Materials Science and Engineering C 48 (2015) 301309

Contents lists available at ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

Adhesion and growth of human bone marrow mesenchymal stem cells

on precise-geometry 3D organicinorganic composite scaffolds for
bone repair
Maria Chatzinikolaidou a,c,, Sima Rekstyte c, Paulius Danilevicius c, Charalampos Pontikoglou b,
Helen Papadaki b, Maria Farsari c, Maria Vamvakaki a,c
a
b
c

Department of Materials Science and Technology, University of Crete, Greece

Hematology Laboratory, School of Medicine, University of Crete, Greece
Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), Greece

a r t i c l e

i n f o

Article history:
Received 5 June 2014
Received in revised form 3 November 2014
Accepted 4 December 2014
Available online 5 December 2014
Keywords:
Human bone marrow mesenchymal stem cells
3D scaffold fabrication
Hybrid material
Cell adhesion and proliferation
Cell variability

a b s t r a c t
Engineering biomaterial scaffolds that promote attachment and growth of mesenchymal stem cells in three dimensions is a crucial parameter for successful bone tissue engineering. Towards this direction, a lot of research
effort has focused recently into the development of three-dimensional porous scaffolds, aiming to elicit positive
cellular behavior. However, the fabrication of three-dimensional tissue scaffolds with a precise geometry and
complex micro- and nano-features, supporting cell in-growth remains a challenge. In this study we report on a
positive cellular response of human bone marrow-derived (BM) mesenchymal stem cells (MSCs) onto hybrid
material scaffolds consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide, and 2(dimethylamino)ethyl methacrylate (DMAEMA). First, we use Direct fs Laser Writing, a 3D scaffolding technology to fabricate the complex structures. Subsequently, we investigate the morphology, viability and proliferation
of BM-MSCs onto the hybrid scaffolds and examine the cellular response from different donors. Finally, we explore the effect of the materials' chemical composition on cell proliferation, employing three different material
surfaces: (i) a hybrid consisting of methacryloxypropyl trimethoxysilane, zirconium propoxide and 50 mol%
DMAEMA, (ii) a hybrid material comprising methacryloxypropyl trimethoxysilane and zirconium propoxide,
and (iii) a purely organic polyDMAEMA. Our results show a strong adhesion of BM-MSCs onto the hybrid material
containing 50% DMAEMA from the rst 2 h after seeding, and up to several days, and a proliferation increase after
14 and 21 days, similar to the polystyrene control, independent of cell donor. These ndings support the potential
use of our proposed cellmaterial combination in bone tissue engineering.
2015 Elsevier B.V. All rights reserved.

1. Introduction
A common objective in bone tissue engineering research is the design of biomaterial scaffolds that support cell and tissue growth [1].
Many synthetic structures have been designed to impart bulk properties
to the construct, such as adequate mechanical strength and sufcient
transport properties for cell inltration and tissue organization. Although the majority of these structures possess similar macroscopic
properties as those of the native tissue, the constructs have failed prior
Abbreviations: BM, bone marrow; MSCs, mesenchymal stem cells; DMAEMA, 2(dimethylamino)ethyl methacrylate; ZPO, zirconium propoxide; MAPTMS,
methacryloxypropyl trimethoxysilane; 3D, three-dimensional; DLW, Direct Laser
Writing; FCS, fetal calf serum; SEM, scanning electron microscopy; ANOVA, analysis of variance; P, passage.
Corresponding author at: University of Crete, Dept. of Materials Science and
Technology & IESL-FORTH, P.O. Box 2208, GR-71003 Heraklio, Greece.
E-mail addresses: mchatzin@materials.uoc.gr, mchatzin@gmail.com
(M. Chatzinikolaidou).

http://dx.doi.org/10.1016/j.msec.2014.12.007
0928-4931/ 2015 Elsevier B.V. All rights reserved.

to full healing [2,3]. An important parameter identied in the failure of

tissue-engineered constructs is insufcient tissue regeneration around
the biomaterial directly after implantation. Since the interaction of
cells with the biomaterial is a vital element in the evaluation of a scaffold, a lot of research effort focuses on designing biomaterial structures
that facilitate favorable interactions and enhance tissue regeneration.
Moreover, the scaffolds designed for tissue engineering applications
must be three-dimensional, highly porous and interconnected to support cell attachment and proliferation [4,5]. The role of the scaffold porosity in bone regeneration has been investigated by Kuboki et al.
using a rat ectopic model and solid porous particles [6]. Pores are necessary for bone tissue formation because they allow migration and proliferation of osteoblasts and mesenchymal cells, as well as angiogenesis
and vascularization. In addition, a porous surface improves mechanical
interlocking between the implant biomaterial and the surrounding natural bone, providing greater mechanical stability at this critical interface
[7]. The minimum pore size required to regenerate mineralized bone is
generally considered to be 100 m after the study of Hulbert and Proc

