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Biomaterials 31 (2010) 3543–3551

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

The osteoblastic differentiation of dental pulp stem cells and bone formation
on different titanium surface textures
Carlo Mangano a,1, Alfredo De Rosa b,1, Vincenzo Desiderio c,1, Riccardo d’Aquino c, Adriano Piattelli d,
Francesco De Francesco c, Virginia Tirino c, Francesco Mangano a, Gianpaolo Papaccio c, *
a
Dipartimento di Scienze dei Biomateriali, Università dell’Insubria-Varese, Italy
b
Dipartimento di Discipline Odontoiatriche, Ortodontiche e Chirurgiche, Secondo Ateneo di Napoli, Italy
c
Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Tissue Engineering and Regenerative Medicine (TERM) Division, Secondo Ateneo di Napoli, 5, via L.
Armanni, Naples 80138, Italy
d
Dipartimento di Discipline Odontostomatologiche, Università degli Studi di Chieti G. D’Annunzio, Chieti, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Bone Tissue Engineering (BTE) and Dental Implantology (DI) require the integration of implanted
Received 15 December 2009 structures, with well characterized surfaces, in bone. In this work we have challenged acid-etched
Accepted 12 January 2010 titanium (AET) and Laser Sintered Titanium (LST) surfaces with either human osteoblasts or stem cells
Available online 1 February 2010
from human dental pulps (DPSCs), to understand their osteointegration and clinical use capability of
derived implants. DPSCs and human osteoblasts were challenged with the two titanium surfaces, either
Keywords:
in plane cultures or in a roller apparatus within a culture chamber, for hours up to a month. During the
Titanium
cultures cells on the titanium surfaces were examined for histology, protein secretion and gene
Surface texturing
Osteodifferentiation expression. Results show that a complete osteointegration using human DPSCs has been obtained: these
DPSCs cells were capable to quickly differentiate into osteoblasts and endotheliocytes and, then, able to produce
Osteoblasts bone tissue along the implant surfaces. Osteoblast differentiation of DPSCs and bone morphogenetic
protein production was obtained in a better and quicker way, when challenging stem cells with the LST
surfaces. This successful BTE in a comparatively short time gives interesting data suggesting that LST is
a promising alternative for clinical use in DI.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction and differentiate into mature osteoblasts, able to secrete extracel-

Commercially, pure titanium and its alloy (Ti–6Al–4V) are the
most widely used materials for Dental Implant (DI). When titanium
is exposed to oxygen, a spontaneous oxidized layer covers the metal
surface, inhibiting the deposition of fibrous tissue around the
implant, thus creating a direct contact between prosthesis and
bone tissue.
Furthermore, excellent mechanical properties, as well as
corrosion resistance, point to the titanium-based implant as the
most suitable device for the substitution of a missing tooth. The
success rate of DI is quite high (about 90%), and an increasing
number of studies have been performed to understand and
improve the process of osteointegration by which osteoblasts and
mesenchymal stem cells can migrate to the healing site, proliferate
* Corresponding author. Tel.: þ39 081 566 6014/7715/7720; fax: þ39 081 566
6015.
E-mail address: gianpaolo.papaccio@unina2.it (G. Papaccio).
1
These authors equally contributed to this research.
lular matrix and calcium hydroxyapatite, embedding the implant
within the new formed bone tissue.
Micro roughness and texturing have been identified as the most
affecting factors on cellular behavior and clinical outcome. Several
reports attest that either cellular differentiation and stability with
matrix deposition and cytoskeletal organization or long-term
stability give best results with rough surfaces, compared with
smooth ones [1–6].
Over the past decades, a considerable number of surface modi-
fications, such as sandblasting, acid-etching, grit-blasting, anod-
ization, plasma-praying, coating with inorganic calcium phosphate
or biological molecules, chemical modification, have been
employed in the attempt to produce better implant surfaces [7–10].
Most of the commercially available titanium implants are produced
by machining titanium rods followed by surface modification.
Direct laser metal sintering (DLMS) is a timesaving and costless
metal forming procedure firstly introduced by Deckard and Beaman
in 1988 [11]. A high power laser beam is directed on a metal powder
bed and programmed to fuse particles according to a CAD file, thus

