins, increases when the RGD ratio

of the MIX gel is[33 % and may be highest when the ratio

is about 67 %. Calcium deposition, however, was not

observed after a 2-week incubation when NHOsts were

cultured on any of the gel types.

On the other hand, NHOsts in the MIX gel showed the

maximum ALP activity after a 3-week incubation and

qualitatively higher calcium deposition than unmodified or

PHSRN-alginate gels after a 4-week incubation, irrespec-

tive of gel composition. Although NHOsts in the MIX gel

with 50 % RGD showed the highest ALP activity, their

calcium deposition might be lower than that observed in

the NHOsts cultured in the MIX gel with 67 % RGD, as

qualitatively assessed on the basis of alizarin red staining

of the gels. The findings from the 2D culture and these

findings suggest that the MIX gel with 67 % RGD ratio is

adequate for enhancing both cell differentiation and cell

proliferation not only in 2D culture but also in 3D culture

of NHOsts when the alginate gels prepared in this study are

utilized. It is probable that the RGD and PHSRN peptides

in the MIX gel have synergistic effects on the number of

NHOsts and their differentiation level even when encap-

sulated in the ge, and that these effects may influence the

calcium deposition level observed after a 4-week incuba-

tion (Fig. 3).

Fig. 3 Photographs of various kinds of alginate gels encapsulating NHOsts after a 4-week incubation before and after alizarin red staining. For a

positive reference, NHOsts cultured on a collagen-coated culture plate is shown in the figure

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The amino acid sequence of fibronectin suggests that the

appropriate RGD to PHSRN ratio in the MIX gel should be

50 %. However, the results of our study indicate that the

number of NHOsts and their differentiation may be at the

maximum when the RGD ratio is between 33 %

(RGD:PHSRN = 1:2) and 67 % (RGD:PHSRN = 2:1),

suggesting that NHOsts interacting with these gels can

simultaneously recognize both RGD and PHSRN peptides.

When the normal 3D structure of fibronectin in taken into

account, the appropriate distance between RGD and

PHSRN should be 3–4 nm. It has been previously reported

that adhesion of human endothelial cells is improved, and

similar to the level of fibronectin-covered surfaces, when

the surface presents RGD and PHSRN domains with a

spacing between 3 and 4 nm [27]. It has also been reported,

however, that the distance between RGD peptides in a 2 %

alginate gel prepared from RGD-alginate containing one

peptide per single alginate chain can be calculated to be

36 nm, assuming that a single alginate chain forms a single

domain in the gel [28]. As we prepared MIX-alginate gels

from RGD- and PHSRN-modified alginates, the MIX-

alginate gels had different total peptide amounts depending

on their RGD ratio. The initial distance between one

PHSRN peptide and a neighboring RGD peptide in the

MIX gels prepared in this study can be assumed to be

36 nm, which is about ninefold longer than the distance

between RGD and PHSRN in fibronectin. PHSRN did not

show any enhancing effects on the differentiation level of

NHOsts, but mixing the RGD with the PHSRN-coupled

alginate chains would increase the average spacing

between RGD peptides, which might offset the advantage

of adding the PHSRN and suppress the differentiation level

of the NHOsts. This analysis is further complicated by past

reports that adherent cells will cluster RGD peptides pre-

sented from the alginate, dramatically altering the initial

spacing [29]. However, the results of our study also suggest

that the differentiation level of NHOsts interacting with the

MIX gels was enhanced when their RGD ratio was[33 %

in the 2D culture. Ochsenhirt et al. [30] reported that

controlling not only the distance but also the secondary

structure of RGD- and PHSRN-modified surfaces is

important when the aim is to modulate cell behavior. It can

be assumed that a distance between RGD and PHSRN in

the gel is regulated by not only the molecular concentration

of the alginate and the extent of crosslinking but also by

interaction of the adhered NHOsts, which may cause

reorganization of alginate chain networks and the

Fig. 4 The cell number percentage ratio estimated from the results of

the Tetracolor One assay against those from NHOsts cultured on

tissue culture plates (a) and ALP activity ratio normalized by the

estimated DNA amounts (b) of NHOsts encapsulated in various kinds

of the MIX-alginate gels and incubated for 1 to 4 weeks. All data are

expressed as the mean value ± SD (n = 6–12)

Fig. 5 The ratio of osteocalcin production normalized by the cell

number ratio after NHOsts were incubated for 2 weeks on various

kinds of MIX-alginate gels composed of RGD- and PHSRN-modified

alginates. All data are expressed as the mean value ± SD (n = 3–4)

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secondary structure of RGD and PHSRN peptides in the

gel. It will be necessary to determine the actual distance

between RGD and PHSRN in this system and, if possible,

observe the reorganization of the network by NHOsts,

which may make the cells recognizing both RGD and

PHSRN initially separate more than 4 nm in the gels (e.g.,

utilizing fluorescence resonance energy transfer (FRET)

techniques [29]). Currently, we are measuring the distance

between RGD and PHSRN in the MIX gel utilizing the

FRET technique to clarify the effect of their spacing on cell

behavior. The results will be reported in the near future.