302

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

[8]. Materials used for fabricating scaffolds for bone tissue-engineering

application must also have sufcient structural integrity matching the
mechanical properties of native tissue, and should offer an ideal and
critical micro-environment to function as an articial extra-cellular matrix (ECM) onto which cells attach, grow, and form new tissues [9,10].
Hybrid materials and composites with tunable mechanical, chemical,
and biological properties exhibit advantageous features and have
attracted particular research attention in recent years [11].
Most available scaffold fabrication methods, such as solvent casting,
ber bonding, phase separation, gas induced foaming, and salt leaching,
are either limited to producing scaffolds with simple geometry, or depend on an indirect casting method for scaffold fabrication [12]. They
are therefore insufcient for the manufacturing of scaffolds with complex, accurate structural architectures, and rather result in structures
of random internal architecture and great structural variation.
Direct Laser Writing (DLW) by multi-photon polymerization (MPP)
[13] enables the design and fabrication of fully three-dimensional (3D),
readily-assembled microstructures with sub-100 nm resolution [14]
and complex internal and external architectures, thereby allowing precise micro-topography control [15,16,41]. Control over characteristics
such as scaffold porosity, pore size, and permeability may enhance cell
inltration and mass transport of nutrients and metabolic waste
throughout the scaffold. Several authors have discussed the advantages
of the DLW technique currently in use [1721].
Recent advances in stem cell research have provided the theoretical background for the development of cell-based therapies for bone
repair. Over the last years, mesenchymal stem cells (MSCs) have
emerged as promising candidates for orthopedic applications. Originally isolated from the bone marrow (BM), MSCs are considered as
the progenitors for skeletal tissues, and both preclinical and clinical
data support the notion that these cells have a great potential for
bone regeneration [2224]. Cells with similar characteristics to BMMSCs have also been isolated from various other tissues [25]. Yet
BM-MSCs represent the most extensively studied population of
adult MSCs and are still considered as the gold standard for MSC clinical application.
In a previous study we showed the positive cellular response of the
pre-osteoblastic cell line MC3T3-E1 on a hybrid material comprising
methacryloxypropyl trimethoxysilane (MAPTMS), zirconium propoxide
(ZPO), and 50 mol% 2-(dimethylamino)ethyl methacrylate (DMAEMA),
and its favorable mechanical properties for scaffold fabrication [11].
In this work, we explore the interactions of BM-MSCs with precise,
complex-geometry 3D scaffolds fabricated by the technology of DLW
and consisting of a hybrid material containing MAPTMS, ZPO and 50%
DMAEMA. For this, we investigate the initial adhesion of BM-MSCs on
the 3D structures within the rst 2 h post-seeding, and up to 7 days. To
explore the inuence of the materials' chemical composition on the cell
proliferation, we use three materials, (i) a hybrid consisting of MAPTMS,
ZPO and 50 mol% DMAEMA, (ii) a hybrid comprising MAPTMS and ZPO,
and (iii) a purely organic polyDMAEMA, to fabricate lms on glass substrates, and we quantify the cell proliferation of BM-MSCs on them. Finally, we report on the variability of the proliferation of BM-MSCs derived
from three different donors.

2. Materials and methods

2.1. Hybrid-50% DMAEMA material
The material used for the fabrication of 2D lms and 3D structures
is an organicinorganic composite comprising MAPTMS (99%), ZPO
(70% in propanol) and DMAEMA (N99%) [11]. 4,4-Bis(diethylamino)
benzophenone (BIS) was used as the photoinitiator. DMAEMA was
copolymerized with MAPTMS upon photopolymerization, whereas ZPO
and the alkoxysilane groups of MAPTMS served as the inorganic network
forming moieties.

2.2. Sample fabrication: thin lms and 3D structures

Three types of specimens, all prepared on round glass substrates
with a diameter of 15 mm and a thickness of 100 m, were employed
in this study: (i) two-dimensional lms for the quantication of the
cell viability and proliferation; (ii) 3D square blocks with dimensions
200 200 10 m (l w h) for the investigation of cell adhesion
and cell morphology by immunocytochemical staining and scanning
electron microscopy (SEM); and (iii) 3D scaffolds with a high precision
cubic geometry made from horizontally and vertically ordered ring
structures for the investigation of cell adhesion by immunocytochemistry and SEM.
For the two-dimensional coatings we used three material compositions: (a) a hybrid material consisting of MAPTMS and ZPO (referred
to as hybrid); (b) a hybrid organicinorganic material of the above
two components supplemented with 50 mol% of DMAEMA (referred
to as hybrid-50% DMAEMA); and (c) a purely organic material based
on DMAEMA (referred to as organic) (see Table 1).
Material specimens used for the adhesion, viability and proliferation
experiments were incubated for 1 h in ethanol, air-dried under sterile
conditions in a laminar ow and rinsed briey with DMEM cell culture
medium prior to cell seeding.
2.2.1. Thin lm preparation
Thin lms of the hybrid and hybrid-50% DMAEMA materials were
prepared by drop-casting or spin-coating onto 100 micron-thick
silanized glass substrates. The samples were heated in an oven at
50 C for 5 min before the photopolymerization, which led to the
condensation of the alkoxide groups and the formation of the inorganic matrix. Next, the methacrylate moieties were polymerized
using a KrF excimer laser, operating at 248 nm, resulting in the formation of irreversible and fully saturated aliphatic CC covalent
bonds that further increase the connectivity of the material. Finally,
the samples were developed for 30 min in a 50:50 solution of 1propanol:isopropanol to remove the non-polymerized material.
Polymer thin lms based on DMAEMA were also prepared on
glass slides using surface initiated atom transfer radical polymerization (ATRP). First, a self-assembled monolayer of the in-house synthesized initiator 3-(2-bromoisobutyramido)-propyl(triethoxy)
silane was formed by immersing the glass substrates in a THF solution of the initiator for 24 h. Next, the substrate was rinsed extensively with THF and ethanol, dried under a stream of nitrogen and
transferred to the polymerization ask. The polyDMAEMA chains
were synthesized by ATRP on the initiator-functionalized glass substrates in a 4:1 methanol:water mixture. The polymerization was
allowed to proceed at room temperature for 5 h, after which the substrate was removed and rinsed thoroughly with water and methanol
and was dried under a nitrogen ow.
2.2.2. Fabrication of 3D hybrid blocks and scaffolds by Direct fs Laser
Writing
The experimental setup employed for 3D structure fabrication has
been described previously [11,26]. A Ti:Sapphire femtosecond laser
(Femtolasers Fusion, 800 nm, 75 MHz, b 20 fs) was focused into the
photopolymerizable composite using a microscope objective lens
(20, N.A. = 0.65 and 40 N.A. = 0.95, Zeiss, Plan Apochromat). Sample movement was achieved using piezoelectric and linear stages, for
ne and step movement, respectively (Physik Instrumente GmbH,
Germany). The whole DLW setup was computer-controlled using the
3DPoli software.
2.3. Isolation and expansion of BM-MSCs
Bone marrow was aspirated from the posterior iliac crest of (n = 3)
of individuals undergoing orthopedic surgery. Institutional ethics committee approval was granted prior to the study and informed consent