0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2010.01.056
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3544 C. Mangano et al. / Biomaterials 31 (2010) 3543–3551
generating a thin metal layer. Apposition of subsequent layers gives
shape to a desired three-dimensional form with the need of
minimal post-processing requirements. This innovative technology
gives not only the chance to directly produce dental implant with
different shape and texture, but also to generate, by varying the
gradient of porosity along the axis of the implant, a prosthesis with
the same stiffness and elastic properties of bone.
In this study a Laser sintered Ti–6Al–4V alloy implant surface
modification (Leader Italy – Milan, Italy) was compared with acid-
etched commercially available (Leader Italy – Milan, Italy) implant
in order to assess the ability to favor proliferation and differentia-
tion of alveolar osteoblasts and dental pulp stem cells. The latter are
mesenchymal stem cells selected from the pulp of both deciduous
and adult teeth. These cells, which share many common features
with bone marrow stem cells such as osseous, adipogenic and
endothelial differentiation, have been showed to yield a fibrous
bone tissue in vitro and to show different performances depending
on the surface scaffold where they are seeded and they have
demonstrated to be able to differentiate into lamellar bone with
vessels, when transplanted into immunosuppressed rats and to
effectively and efficiently repair bone defects in humans [12–15].
2. Materials and methods
2.1. Titanium surface preparation
All test specimens were prepared by Selective Laser Sintering (SLS) as described
in Traini et al. [16]. Master alloy powder (Ti–6Al–4V) with a particle size of 1–10 mm
was used as basic material. Processing was carried out in an argon atmosphere using
a powerful Yb (Ytterbium) fiber laser system (EOS GmbH Munchen, Germany) with
the capacity to build a volume up to 250 mm  250 mm  215mm using a wave-
length of 1054 nm with a continuous power of 200 W and a scanning rate of 7 m/s.
The size of the laser spot was 0.1 mm.
Acid-etched titanium specimens were prepared as control in according with
procedure for Implus implant Leader Italy, Milan, Italy. The samples were immersed
in NaOH (20 g/l) and hydrogen peroxide (20 g/l) at 80  C for 30 min and then further
sonicated for 5 min in distilled water. Acid-etching was carried out by immersion of
the samples in a mixture of 50% oxalic acid and 50% maleic acid at 80  C for 45 min,
washed for 5 min in distilled water in a sonic bath.
2.2. Dental pulp extraction and digestion
Human dental pulp was extracted from teeth of healthy adults following our
protocol [13,15]. Before extraction, each subject was checked for systemic and oral
infection or diseases. Only disease-free subjects were selected. Each subject was
pretreated for a week with professional dental hygiene. Before extraction, the dental
crown was covered with 0.3% chlorexidin gel (Forhans, NY, USA) for 2 min and then
pulp was extracted with a dentinal excavator or a Gracey curette.
2.3. Cell cultures
Once removed, the pulp was immersed in a digestive solution (type I collagenase
3 mg/ml plus dispase 4 mg/ml in phosphate buffer saline, PBS, containing 100 U/ml
penicillin, 100 mg/ml streptomycin) for 1 h at 37  C in agitation. The solution was
then filtered by 70 mm Falcon strainers (Becton and Dickinson, Franklin Lakes, NJ,
USA). After filtration, cells were immersed in MegaCell culture medium (Sigma,
Milan, Italy) supplemented with 10% FBS, 100 mM 2P-ascorbic acid, 2 mM L-gluta-
mine, 100 U/ml penicillin, 100 mg/ml streptomycin (all purchased from Invitrogen,
San Giuliano Milanese, Milan, Italy) and placed in 75 ml flasks with filtered valves.
Flasks were incubated at 37  C and 5% CO2 and the medium changed twice a week.
Just before cells become confluent, they were subdivided into new flasks. From 16 to
20 passages were performed. Stem cells were sorted (see below) only when their
number was at least 1,000,000 cells per flask. This number was achieved around day
22, when they were still undifferentiated. Differentiated cells were obtained from
sorted stem cells cultured for at least 30 days in a-MEM culture medium at 20% FBS
(all purchased from Invitrogen, San Giuliano Milanese, Milan, Italy); in fact, FBS
exerts a differentiation activity favoring osteoblastic differentiation when used in
high percentage, as previously demonstrated by us [13,15].
2.4. Tissue engineering and rotating cultures
In order to achieve a 3-dimensional tissue formation, we have challenged the
DPSCs in a roller apparatus with different scaffold surfaces, in order to verify the
influence of texturing on bone tissue formation.
At least 500,000 cells after sorting were gently plated onto 3-dimensional
scaffolds made of titanium dental implant. Samples were placed in a roller apparatus
(Wheaton, Millville, NJ) and were left for 30 days at a speed of 6 rpm in incubator at