The 3D culture of cells has been reported to affect cell

shape, gene expression, and protein and extracellular

matrix production and to lead to significant differences in

2D culture [31–33]. When NHOsts were cultured in algi-

nate gels, their behaviors, such as shape, proliferation, ALP

activity profiles, and calcium deposition, were different

from those observed in cells cultured on a normal culture

dish (2D culture). Although it was very difficult to clearly

observe NHOsts encapsulated in alginate gels by light

microscopy, our microscopic observation revealed that the

encapsulated NHOsts in all of the gels had a round shape,

indicating that it is very difficult for them to take on a

spreading shape as normally seen in 2D culture. This

finding suggests that encapsulated cells tend to acquire a

round shape because they are surrounded by alginate gels

that they can utilize as their scaffold, especially when the

gels are prepared with RGD or MIX-alginate. Since they

cannot take on a spreading shape in the gels, it is probable

that they have different proliferation profiles as compared

to those observed in our 2D culture study. In addition,

taking into consideration the ALP activity profiles (Fig. 4b)

and calcium deposition after the 4-week incubation

(Fig. 3), a decrease in the cell number ratio at the 3-week

culture time point suggests that changes in NHOsts, such as

osteoblastic maturation, may occur at this time point. This

may also be one explanation of why the proliferation

profiles of NHOsts in 3D culture differ from those of

NHOsts in the 2D culture.

Not only did the proliferation of NHOsts in 3D and 2D

culture differ, but also the ALP activity profiles. The

highest ALP activities of the encapsulated NHOsts were

observed after a 3-week incubation, while the ALP activity

of the 2D-cultured NHOsts showed the highest value after

a 2-week incubation. This difference in the ALP profiles

may be ascribed to a difference in the culture condition,

namely, 2D versus 3D. Irrespective of the ALP activity

profile, calcium deposition was observed in unmodified,

RGD-modified, and MIX gels which encapsulated NHOsts,

although a small level of calcium deposition was also

observed on the same gels with 2D-cultured NHOsts.

When gels were prepared from a MIX-alginate, the gels

showed a potential to enhance the differentiation level of

NHOsts in a 3D culture system, irrespective of the specific

gel composition tested. When NHOsts are cultured on a

gel, they can recognize the modified peptides only from the

one surface they utilize to adhere, whereas in a 3D culture

system they can recognize the peptides on all surfaces since

they are surrounded by the gel. The lack of calcium

deposition in PHSRN-modified alginate gels encapsulating

NHOsts suggests that PHSRN does not enhance the dif-

ferentiation level of the encapsulated NHOsts and may

suppress differentiation in 3D culture. Given the higher

ALP activities of NHOsts encapsulated in MIX gels com-

pared to those in RGD-alginate gels (Fig. 4b), these results

suggest that synergistic effects of RGD and PHSRN pep-

tides on osteoblastic differentiation occur even in the 3D

culture system. NHOsts are able to recognize the modified

peptides only from the one surface they utilize for adher-

ence in the 2D culture system, whereas they can recognize

the peptides on all surfaces in the 3D culture system since

they are surrounded by the gel. This difference between the

2D and 3D culture system potentially results in a different

synergistic effect of the RGD and PHSRN peptides on

osteoblastic differentiation as well as on their proliferation.

Interestingly, even when encapsulated in unmodified algi-

nate gels, NHOsts demonstrated calcium deposition after a

4-week culture (Fig. 3), while few cells were observed

when NHOsts were cultured on the same gel (Fig. 1). This

3D culture effect on the differentiation must be further

clarified, as must the synergistic effect of the two peptides.

It was surprising that no osteocalcin was detected from

culture medium of the 3D cultures. The ELISA kit used in

this study is for detecting active osteocalcin (gamma-car-

boxylated formed), and it is known that active osteocalcin

can bind hydroxyapatite. As calcium deposition was

observed in 3D cultures, we hypothesize that all secreted

active osteocalcin bound to the deposited calcium. Addi-

tional studies, such as RT-PCR measurements of osteo-

calcin mRNA, are needed to test this hypothesis. Moreover,

analysis of the mechanical properties of the mineralizing

alginate gels encapsulating the NHOsts (Fig. 3) should be

performed.