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

303

Table 1
Organic and hybrid lm characteristics [11].
Film

Composition

Thickness by ellipsometry (nm)

Roughness by AFM (nm)

Static contact angle []

Organic
Hybrid
Hybrid-50% DMAEMA

PDMAEMA
MAPTMS + ZPO
50 mol% MAPTMS + ZPO + 50 mol% DMAEMA

50 2
6300 2
2365 2

0.5
0.3
0.4

70 2
71 2
85 2

according to Helsinki Declaration was provided from all subjects included in the study. BM cells were subsequently diluted 1:1 in Dulbecco's
Modied Eagle Medium-Low Glucose (DMEM-LG; Gibco Invitrogen,
Paisley Scotland) supplemented with penicillinstreptomycin (PS;
Gibco) and preservative-free heparin (Sigma, Saint Louis MO). BM
mononuclear cells (BMMNCs) were obtained following gradient centrifugation on Histopaque-1077 (Sigma) and were cultured in DMEM-LG/
10% fetal calf serum (FCS; Hyclone, Logan, Utah, USA)/100 IU/ml Pen/
Strep (thereafter referred as MSC medium) at a concentration of
2 105 cells/cm2 in 25 cm2 culture asks at 37 C/5%CO2 fully humidied atmosphere. One to two days post-seeding, non-adherent cells
were removed and from then on the medium was replaced twice per
week. Upon 7090% conuence, the cells were detached using
0.25% trypsin/0.1 mM EDTA (Gibco) and re-seeded at a concentration
of 2 103 cells/cm2 (passage 1, P1).
2.4. MSC differentiation assays
Trypsinized MSCs from passage 2 were induced to differentiate into
adipocytes and osteoblasts as previously described [2729]. Briey, for
the adipogenic differentiation, cells were cultured for 3 weeks in MSC medium, supplemented with 10% FCS, 0.5 mM 1-methyl-3-butylisoxanthine,
1 mM dexamethasone, 0.2 mM indomethacin, and 10 mg/mL insulin.
Lipid vacuoles were revealed by Oil Red O staining and were visualized
under a microscope (Zeiss). For the osteogenic differentiation, cells
were cultured in MSC medium supplemented with 0.1 mM dexamethasone, 0.15 mM ascorbate-2-phosphate, and 3 mM NaH2PO4. Osteogenesis
was assessed by Von Kossa staining. All reagents for adipogenic and osteogenic induction were purchased from Sigma.
2.5. MSC immunophenotypic characteristics
Trypsinized MSCs from P2 were washed twice with PBS containing 1%
FCS and then labeled with anti-CD29 (4B4; Cyto-Stat/Beckman-Coulter,
Florida, USA), anti-CD73 (AD2; Pharmingen, San Diego, CA), anti-CD90
(F15.42; Immunotech/Coulter, Marseille, France), anti-CD105 (SN6;
Caltag, Burlingame, CA), anti-CD45 (IMMU19.2; Immunotech/Coulter),
anti-CD14 (RMO52; Immunotech/Coulter) and anti-CD34 (QBend10;
Beckman-Coulter, Florida, USA) monoclonal antibodies (mAbs). A
mouse anti-human IgG1 mAb purchased from Caltag (MOPC31C) was
used as isotypic control. Following 30 min incubation on ice, in the dark,
cells were washed twice with PBS prior to ow cytometric analysis.
Data were processed in Beckman Coulter Cytomics FC500 (Coulter,
Miami, FL, USA).
2.6. Scanning electron microscopy (SEM)
2 104 BM-MSCs from passages 23 in DMEM-LG were seeded on
specimens with 3D scaffolds consisting of the hybrid-50% DMAEMA material and placed in the cell culture incubator at 37 C for 2 h. Specimens
were then removed from the incubator and rinsed three times with PBS,
xed with 2% paraformaldehyde for 1 h, post-xed with 1% osmium tetroxide, and dehydrated in increasing concentrations (from 30100%)
of ethanol. The specimens were then dried in a critical point drier
(Baltec CPD 030), sputter-coated with a 15 nm thick layer of gold
(Baltec SCD 050) and observed under a scanning electron microscope
(JEOL JSM-6390 LV) at an accelerating voltage of 15 kV.