37  C and 5% CO2. At the end of the experiments, all the specimens were immersed
in a fixative solution of 10% buffered formalin at pH 7.2 in PBS, for 4 h at room
temperature and left overnight at 48  C. They were then dehydrated in graded
alcohols and embedded in LR White resin (London Resin, Berkshire, UK). Unde-
calcified cut and ground sections were prepared by using the Precise automated
system 1 (Assing, Rome, Italy). Collected sections were ground to a final thickness of
40  5 mm. Experiments were made in quadruplicates.
2.5. FACScanning, sorting and differentiation
Cells were sorted using both morphological traits (high side scatter and low
forward scatter) and antigenic criteria (firstly using CD117 and CD34, and then flk-1),
as specified in our previously published papers [12,13,15,17]. Only cells that
expressed all these markers were selected in order to obtain a homogeneous
population, called DPSC. Briefly, cells were detached using 0.02% EDTA in PBS and
pelleted (10 min at 1000 rpm), washed in PBS at 0.1% BSA at 4  C and incubated in
a solution of 1 ml antibody/9 ml 0.1% BSA in PBS. Cells were washed in the same
solution once and were processed for sorting (FACsorter ARIA II, Becton & Dickinson,
Franklin Lakes, NJ, USA). The mouse anti-human antibodies CD117 (c-kit), CD34, and
flk-1 were purchased from DBA, Segrate, Milan, Italy. Isotypes were used as controls.
Osteogenic differentiation was achieved as reported before by us [13,15]. Briefly,
SBP-DPSCs were cultured with 20% FBS for 15 days without passaging, after which
cells were cultured with 20% FBS for the rest of the experiment.
To monitor differentiation, the cells were examined using mouse anti-human
antibodies to CD44, the transcription factor Runx-2 (all from Santa Cruz, CA, USA).
For Runx-2 analysis, cells were processed using the Caltag Fix& Perm Kit (Invitrogen,
Milan, Italy) following the manufacturer’s guidelines. Isotypes were used as controls.
All data were analyzed using a CellQuest software.
2.6. Adhesion assays
In order to assess the adhesion potential of implants, 10,000 cells were placed on
a titanium disk of 4 mm of diameter with both sintered and machined surfaces. The
disks were seeded onto a microwell in which the gap between the disk and the
plastic wall was filled with silicon. With this technique we allow to seed all the cells
on the titanium surface. Culture plates were taken as control surfaces. Media were
collected after 4 h, 8 h, 24 h, 48 h and the cells were counted in order to assess the
number of cells which remained unattached.
2.7. Scanning electron microscopy
Cells were fixed in 2.5% glutaraldehyde (EM grade) in 0.1 M phosphate buffer,
postfixed in 0.1% OsO4 in the same buffered solution for 1 h and, after critical point
drying and gold–palladium coating, were observed under a SEM microscope
(JEOL-6700F, Tokyo, Japan).
2.8. Histological evaluations
As specified above, we performed either plan cultures or cultures in a rotating
apparatus, the latter, in order to achieve three-dimensional (3D) tissue formation.
Both DPSCs and osteoblasts were cultured in Petri dishes in normal conditions or in
a roller apparatus. In addition, different scaffold surfaces were challenged in order to
verify the influence of texturing on bone tissue formation. At least 500,000 cells
were gently plated onto normal (plane) and 3-dimensional scaffolds made of SLS
and machined titanium. Samples placed in a roller apparatus (Wheaton, Millville, NJ)
were left for 30 days at a speed of 6 rpm. At the end of the experiments, all the
specimens were immersed in a fixative solution of 10% buffered formalin at pH 7.2
with 0.1 M sodium phosphate, for 4 h at room temperature and left overnight at
48  C. They were then dehydrated in graded alcohols and embedded in LR White
resin (London Resin, Berkshire, UK). Undecalcified cut and ground sections were
prepared by using the Precise automated system 1 (Assing, Rome, Italy). Collected
sections were ground to a final thickness of 40  5 mm and counterstained with
alizarin red for bone. Experiments were made in quadruplicates.
2.9. RT-PCR analysis
Total RNA was extracted from specimens at 7 and 15 days by homogenization in
TRI Reagent (Sigma, Milan, Italy), following the manufacturer’s instructions, treated
with DNAse (Promega) to exclude DNA contamination and stored at 70  C until the
assays. cDNA synthesis was carried out from total RNA using Superscript II reverse
transcriptase (Invitrogen Celbio Italy, San Giuliano Milanese, Milan, Italy), using
oligo (dT) 12–18 and Moloney murine leukemia virus RT (10 U/l) in 20 l at 42  C for
50 min. PCR analyses were made in triplicates using a TC-312 thermal cycler
(Techne, Burlington, NJ, USA), in which samples underwent a 2-min denaturing step
to 94  C, followed by 35 cycles of 94  C for 30 s, 54  C for 45 s, 72  C for 1 min, and
a final extension step at 72  C for 4 min. The PCR mixture contained 0.2 mM of each
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C. Mangano et al. / Biomaterials 31 (2010) 3543–3551 3545