Conclusion

The results of this study demonstrate that co-presentation

of RGD and PHSRN peptides can be utilized to improve

various cell behaviors, especially differentiation. Mixed

gels formed from a combination of RGD-modified and

PHSRN-modified alginates enhanced the differentiation of

NHOsts when the RGD ratio was[33 %. In 3D culture, the

differentiation level of NHOsts was qualitatively enhanced

when they were encapsulated in the MIX gel with a 50 or

67 % RGD ratio. Synthesizing biomaterials mimicking the

J Artif Organs

123

chemistry of natural cell adhesive proteins, such as fibro-

nectin, may be a useful way to develop scaffolds for bone

tissue engineering. In addition, our results suggest that the

effects of 3D culture on cell behaviors should be studied in

more detail, since the differentiation level of the cell in 3D

culture may be more highly enhanced than that observed in

2D culture based on qualitative results of calcium deposi-

tion, whereas the cell number in 3D culture may be similar

to that in 2D culture. Further studies are necessary to

clarify the mechanism by which 3D culture utilizing the

MIX-alginate gel consisting of RGD- and PHSRN-modi-

fied alginates enhances cell differentiation and

proliferation.

Acknowledgments This work was partly supported by Health and

Labour Sciences Research Grants for Research on Regulatory Science

of Pharmaceuticals and Medical Devices by Ministry of Health,

Labour and Welfare (H24-Iyaku-Shitei-018). The authors are also

grateful to Dr. Kuen Yong Lee (Hanyang University, South Korea),

Dr. Hyun-Joon Kong (University of Illinois, USA), and Dr. Takuya

Matsumoto (Okayama University, Japan) for many helpful

discussions.

References

1. Shoichet MS, Li RH, White ML, Winn SR. Stability of hydrogels

used in cell encapsulation: an in vitro comparison of alginate and

agarose. Biotechnol Bioeng. 1996;50:374–81.

2. Jen AC, Wake MC, Mikos AG. Hydrogels for cell immobiliza-

tion. Biotechnol Bioeng. 1996;50:357–64.

3. Mooney DJ, Mikos AG. Growing new organs. Sci Am. 1999;280:

38–43.

4. Lee KY, Mooney DJ. Hydrogel for tissue engineering. Chem

Rev. 2001;101:1869–80.

5. Griffith LG, Naughton G. Tissue engineering—current challenges

and expanding opportunities. Science. 2002;295:1009–14.

6. Langer R, Tirrell DA. Designing materials for biology and

medicine. Nature. 2004;428:487–92.

7. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive

extracellular microenvironments for morphogenesis in tissue

engineering. Nat Biotech. 2005;23:47–55.

8. Smidsrød O, Skjak-Bræk G. Alginate as immobilization matrix

for cells. Trends Biotechnol. 1990;8:71–8.

9. Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as

synthetic extracellular matrix materials. Biomaterials. 1999;20:

45–53.

10. Alsberg E, Anderson KW, Albeiruti A, Rowley JA, Mooney DJ.

Engineering growing tissues. Proc Natl Acad Sci USA. 2002;99:

12025–30.

11. Alsberg E, Kong HJ, Hirano Y, Smith MK, Albeiruti A, Mooney

DJ. Regulating bone formation via controlled scaffold degrada-

tion. J Dent Res. 2003;82:903–8.

12. Comisar WA, Hsiong SX, Kong HJ, Mooney DJ, Linderman JJ.

Multi-scale modeling to predict ligand presentation within RGD

nanopatterned hydrogels. Biomaterials. 2006;27:2322–9.

13. Kong HJ, Boontheekul T, Mooney DJ. Quantifying the relation

between adhesion ligand-receptor bond formation and cell phe-

notype. Proc Natl Acad Sci USA. 2006;103:18534–9.

14. Ruoslahti E, Pierschbacher MD. New perspectives in cell adhe-

sion: RGD and integrins. Science. 1987;238:491–7.

15. Hirano Y, Okuno M, Hayashi T, Goto K, Nakajima A. Cell-

attachment activities of surface immobilized oligopeptides RGD,

RGDS, RGDV, RGDT, and YIGSR toward five cell lines.

J Biomater Sci Polym Ed. 1993;4:235–43.

16. Masters KS, Shah DN, Walker G, Leinwand LA, Anseth KS.

Designing scaffolds for valvular interstitial cells: cell adhesion

and function on naturally derived materials. J Biomed Mater Res.