2.7. Laser scanning confocal uorescence microscopy

A suspension of 2 104 BM-MSCs from passages 23 in DMEM-LG
was seeded on specimens with 3D scaffolds consisting of the hybrid50% DMAEMA material and placed in the cell culture incubator at 37 C
for 24 h. Cells on specimens were rinsed with PBS, xed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100/PBS for
5 min on ice. The non-specic binding sites were blocked with a 2%
BSA solution in PBS for 60 min. Actin and vinculin focal adhesion complexes were stained by incubating cells-on-specimens in 100 l diluted
uorescein isothiocyanate-conjugated anti-vinculin primary antibody
(FITC-conjugated anti-vinculin, Sigma-Aldrich Chemie GmbH, Munich,
Germany) in blocking solution (1:100) for 1 h and subsequently staining
them with simultaneous incubation using 100 l 1 mg/ml tetramethyl
rhodamine isothiocyanate-conjugated phalloidin (TRITC-phalloidin conjugate, Sigma-Aldrich Chemie GmbH, Munich, Germany) for 15 min.
The samples were then washed with PBS and observed under a Leica
DM IRBE laser scanning confocal microscope.
2.8. Cell proliferation on the materials
Focusing on the hybrid-50% DMAEMA material for the fabrication of
the high-geometry scaffolds, we address the question of whether the
chemical composition of the hybrid affects cell proliferation. We therefore fabricated separately coatings consisting of the different components used in the hybrid-50% DMAEMA material, and explored the cell
growth on them at various time points, and for cells derived from the
three donors.
10 103 BM-MSCs from passages 24 in DMEM-LG were seeded on
specimens with lm coatings consisting of our testing materials and
placed in the cell culture incubator at 37 C. On days 7, 14 and 21
post-seeding cell viability and proliferation assay was performed with
the resazurin-based PrestoBlue reagent (Invitrogen) according to
the manufacturer's instructions. The reagent was incubated on the
cells at 37 C for 60 min. The absorbance was measured in a spectrophotometer (Molecular Devices SpectraMax M2) and cell number quantication was performed by means of a calibration curve. Error bars
represent the average of triplicates STDV in three independent experiments. The percent viability of the cells on the examined surfaces was
calculated from the mean values of proliferation, related to the polystyrene tissue culture treated surface.
2.9. Statistical analysis
Statistical analysis was performed using the one-way ANOVA
Dunnett's test. To statistically evaluate the difference in cell viability
and proliferation after certain time points (7, 14, and 21 days), we compared all three material chemical compositions at each time point
against the control tissue culture treated polystyrene surface.
3. Results
3.1. Samples preparation and characterization
For the biological investigation of the materials we fabricated two
types of samples: thin lms and 3D structures. The physicochemical
characteristics of the coated materials related to lm roughness,

304

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

Fig. 1. SEM images of high-precision 3D scaffolds with complex geometry fabricated by Direct fs Laser Writing. (A) A cubic porous scaffold consisting of horizontal and vertical ring structures in 170-fold magnication and (B) a scaffold cell unit in 550-fold magnication.

thickness and hydrophilicity have been reported previously [11] and are
summarized in Table 1. For the 3D structuring of the hybrid composite,
the DLW parameters were rst optimized for this material. The 3D
structures were written with a 40 plan apochromat lens (N.A. =
0.95), with a typical laser power of 30 mW and a writing speed of
2 mm/s. Fig. 1 shows a 3D structure fabricated by DLW (A) and a single
unit of the structure in a higher magnication (B). They consist of 3D

cubes, whose each side is a circle with an inner diameter of 100 m.

While it is possible to structure this material to sub-100 nm resolution
[30,31], in this case the feature size is in the order of 10 m, to give
the structure the mechanical strength required for a scaffold. The fabricated 3D scaffolds with a cubic geometry made from horizontally and
vertically ordered ring structures have inner diameters of 100 m and
a total dimension of 450 450 150 m. Their calculated porosity (P)

Fig. 2. Morphologic and immunophenotypic characteristics, and differentiation potential of BM-derived MSCs. (A) P2 BM-MSCs display an extended morphology. (B) Representative ow
cytometric characterization of P2 BM-MSCs. Gray-lled histograms depict the expression of CD73, CD90 and CD105 and the lack of CD14, CD34 and CD45. Open histograms depict isotypic
control. (C) Osteogenic differentiation evaluated by von Kossa staining (left) and adipogenic differentiation assessed by Oil Red O staining (right) of culture-expanded P2 BM-MSCs from a
representative donor.

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

305

Fig. 3. Scanning electron microscopy images showing adhered BM-MSCs on block structures (A) 24 h, (B) 3 days and (C) 7 days after seeding.

void
is 84%. It is calculated from CAD models asP VV total
 100, where Vtotal =
Vvoid + Vmaterial is the total volume of the rectangular space occupied by
a scaffold, Vvoid is the volume of an empty space, and Vmaterial is the volume occupied by the material.

3.2. Morphologic and immunophenotypic properties of BM-MSCs

MSCs were successfully expanded from all subjects included in
the present study. Adherent cells displayed the characteristic extended morphology (Fig. 2A), and immunophenotypic analysis at
the end of P2 demonstrated that cultures constituted of a homogeneous cell population expressing CD73, CD90 and CD105, surface antigens but lacking the hematopoietic markers CD45, CD14 and CD34
(Fig. 2B).