Table 1 seeded on LST as well as on AST in static cultures and then

Percentage of adhered cells. processed for SEM analysis at 4, 8, 24 and 48 h. Cell density and
Cells 4h 8h 24 h 48 h morphology on both surfaces were compared. On acid-etched
A) Laser sintered titanium (LST) implant surfaces we were able to find only a very small number of
DPSCs 61.5% 80.8% 87.5% 95.5% cells at all the times considered. These cells were attached to the
DPSCs contr 43.8% 89.0% 97.0% 90.8% titanium surface and did not show a flattened or spherical shape
Osteoblasts 99.2% 95.8% 96.8% 99.6%
(Fig. 2A, B). LST surface presented [16,18] with narrow inter-
Osteoblasts contr 98.8% 94.6% 94.6% 95.4%
communicating crevices, shallow depressions and deep, rounded
B) Acid-etched titanium (AET) pits of widely variable shape and size. On this surface cells were
DPSCs 60.0% 88.8% 97.8% 99.8%
DPSCs contr 52.0% 83.3% 96.0% 94.0%
found to be numerous, scattered through the whole accessible
Osteoblasts 99.4% 97.2% 98.2% 98.4% space. Most of them were flattened on the wall of a crevice and
Osteoblasts contr 99.4% 98.0% 97.8% 88.0% showed long protrusions, attached to the opposite façade (Fig. 2C,
D, E, F). No appreciable differences at different times were detected,
and cells seemed to reach a stabilized shape even after 4 h of
dNTP, 1.5 mM MgCl2, and 0.2 M of each primer. The primer sequences were as culture, although DPSCs displayed a clear osteoblastic feature and
follows: RUNX-2 forward, 5-CACTCACTACCACACCTACC-3, reverse 5-TTCCAT- stability by 24 h.
CAGCGTCAACACC-3; Osterixforward-50 -GCAAAGCAGGCACAAAGAAG-30 , reverse 50 -
AGGGAATGAGTGGGAAAAGG-30 ; Osteocalcin forward 50 -catgagagccctcaca-30 ,
reverse 50 -agagcgacaccctagac-30 ; VEGF forward 50 -TGACAGGGAAGAGGAGGAGA-30 ,
3.3. Rotating cultures and histology
reverse 50 -CGTCTGACCTGGGGTAGAGA-30 . The amplification products were sepa-
rated on a 2% agarose gel in Tris–acetate EDTA (TAE) buffer. Cultures were performed in a rotating apparatus, in order to
achieve three-dimensional (3D) tissue formation, using on AET and
2.10. ELISA LST both sorted DPSCs, as above specified, and osteoblasts. After 30
days of rotating cultures, DPSCs differentiated and expressed CD44
In order to evaluate BMP-2 and VEGF levels in the culture medium, the complete
supernatant medium was collected from cultures after 24, 48, 72 and 7, 15, 30 days and Runx-2 antigens (Fig. 3). Rotating cultures demonstrated that
from DPSCs and Osteoblast cultured on different surfaces. After centrifugation to both DPSCs and osteoblasts left for 30 days at a speed of 6 rpm,
remove particulates, aliquots of 2 ml were stored at 20  C. After thawing at room showed a diffuse bone formation on the surfaces (Fig. 4A, B). In
temperature, 0.5 ml were collected from aliquots and analyzed with ELISA kit for
particular the bone deposition, positive for alizarin staining, was
BMP-2 or anti-VEGF (R&D, Milan, Italy).
slightly more evident in the samples where cells were challenged
2.11. Statistical analysis with LST (Fig. 4C) than those challenged with the acid-etched
titanium. This could be mainly because in the case of LST micro
Student t-test (two-tailed) was used for statistical evaluation. Level of signifi- concavities were numerous and, therefore, bone was secreted and
cance was set at p < 0.05. found within these (Fig. 4C). In addition, DPSCs showed a signifi-
cant better bone formation with respect to human osteoblasts.
3. Results
3.4. PCR analysis
3.1. Adhesion assay
PCR analyses for the most specific and common markers of the
With this assay, we did not find a significant difference in osteogenic lineage were performed on DPSCs and human osteo-
adhesion potential between the surfaces regardless of DPSC or blasts gown on LST and AET implants at 7 and 15 days (Fig. 5).
osteoblasts. Both surfaces showed a considerable fitness for cell Osterix and Runx-2 are the two main key factors involved in bone
adhesion at all times, compared to control surface (see Table 1). differentiation. In Osterix-null mice no bone formation occurs,
mesenchymal cells do not deposit mineralized matrix and are not
3.2. Plane cultures and scanning electron microscopy able to differentiate into osteoblasts. Runx-2 is a transcription
factor involved in cell fate, growth and proliferation control.
DPSCs isolated from the dental pulps of healthy individuals were It interacts with Rb1-p27 mechanism to establish a terminal
sorted for c-kit/CD34/flk-1 (Fig. 1). Both osteoblasts and DPSCs were differentiate state into osteoblasts and it is able to induce

Fig. 1. Fluorescence activated cell sorting. Image showing DPSCs sorted for c-kit (CD117), CD34 and flk-1. The green line shows the isotype control.
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3546 C. Mangano et al. / Biomaterials 31 (2010) 3543–3551

Fig. 2. Scanning Electron Microscope analyses of DPSCs and osteoblasts challenged, in culture, on titanium surfaces (AET ¼ Acid-Etched Titanium; LST ¼ Laser Sintered Titanium).
The images show that, after a few hours of culture (4 or 8) osteoblasts (A) or DPSCs (B) on AET surfaces display a morphology indicating quiescent cells, while on LST surfaces those
cells display better performances (C) (bars ¼ 10 mm) and, in particular, DPSCs at 48 h (D) (bar ¼ 1 mm) project several bridges and ramificate, leading the cells to a different
morphology (E, F) (bars ¼ 10 mm), typical of a differentiated osteoblast.
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C. Mangano et al. / Biomaterials 31 (2010) 3543–3551 3547