2004;71A:172–80.

17. Lee MH, Adams CS, Boettiger D, DeGrado WF, Shapiro IM,

Composto RJ, Ducheyne P. Adhesion of MC3T3-E1 cells to RGD

peptides of different flanking residues: detachment strength and

correlation with long-term cellular function. J Biomed Mater Res.

2007;81A(150–60):2007.

18. Aota S, Nomizu M, Yamada KM. The short amino-acid-sequence

Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhe-

sive function. J Biol Chem. 1994;269:24756–61.

19. Ohyama M, Suzuki N, Yamaguchi Y, Maeno M, Otsuka K, Ito K.

Effect of enamel matrix derivative on the differentiation of

C2C12 cells. J Periodontol. 2002;73:543–50.

20. Redick SD, Settles DL, Briscoe G, Erickson HP. Defining

fibronectin’s cell adhesion synergy site by site-directed muta-

genesis. J Cell Biol. 2000;149:521–7.

21. Moursi AM, Globus RK, Dmasky CH. Interactions between

integrin receptors and fibronectin are required for calvarial

osteoblast differentiation in vitro. J Cell Sci. 1997;110:2187–96.

22. Fittkau MH, Zilla P, Bezuidenhout D, Lutolf MP, Human P,

Hubbell JA, Davies N. The selective modulation of endothelial

cell mobility on RGD peptide containing surfaces by YIGSR

peptides. Biomaterials. 2005;26:167–74.

23. Petrie TA, Capadona JR, Reyes CD, Garcıa AJ. Integrin speci-

ficity and enhanced cellular activities associated with surfaces

presenting a recombinant fibronectin fragment compared to RGD

supports. Biomaterials. 2006;27:5459–70.

24. Shibasaki Y, Hirohara S, Terada K, Anod T, Tanihara M. Col-

lagen-like polypeptide Poly(Pro-Hyp-Gly) conjugated with Gly-

Arg-Gly-Asp-Ser and Pro-His-Ser-Arg-Asn peptides enhances

cell adhesion, migration, and stratification. Biopolymers (Pept

Sci). 2011;96:302–15.

25. Kao WJ, Lee D, Schense JC, Hubbell JA. Fibronectin modulates

macrophage adhesion and FBGC formation: The role of RGD,

PHSRN, and PRRARV domains. J Biomed Mater Res. 2001;55:

79–88.

26. Benoit DSW, Anseth KS. The effect on osteoblast function of

colocalized RGD and PHSRN epitopes on PEG surfaces. Bio-

materials. 2005;2005(26):5209–20.

27. Mardilovich A, Craig JA, McCammon MQ, Garg A, Kokkoli E.

Design of a novel fibronectin-mimic peptide—amphiphile for

functionalized biomaterials. Langmuir. 2006;22:3259–64.

28. Lee KY, Alsberg E, Hsiong S, Comisar W, Linderman J, Ziff R,

Mooney D. Nanoscale adhesion ligand organization regulates

osteoblast proliferation and differentiation. Nanoletters. 2004;4:

1501–6.

29. Kong HJ, Polte TR, Alsberg E, Mooney DJ. FRET measurements

of cell-traction forces and nano-scale clustering of adhesion

ligands varied by substrate stiffness. Proc Natl Acad Sci USA.

2005;102:4300–5.

30. Ochsenhirt SE, Kokkoli E, McCarthy JB, Tirrell M. Effect of

RGD secondary structure and the synergy site PHSRN on cell

adhesion, spreading and specific integrin engagement. Biomate-

rials. 2006;27:3863–74.

31. Gruber HE, Hanley Jr EN. Human disc cells in monolayer vs 3D

culture: cell shape, division and matrix formation. BMC

J Artif Organs

123

Musculoskelet. Disord. 2000; 1:1. doi:10.1186/1471-2474-1-1.

Available at: http://www.biomedcentral.com/1471-2474/1/1.

32. Hishikawa K, Miura S, Marumo T, Yoshioka H, Mori Y, Takato

T, Fujita T. Gene expression profile of human mesenchymal stem

cells during osteogenesis in three-dimensional thermoreversible

gelation polymer. Biochem Biophys Res Commun. 2004;

317:1103–7.

33. Winters BS, Raj BK, Robinson EE, Foty RA, Corbett SA. Three-

dimensional culture regulates Raf-1 expression to modulate

fibronectin matrix assembly. Mol Biol Cell. 2006;17:3386–96.

J Artif Organs

123