3.3. MSC differentiation potential

To evaluate the multilineage differentiation capacity of the cultureexpanded MSCs used in the present study, P2 cells from all three investigating donors were induced to differentiate into osteoblasts and adipocytes. Respective cytochemical staining (Fig. 2C) demonstrated that
MSC populations from every single donor had both osteogenic and
adipogenic differentiation potentials.
3.4. BM-MSCs' morphology on composite materials and scaffolds
We examined the adhesion potential and morphology of BM-MSCs
on block structures and complex-geometry scaffolds both made from
the same hybrid-50% DMAEMA material, by means of optical microscopy, scanning electron microscopy, and confocal laser uorescence

Fig. 4. Confocal uorescence microscopy images on block structures 24 h (top panel) and 7 days (bottom panel) after seeding. Actin staining is shown in red (A and D), vinculin in green (B
and E), whereas an overlay of both stains is shown in C and F. Scale bar in top panel is 40 m and in bottom panel 20 m.

306

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

microscopy at different time points after cell seeding. First, we investigated the adhesion of cells and their morphology on block structures
with plane surface to study the cellmaterial interactions (Figs. 3 and
4), and then we employed more complex geometry scaffolds to investigate the impact of the structure on cell adhesion and morphology
(Figs. 5 and 6).
Representative SEM images (Figs. 3 and 5) demonstrate BM-MSCs
that adhere on 3D block structures and complex-geometry scaffolds
fabricated from the hybrid-50% DMAEMA material. Cells extend protrusions on the material within the rst hours after seeding, and proliferate
after 7 days. Specically, Fig. 3A shows that cells adhered after 24 h at
the edge of a block structure and the substrate glass surface. The observed fully spread cell morphology with extending cytoplasmic protrusions signals a strong initial cell adhesion within the rst hours after
seeding on the material. After one day, cells adhered well on the block
structure showing a attened morphology, and covered the edge of
the block structure by extending more elongated protrusions and
expanding to connect the space between the material and the substrate.
This is a characteristic morphology for cells seeded on a preferable, biocompatible material surface. After 3 days in culture, BM-MSCs indicate a
attened morphology as shown in Fig. 3B. After 7 days in culture, BMMSCs proliferate and cover completely the block structure with a

dense cell layer as shown in Fig. 3C. The observed strong initial adhesion, and the consequent proliferation increase validate the biocompatibility of the hybrid material.
The SEM images in Fig. 5 demonstrate a good initial attachment of
the BM-MSCs onto the high-precision 3D scaffolds from the rst two
hours post-seeding and up to 7 days. Only 2 h after seeding, cells at different scaffold locations appear to develop cytoplasmic extensions, facilitating their 3D attachment into the materials surface (Fig. 5A, B, C).
We observe characteristic spread cytoplasmic extensions of the cells
into the scaffold after 24 h in culture (Fig. 5D, E, F). The same spread
cell morphology can be observed 3 days after seeding (Fig. 5G, H, I).
After one week in culture, BM-MSCs proliferate and develop adhered
cell layers at different levels within the 3D scaffold, and form connections for their organization into tissue (Fig. 5I, K, L).
The morphology of the adhered BM-MSCs is visualized by confocal
uorescence microscopy on the hybrid-50% DMAEMA block structures
(Fig. 4) with plane surface and on scaffolds with a complex-geometry
(Fig. 6) at different time points. BM-MSCs display a well-organized cytoskeleton with a attened morphology 24 h after seeding on the block
structures, as reected by the actin staining shown in red (Fig. 4A).
The visualization of vinculin participating in focal adhesion points is
demonstrated in green in Fig. 4B. Fig. 4C depicts an overlay of the

Fig. 5. Scanning electron microscopy images showing adhered BM-MSCs onto different locations of the scaffolds' ring structures at various time points: (A, B, C) 2 h, (D, E, F) 24 h, (G, H, I)
3 days and (J, K, L) 7 days after seeding. For comparison with the ring structure without cells, refer to Fig. 1.

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

double-stain. After 7 days in culture, we observe a proliferation increase

of fully spread BM-MSCs, exhibiting an excellent morphology, and the
formation of a dense cell layer covering completely the surface of the
block structure as shown in the images after actin staining (Fig. 4D), vinculin staining (Fig. 4E) and in the overlay image of both stains (Fig. 4F).
The scaffolds exhibit a strong auto-uorescence due to the initiator used
for the material polymerization [32].
Our investigations on the BM-MSCs morphology onto the 3D scaffolds within the rst 2 h show the actin laments (Fig. 6A) and vinculin
(Fig. 6B) in the protrusions of a few adhered cells onto the scaffolds' ring
structures. An overlay image clearly depicts the cell attachment with the
elongated cytoplasmic formations 2 h after seeding (Fig. 6C). 24 h after
seeding, cells show an elongated morphology and develop connections
onto the inner walls of the scaffolds' ring structures (Fig. 6D, E, F). The
observed spread cell morphology and cytoplasmic extensions signal
good cell adhesion 24 h post-seeding on the material. After 3 days in culture, cells retain a strong adhesion prole, and they appear to partially
ll the space between the scaffold's ring structures with their connections. Images 6G and 6H show a well-organized cytoplasmic network

307

and the vinculin adhesion points, whereas Fig. 6I displays an overlay

of both stains. After 7 days in culture, strongly attached BM-MSCs proliferate and successively ll part of the volume inside the ring structures of
the scaffold, as shown from the actin stain (Fig. 6J), the vinculin stain
(Fig. 6K) and the overlay image (Fig. 6L).