Fig. 3. Fluorescence activated cell sorting. Image showing differentiated DPSCs, positive for CD44 and Runx-2. The green line shows the isotype control.

epigenetic modification of some cell cycle associated gene by
histone acetylation and methylation. In Fig. 3, the expression
pattern of both Osterix and Runx-2 is shown. DPSCs express normal
levels of Runx-2 when differentiating into osteoblasts at day 7 and
15, both in control and titanium surfaces, while human osteoblasts
expressed a comparable amount of Runx-2 mRNA transcripts in
control and AET surface, but a lower amount on LST surface even at
day 7. The expression of Osterix mRNA in osteoblasts was
comparable with Runx-2 pattern, giving less intense bands on AET
with respect to control and barely visible bands on LST. DPSCs
display comparable gene expressions when challenged to both AET
and LST surfaces, although both of them showed a lesser expression
than the internal control. Osteocalcin is an ECM protein produced
only by cells differentiating toward the osteogenic phenotype.
Osteocalcin is constitutively produced by osteoblasts: in our hands
no appreciable difference of its expression on the examined

Fig. 4. Image showing alizarin red positive surfaces on different implants, challenged with human osteoblasts and DPSCs for 30 days in a roller apparatus. The results show that the
surfaces are positively stained for alizarin red, demonstrating bone deposition both on AET (A, B) (Original magnification 200) and LST (C) (Original magnification 100) surfaces.
In the latter case a better performance of cells, and DPSCs in particular, was noticed, because the concavities, being numerous, were completely filled of bone, as shown in the figure.
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3548 C. Mangano et al. / Biomaterials 31 (2010) 3543–3551

Fig. 5. The figure shows the PCR expression pattern of both Osterix and Runx-2 genes, which are both transcription factors necessary for bone deposition. DPSCs do not exhibit
alterations of Runx-2 expression at 7 and 15 day both in control and titanium surfaces, while human osteoblasts expressed a comparable amount of Runx-2 mRNA transcripts in
control and AET (acid-etched titanium) surface, but a considerable less amount on LST (laser sintered titanium) surface, also even at day 7. The expression of Osterix mRNAs in the
osteoblasts is comparable with Runx-2 pattern giving less intense bands on AET with respect to control and barely visible bands on LST. DPSCs display comparable gene expressions
between AET and LST, although both surfaces showed a lesser expression of the control. DPSCs expressed osteocalcin only during their differentiation into osteoblasts. On day 7,
osteocalcin levels were low in all the cases, while at day 15, cells grown on LST surface showed a higher expression of osteocalcin. VEGF mRNAs are highly expressed by both DPSCs
and osteoblast upon all the examined surfaces, demonstrating that all the surfaces lead to the formation of vessels with bone.

surfaces was found. Of interest, DPSCs expressed osteocalcin only 4. Discussion

during their differentiation into osteoblasts; therefore we can
assume that an increased osteocalcin expression means that more
cells have switched toward the osteogenic lineage.
On day 7, osteocalcin levels were low in all cases, while at day
15, cells grown on LST surface showed a higher expression of
osteocalcin, with respect to those challenged with AET surfaces.
VEGF is a growth factor essential for endothelium development,
and, in addition, a crosstalk between osteoblasts and endothelial
cells is a fundamental step for bone formation. We found that VEGF
mRNAs are highly expressed by both DPSCs and Osteoblasts upon
all the examined surfaces, demonstrating that all the surfaces lead
to the formation of blood vessels in association with bone.
3.5. ELISA
Quantitative evaluation of morphogens by means of ELISA for
VEGF and BMP-2 was performed on culture media in two steps: the
first was done quite early (after 8, 24 and 48 h of culture), while the
second much later (7, 15 and 30 days after culture). This assay
permits an accurate evaluation of secreted proteins amounts, at pg
levels.
3.5.1. BMP-2
The levels of BMP-2 produced by osteoblasts rise from 8 h to 7
days and then drop through 15 and 30 days on LST, in contrast on
AET the production of protein is high at 8 h, decreases at 24 and
48 h and keeps stable till 30 days (Fig. 6A). DPSCs secretion levels of
BMP-2 rise from 8 to 48 h for both LST and AET, with a higher
production on LST. Between 7 and 30 days BMP-2 production drops
gradually on LST while it stays high on AET till 30 days (Fig. 6A).
3.5.2. VEGF
At nearly time, the VEGF secretion is very similar for both DPSCs
and Osteoblasts. In particular, the VEGF level considerably rises
from 8 to 48 h and from 7 to 15 days on both the compared surfaces
(Fig. 6B). In addition, on LST surface, the secretion of VEGF is
significantly higher with respect to AET, while the concentration of
protein in the medium then tends to average between 15 and 30
days (Fig. 6B). This pattern of expression is rather characteristic of
mesenchymal stem cells undergoing differentiation into an osteo-
genic phenotype.
Bone integration is favored by porous implants that improve
fixation by creating a mechanical interlock via the growth of bone
into the porous structure [19,20]. Porous structures also help to
reduce the stiffness mismatch between implant and bone tissue,
thus reducing ‘stress shielding’ and achieving stable long-term
fixation [21]. The properties and requirements for the success of
porous scaffolds have been studied extensively, and include
mechanical properties, porosity, pore size, pore shape and distri-
bution. Porosity and pore size both play a critical role in bone
ingrowth [22]. The minimum requirement for pore size is consid-
ered to be 100 mm due to cell size migration and transport, and
higher porosity and larger pore size result in greater bone ingrowth
[22]. Pore shape and distribution are also important for osteointe-
gration of Ti implants. Open-cellular pores are necessary for bone
ingrowth, and extensive body fluid transport through the porous
scaffold matrix is possible, which can trigger bone growth if
substantial pore interconnectivity is established [20].
Since the 1980s RP technologies have emerged as a revolu-
tionary manufacturing process with inherent capability to rapidly
make objects in virtually any shape.
Rapid Prototyping (RP) techniques are considered as a viable
alternative for achieving extensive and detailed control over
scaffold architecture [23,24] by combining computer-aided design
(CAD) with computer-aided manufacturing (CAM). RP makes it
possible to build objects with predefined microstructure and
macrostructure and provides the potential for making scaffolds
with controlled hierarchical structures [25]. The transfer of RP
technologies to metallic materials for tissue engineering and dental
implants poses a significant challenge.
A key requirement for such RP technologies is control over the
scaffolds’ pore structure, including pore size, shape, volume and
interconnectivity [26]. It is generally accepted that pore sizes
between 100 and 400 mm are optimal for bone ingrowth [27]. High
pore interconnectivity greatly affects mass transport through the
scaffold and is necessary to ensure adequate delivery of cells and
nutrient supply during subsequent culture throughout the
complete porous scaffold [28,29]. Porosity and pore geometry also
influences the scaffolds’ mechanical properties and affect stress
shielding and fatigue strength [30,31]. Tight control over the
scaffolds’ porous architecture requires that reliable methods are
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C. Mangano et al. / Biomaterials 31 (2010) 3543–3551 3549