3.5. BM-MSCs' proliferation on three material coatings and donor variability

We described above a qualitative study of the morphology of cells
adhered on the material. Here, we quantify the cell number cultured
on different material surfaces at different time periods. Fig. 7 A, B, C
shows the number of cells derived from three donors on days 7, 14,
and 21 in culture and on three material compositions, organic, hybrid
and hybrid-50% DMAEMA, compared to the polystyrene control surface.
BM-MSCs cultured on all three materials of different chemical compositions show a proliferation increase after 14 and 21 days, without any
statistically signicant difference to the control tissue culture treated
polystyrene surface. BM-MSCs cultured on the examined material

Fig. 6. Confocal uorescence microscopy images on the scaffolds' ring structures. Cell morphology is shown following double-staining with TRITC-phalloidin (red) and FITC-conjugated
anti-vinculin antibody (green). Images A, B, and C are taken 2 h, D, E, and F 24 h, G, H, and I 3 days and J, K, and L 7 days after cell seeding. The actin staining is visualized in A, D, G,
and J, the vinculin staining in B, E, H, and K, and the overlay images of double stains in C, F, I, and L. Scaffolds exhibit a strong auto-uorescence. Scale bar represents 20 m.

308

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

surfaces indicate at least 90% viability compared to the control polystyrene surface.
Regarding the variability in cellular response, which occurs when
the cells are derived from different donors, we investigated the viability
and proliferation of BM-MSCs from three different donors. The results
indicate that the cell viability and growth on the three material surfaces
are similar, and without any signicant differences, independent of the

Fig. 7. Bone marrow MSC proliferation on the three material coatings investigated by
means of the PrestoBlue assay after 7, 14 and 21 days in culture. Cells from three different human donors are investigated: (A) Donor #1; (B) donor #2; (C) donor #3. Error bars
represent the average of triplicates STDV in three independent experiments. Optical
density values are normalized to the cell number according to a calibration curve. Tissue
culture treated polystyrene surface was used as control.

cell donor. Error bars represent the average of triplicates STDV in

three independent experiments.
4. Discussion
The main objective of this study was to explore the interactions of BMMSCs, in terms of adhesion and proliferation, with hybrid material structures fabricated by Direct Laser Writing. Since our aim is to show the suitability of these materials for bone tissue repair, in which interconnected
pores are essential for nutrients and oxygen supply, we designed structures, such as those shown in Fig. 1, with specic architectural characteristics. Cells on a at surface grow by forming a monolayer, while a 3D cell
culture can only be achieved via cell growth in a 3D environment or scaffold [33]. Scaffold parameters including pore size, porosity, interconnectivity, and mechanical strength are considered important in order to
achieve a functional cell/scaffold construct for bone tissue engineering
[34]. Our hybrid scaffolds possess a complex 3D geometry with interconnected pores of diameters ranging from 100150 m, and a porosity of
84%.
Cellular populations used in the study were dened as BM-MSCs
based on their morphology, immunophenotype and differentiation potential, according to previously established criteria [35]. Since cell adhesion is a prerequisite for any further cellular function, we rst examined
the cell morphology on simple geometry block structures with plane
surface to investigate the initial and long-term interactions of the BMMSCs with the hybrid material surfaces. It is demonstrated that BMMSCs display a well-organized cytoskeleton with spread morphology
24 h after seeding on the block structures. The integration of scaffold
computational design and fabrication techniques could prove highly
useful for the construction of scaffolds that have anatomy specic exterior architecture and an interior porous architecture [36]. We therefore
focused next on designing and fabricating more complex geometry
cubic structures consisting of rings with a diameter of 100 m, which
has been reported to be the appropriate scaffold porosity for bone ingrowth [8]. Our observations regarding cell adhesion, both in the initial
stages and also in the long-term, validate the biocompatibility of the
hybrid-50% DMAEMA material, and its suitability to support tissue
formation. The confocal microscopy data in Fig. 6 depict the evolution
of cell attachment onto the scaffold pores over time. Cells exhibit a
well-organized actin cytoskeleton and focal adhesion points, which
evolved into a dense cell network within the scaffold pores after
7 days in culture. The interconnectivity between the cells, and also between the cells and the material evidenced by confocal uorescence
microscopy, together with the maintenance of an excellent cytoplasmic
morphology, indicate the potential of the hybrid-50% DMAEMA material to promote the in-growth of BM-MSCs, and therefore, to support tissue organization in three dimensions. Additional investigations on the
proliferation of BM-MSCs onto scaffolds made from the same material
comprising the 50% DMAEMA hybrid, with similar porosity and pore
size, and having hexagonal geometry of units instead of rings, revealed
a three-fold proliferation increase after 10 days in culture (data not
shown). In the same experimental set-up we determined a two-fold collagen production increase in the ECM after 14 days in the presence of an
osteogenic medium. Although these data support the potential of the
hybrid-50% DMAEMA material for its possible application in bone tissue
engineering, we do not consider them as subject of this work since we
focus on the initial cell adhesion onto complex geometry scaffolds and
the inuence of the chemical composition on the proliferation.
As mentioned before, the mechanical properties of the scaffold
should match the ones of the native tissue. We therefore investigated
and reported recently the nanomechanical properties of the hybrid50% DMAEMA material by means of nanoindentation using a pyramidal
Berkovich diamond indenter [37]. Although nanoindentation is widely
used for the evaluation of the materials' mechanical properties, it is a
relatively new tool for testing biological materials [38]. The obtained
pertinent parameters, which have previously been quantied in bone,