Fig. 6. (A) ELISA BMP-2 assays. The levels of BMP-2 produced by osteoblasts rise from 8 h up to 7 days and then drop through 15 and 30 days on LST, while on AET the production of
protein is high at 8 h, decreases at 24 and 48 h and then keeps stable until 30 days. DPSCs secretion levels of BMP-2 rise from 8 to 48 h for both LST and AET, with an higher
production on LST can be detected. Between 7 and 30 days BMP-2 production drop gradually on LST while keep high on AET until 30 days. (*p < 0.001; **p < 0.5) (B) ELISA VEGF
assays. VEGF secretion is very similar for both DPSCs and Osteoblasts when the assays are performed at the beginning (earlier times). VEGF level considerably rises from 8 to 48 h
and from 7 to 15 days on both the considered surfaces. On LST surfaces, the secretion of VEGF is significantly higher with respect to AET, while the concentration of protein in the
medium leads then to average between 15 and 30 days. (*p < 0.001; **p < 0.5).
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3550 C. Mangano et al. / Biomaterials 31 (2010) 3543–3551
essential for its characterization following the design and fabrica-
tion processes. This enables an accurate assessment of the level of
precision that the fabrication process can deliver.
Direct Metal Laser Sintering is a solid freeform fabrication
process, where material is deposited layer by layer directly based
on computer-aided design (CAD) data, to build a fully functional
three-dimensional part. As a CAD driven process, DMLS opens the
way for mass customization of metallic parts, such as bone
implants, producing Ti parts that have a natural rough surface with
a macroporous structure. Moreover, this technique allows us to
control porosity, pore size and distribution to meet a variety of
different application needs. The revolutionary precision of these
techniques, by which it is possible to join very thin sections
(from 0.02 to 0.06 mm) together, permits very complex geometries
to be created, with a gradient of porosity perpendicular to the long
axis of the implant. The difference of material density thus intro-
duced brings to implant dentistry a new functionally graded
material which has the potential to have the same stiffness of the
bone tissue at the implant-bone interface. This introduces the
concept of isoelastic-dental implants.
With appropriate process parameters, DMLS can fabricate fully
functional net shape Ti implants with micro- and macroporosity of
implant surface. Porous structure is fabricated based on CAD-model
and tool path modifications to introduce large porosity, to create
porous implants with controlled porosity, pore size and distribu-
tion. Design and fabrication of macroporous structures are impor-
tant for a porous implant to enable capillary tissue and
osteoprogenitor cell migration into porous spaces [24]. Porous
structures help to reduce the density of metallic implants and
stiffness mismatches between implant and host tissue. Ingrowth of
bone tissue into pores is critical for a porous implant to obtain
successful osteointegration. One critical factor for bone ingrowth is
pore size, in order to optimize capillary tissue and osteoprogenitor
cell migration into porous spaces, pore sizes greater than 100 mm
are necessary [24]. The scaffold attempts to mimic the function of
the natural extracellular matrix. The primary roles of scaffold are:
(1) to serve as an adhesion substrate for the cell, facilitating the
localization and delivery of cells when they are implanted; (2) to
provide temporary mechanical support to the newly grown tissue
by defining and maintaining a 3D structure; (3) to guide the
development of new tissues with the appropriate function [31].
A successful scaffold should possess the following characteristics
[32]: (1) a suitable macrostructure to promote cell proliferation and
cell-specific matrix production; (2) an open-pore geometry with
a highly porous surface and microstructure that enables cell
ingrowth; (3) optimal pore size employed to encourage tissue
regeneration and to avoid pore occlusion; (4) suitable surface
morphology and physiochemical properties to encourage intracel-
lular signalling and recruitment of cells.
DMLS can fabricate macroporous structures with a controlled
pore size. Cells showed different responses to pores of different
sizes. For larger pores (>200 mm), ingrowth of cells was observed in
the interior of these large pores. The results obtained by SEM
showed clearly that in static cultures the LST surface induces
a quick and efficient differentiation of DPCSs allowing them to
produce an extracellular secretion of morphogenetic proteins, as
substantiated by the ELISA.
Moreover the PCR mRNA transcripts confirm those data,
showing that this sintered surface leads to a fast osteodiffer-
entiation and then integration, with respect to the AET surface. In
the rotating 3D cultures results show that both surfaces show
comparable results, allowing both to a similar rate of osteodiffer-
entiation, integration and bone morphogens secretion, although
the presence of a major quantity of micro concavities allow cells to
deposit increased quantities of bone into them. The latter has been
previously shown to happen by us in rotating cultures [33].
Regarding the cytotypes, we have shown that dental pulp stem cells
can grow into the porous structures and allow to an extremely
quick osteodifferentiation and secretion of significant amounts of
morphogens such as VEGF and BMP-2. In addition these cells are
capable of expressing significantly higher amounts of transcription
factors for bone secretion with respect to human osteoblasts,
demonstrating a good performance toward osteogenesis of these
stem cells, thanks to the surfaces who drive their differentiation.
Of interest, the VEGF expression both as gene transcripts and
protein quantity suggests that a vascularised bone is formed. This is
of noticeable importance taking into consideration that minerali-
zation takes place only in association with vessels.
5. Conclusions
Our results, in conclusion, demonstrate that challenging two
titanium surfaces, namely the laser sintered titanium (LST) with
respect to the acid-etched titanium (AET) surfaces with stem cells
from human dental pulp, we are capable to obtain osteoblast
differentiation of DPSCs, production of appreciable amounts of
bone morphogenetic proteins as well as vascular endothelial
growth factor and specific bone proteins. Therefore, a complete