M. Chatzinikolaidou et al. / Materials Science and Engineering C 48 (2015) 301309

include hardness and/or reduced elastic modulus. For the hybrid-50%

DMAEMA material the hardness values (H) are reported to be
0.53 GPa for the dry state and 0.64 GPa in the wet state [11], whereas
for trabecular bone they were determined to be 0.72 GPa, and for cortical bone 0.78 GPa using a cube corner diamond tip [39]. The reported
data from the literature are in accordance with our values, taking into
account the specic features of the method used for each investigation.
These nanomechanical data support the potential use of the hybrid material in bone repair applications.
Another important parameter to be investigated when employing
human mesenchymal stem cells is the donor-to-donor variability [40].
Our results indicate that cell proliferation on the three different materials compared to the polystyrene control, is in a similar range, without
any signicant differences, independent of the human cell donor.

[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]

5. Conclusions
[17]

The present work demonstrates the successful fabrication of highprecision scaffolds with complex 3D geometry by Direct fs Laser Writing. The scaffolds were fabricated using a hybrid material comprising
MAPTMS, ZPO, and 50 mol% DMAEMA. Isolated bone marrow derived
mesenchymal stem cells cultured on the hybrid-50% DMAEMA scaffolds
showed excellent cellular response, independent of the donor. A good
cell adhesion onto the 3D scaffolds with spread cell morphology from
the rst hours of observation and up to several days, together with a
proliferation increase after 14 and 21 days validate the biocompatibility
of the developed composite material. These results establish the basis
for the potential use of this cellmaterial combination in bone tissue regeneration. Ongoing experiments focusing on the differentiation of the
BM-MSCs onto the hybrid material structures are expected to reinforce
the applicability of the scaffolds for the intended application.
Disclosure
The authors indicate no potential conicts of interest.
Acknowledgments

[18]
[19]
[20]
[21]

[22]
[23]
[24]
[25]
[26]

[27]
[28]

[29]
[30]
[31]
[32]

This work was partly funded by the Special Account for Research
Fund of the University of Crete (3090) and by the THALIS 3DSET Program (MIS380278) of the Hellenic Ministry of Education. PD and MF acknowledge nancial support from the FP7 Marie Curie ITN program
TopBio (PITN-GA-2010-264362). Konstantina Terzaki is acknowledged
for the block structures, Maria Kissamitaki for the thin lms preparation,
and Prof. Pavlos Katonis is acknowledged for the donation of the bone
marrow tissues. We would also like to thank Ms. Alexandra Siakouli
for expert technical assistance with SEM.
References
[1] E.M. Christenson, K.S. Anseth, L.J.J.P. van den Beucken, C.K. Chan, B. Ercan, J.A. Jansen,
C.T. Laurencin, W.J. Li, R. Murugan, L.S. Nair, S. Ramakrishna, R.S. Tuan, T.J. Webster,
A.G. Mikos, J. Orthop. Res. 25 (2007) 1122.
[2] J.A. Burdick, K.S. Anseth, Biomaterials 23 (2002) 43154323.

[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]

309

R. Murugan, S. Ramakrishna, Compos. Sci. Technol. 65 (2005) 23852406.

L. Shor, S. Guceri, X.J. Wen, M. Gandhi, W. Sun, Biomaterials 28 (2007) 52915297.
V. Karageorgiou, D. Kaplan, Biomaterials 26 (2005) 54745491.
Y. Kuboki, H. Takita, D. Kobayashi, E. Tsuruga, M. Inoue, M. Murata, N. Nagai, Y. Dohi,
H. Ohgushi, J. Biomed. Mater. Res. 39 (1998) 190199.
B.J. Story, W.R. Wagner, D.M. Gaisser, S.D. Cook, A.M. Rust-Dawicki, Int. J. Oral
Maxillofac. Implants 13 (1998) 749757.
S.F. Hulbert, Fed Proc, 30 (1971) A705.
D.W. Hutmacher, Biomaterials 21 (2000) 25292543.
S.J. Hollister, R.D. Maddox, J.M. Taboas, Biomaterials 23 (2002) 40954103.
K. Terzaki, M. Kissamitaki, A. Skarmoutsou, C. Fotakis, C.A. Charitidis, M. Farsari, M.
Vamvakaki, M. Chatzinikolaidou, J. Biomed. Mater. Res. A 101A (2013) 22832294.
W. Sun, A. Darling, B. Starly, J. Nam, Biotechnol. Appl. Biochem. 39 (2004) 2947.
S. Maruo, J.T. Fourkas, Laser Photonics Rev. 2 (2008) 100111.
I. Sakellari, E. Kabouraki, D. Gray, V. Purlys, C. Fotakis, A. Pikulin, N. Bityurin, M.
Vamvakaki, M. Farsari, ACS Nano 6 (2012) 23022311.
M.T. Raimondi, S.M. Eaton, M.M. Nava, M. Lagana, G. Cerullo, R. Osellame, J. Appl.
Biomater. Funct. 10 (2012) 5666.
J. Torgersen, A. Ovsianikov, V. Mironov, N. Pucher, X.H. Qin, Z.Q. Li, K. Cicha, T.
Machacek, R. Liska, V. Jantsch, J. Stamp, J. Biomed. Opt. 17 (10) (2012) 105008,
http://dx.doi.org/10.1117/1.JBO.17.10.105008.
S. Turunen, E. Kapyla, K. Terzaki, J. Viitanen, C. Fotakis, M. Kellomaki, M. Farsari,
Biofabrication 3 (2011) 045002, http://dx.doi.org/10.1088/1758-5082/3/4/045002.
E.T. Ritschdorff, J.B. Shear, Anal. Chem. 82 (2010) 87338737.
S.K. Seidlits, C.E. Schmidt, J.B. Shear, Adv. Funct. Mater. 19 (2009) 35433551.
F. Klein, B. Richter, T. Striebel, C.M. Franz, G. von Freymann, M. Wegener, M.
Bastmeyer, Adv. Mater. 23 (2011) 13411345.
P. Danilevicius, S. Rekstyte, E. Balciunas, A. Kraniauskas, R. Jarasiene, R. Sirmenis, D.
Baltriukiene, V. Bukelskiene, R. Gadonas, M. Malinauskas, J. Biomed. Opt. 17 (8)
(2012) 081405, http://dx.doi.org/10.1117/1.JBO.17.8.081405.
I. Pountos, P.V. Giannoudis, Injury 36 (2005) 812.
E. Gomez-Barrena, P. Rosset, I. Muller, R. Giordano, C. Bunu, P. Layrolle, Y.T.
Konttinen, F.P. Luyten, J. Cell. Mol. Med. 15 (2011) 12661286.
A.H. Undale, J.J. Westendorf, M.J. Yaszemski, S. Khosla, Mayo Clin. Proc. 84 (2009)
893902.
C. Pontikoglou, B. Delorme, P. Charbord, Regen. Med. 3 (2008) 731741.
I. Sakellari, A. Gaidukeviciute, A. Giakoumaki, D. Gray, C. Fotakis, M. Farsari, M.
Vamvakaki, C. Reinhardt, A. Ovsianikov, B.N. Chichkov, Appl. Phys. A-Mater. 100
(2010) 359364.
B. Delorme, P. Charbord, Methods Mol. Med. 140 (2007) 6781.
M.C. Kastrinaki, P. Sidiropoulos, S. Roche, J. Ringe, S. Lehmann, H. Kritikos, V.M.
Vlahava, B. Delorme, G.D. Eliopoulos, C. Jorgensen, P. Charbord, T. Haupl, D.T.
Boumpas, H.A. Papadaki, Ann. Rheum. Dis. 67 (2008) 741749.
C. Pontikoglou, M.C. Kastrinaki, M. Klaus, C. Kalpadakis, P. Katonis, K. Alpantaki, G.A.
Pangalis, H.A. Papadaki, Stem Cells Dev. 22 (2013) 13291341.
K. Terzaki, N. Vasilantonakis, A. Gaidukeviciute, C. Reinhardt, C. Fotakis, M.
Vamvakaki, M. Farsari, Opt. Mater. Express 1 (2011) 586597.
N. Vasilantonakis, K. Terzaki, I. Sakellari, V. Purlys, D. Gray, C.M. Soukoulis, M.
Vamvakaki, M. Kafesaki, M. Farsari, Adv. Mater. 24 (2012) 11011105.
V. Melissinaki, A.A. Gill, I. Ortega, M. Vamvakaki, A. Ranella, J.W. Haycock, C. Fotakis,
M. Farsari, F. Claeyssens, Biofabrication 3 (2011) 045005, http://dx.doi.org/10.1088/
1758-5082/3/4/045005.
A. Abbott, Nature 424 (2003) 870872.
K. Kim, D. Dean, J. Wallace, R. Breithaupt, A.G. Mikos, J.P. Fisher, Biomaterials 32
(2011) 37503763.
M. Dominici, K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R.
Deans, A. Keating, D. Prockop, E. Horwitz, Cytotherapy 8 (2006) 315317.
J.M. Williams, A. Adewunmi, R.M. Schek, C.L. Flanagan, P.H. Krebsbach, S.E. Feinberg,
S.J. Hollister, S. Das, Biomaterials 26 (2005) 48174827.
A. Skarmoutsou, G. Lolas, C.A. Charitidis, M. Chatzinikolaidou, M. Vamvakaki, M.
Farsari, J. Mech. Behav. Biomed. 25 (2013) 4862.
M.L. Oyen, R.R. Cook, J. Mech. Behav. Biomed. 2 (2009) 396407.
H. Isaksson, S. Nagao, M. Malkiewicz, P. Julkunen, R. Nowak, J.S. Jurvelin, J. Biomech.
43 (2010) 24102417.
M.R. Koller, I. Manchel, D.A. Brott, B.O. Palsson, Exp. Hematol. 24 (1996) 14841493.
S. Skoog, A.K. Nguyen, G. Kumar, J. Zheng, P.L. Goering, A. Koroleva, B.N. Chichkov,
R.J. Narayan, Biointerphases 9 (2014) 029014, http://dx.doi.org/10.1116/1.4873688.