osteointegration is obtained.
This happens in a better and quicker way when challenging
sintered titanium with respect to the acid-etched titanium surface.
This successful bone tissue engineering gives interesting data
for consideration when evaluating the technique as an alternative
to standard methods for producing implants for clinical dental
implantology.
Moreover, using stem cells of dental pulp (DPSCs), which are
capable of producing woven bone, we can increase the chance to
accelerate the time for implant loading.
Acknowledgment
Funding: This project has been funded by Leader Italia (Cinisello
Balsamo, Milan, Italy) to CM and GP and by FIRB-SIRIO (Italian
Council for Strategic University Projects) project #RBIP06FH7J to GP.
References
[1] Grassi S, Piattelli A, de Figueiredo LC, Feres M, de Melo L, Iezzi G, et al.
Histologic evaluation of early human bone response to different implant
surfaces. J Periodontol 2006;77:1736–43.
[2] Ivanoff CJ, Hallgren C, Widmark G, Sennerby L, Wennerberg A. Histologic
evaluation of the bone integration of TiO(2) blasted and turned titanium
microimplants in humans. Clin Oral Implants Res 2001;12:128–34.
[3] Ivanoff CJ, Widmark G, Johansson C, Wennerberg A. Histologic evaluation of
bone response to oxidized and turned titanium micro-implants in human
jawbone. Int J Oral Maxillofac Implants 2003;18:341–8.
[4] Lumbikanonda N, Sammons R. Bone cell attachment to dental implants of
different surface characteristics. Int J Oral Maxillofac Implants 2001;16:627–36.
[5] Roach P, Eglin D, Rohde K, Perry CC. Modern biomaterials: a review – bulk
properties and implications of surface modifications. J Mater Sci Mater Med
2007;18:1263–77.
[6] Shibli JA, Grassi S, de Figueiredo LC, Feres M, Marcantonio Jr E, Iezzi G, et al.
Influence of implant surface topography on early osseointegration: a histo-
logical study in human jaws. J Biomed Mater Res B Appl Biomater
2007;80:377–85.
[7] Buser D. Titanium for dental application. In: Brunette DM, Tengval P, Textor M,
Thomsen P, editors. Titanium in medicine. Material science, surface science,
engineering, biological responses and medical applications. Berlin: Springer;
2001. p. 875–88.
[8] Esposito M. Titanium for dental application(I). In: Brunette DM, Tengval P,
Textor M, Thomsen P, editors. Titanium in medicine. Material science, surface
science, engineering, biological responses and medical applications. Berlin:
Springer; 2001. p. 827–74.
[9] Shalabi MM, Gortemaker A, Van’t Hof MA, Jansen JA, Creugers NH. Implant
surface roughness and bone healing: a systematic review. J Dent Res
2006;85:496–500.
 Author's personal copy
C. Mangano et al. / Biomaterials 31 (2010) 3543–3551
[10] Sykaras N, Iacopino AM, Marker VA, Triplett RG, Woody RD. Implant materials,
designs, and surface topographies: their effect on osseointegration. A litera-
ture review. Int J Oral Maxillofac Implants 2000;15:675–90.
[11] Deckard C, Beaman JJ. Process and control issues in selective laser sintering.
ASME Prod Eng Div 1988;33:191–7.
[12] d’Aquino R, De Rosa A, Lanza V, Tirino V, Laino L, Graziano A, et al. Human
mandible bone defect repair by the grafting of dental pulp stem/progenitor
cells and collagen sponge biocomplexes. Eur Cell Mater 2009;18:75–83.
[13] d’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al.
Human postnatal dental pulp cells co-differentiate into osteoblasts and
endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell
Death Differ 2007;14:1162–71.
[14] Graziano A, d’Aquino R, Cusella-De Angelis MG, Laino G, Piattelli A, Pacifici M,
et al. Concave pit-containing scaffold surfaces improve stem cell-derived
osteoblast performance and lead to significant bone tissue formation. PLoS
One 2007;2:e496.
[15] Laino G, d’Aquino R, Graziano A, Lanza V, Carinci F, Naro F, et al. A new
population of human adult dental pulp stem cells: a useful source of living
autologous fibrous bone tissue (LAB). J Bone Miner Res 2005;20:1394–402.
[16] Traini T, Mangano C, Sammons RL, Mangano F, Macchi A, Piattelli A. Direct
laser metal sintering as a new approach to fabrication of an isoelastic func-
tionally graded material for manufacture of porous titanium dental implants.
Dent Mater 2008;24:1525–33.
[17] Laino G, Graziano A, d’Aquino R, Pirozzi G, Lanza V, Valiante S, et al. An
approachable human adult stem cell source for hard-tissue engineering. J Cell
Physiol 2006;206:693–701.
[18] Mangano C, Raspanti M, Traini T, Piattelli A, Sammons R. Stereo imaging and
cytocompatibility of a model dental implant surface formed by direct laser
fabrication. J Biomed Mater Res A 2009;88:823–31.
[19] Head WC, Bauk DJ, Emerson Jr RH. Titanium as the material of choice for
cementless femoral components in total hip arthroplasty. Clin Orthop Relat
Res 1995:85–90.
[20] Ryan G, Pandit A, Apatsidis DP. Fabrication methods of porous metals for use
in orthopaedic applications. Biomaterials 2006;27:2651–70.
[21] Kroger H, Venesmaa P, Jurvelin J, Miettinen H, Suomalainen O, Alhava E. Bone
density at the proximal femur after total hip arthroplasty. Clin Orthop Relat
Res 1998:66–74.
3551
[22] Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteo-
genesis. Biomaterials 2005;26:5474–91.
[23] Hutmacher DW, Sittinger M, Risbud MV. Scaffold-based tissue engineering:
rationale for computer-aided design and solid free-form fabrication systems.
Trends Biotechnol 2004;22:354–62.
[24] Yeong WY, Chua CK, Leong KF, Chandrasekaran M. Rapid prototyping in
tissue engineering: challenges and potential. Trends Biotechnol
2004;22:643–52.
[25] Sachlos E, Czernuszka JT. Making tissue engineering scaffolds work.
Review: the application of solid freeform fabrication technology to the
production of tissue engineering scaffolds. Eur Cell Mater 2003;5:29–39.
discussion – 40.
[26] Cameron HU, Pilliar RM, Macnab I. The rate of bone ingrowth into porous
metal. J Biomed Mater Res 1976;10:295–302.
[27] Gomes ME, Sikavitsas VI, Behravesh E, Reis RL, Mikos AG. Effect of flow
perfusion on the osteogenic differentiation of bone marrow stromal cells
cultured on starch-based three-dimensional scaffolds. J Biomed Mater Res A
2003;67:87–95.
[28] Cartmell SH, Porter BD, Garcia AJ, Guldberg RE. Effects of medium perfusion
rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Eng
2003;9:1197–203.
[29] Hollister SJ, Maddox RD, Taboas JM. Optimal design and fabrication of scaf-
folds to mimic tissue properties and satisfy biological constraints. Biomaterials
2002;23:4095–103.
[30] Fang Z, Starly B, Sun WE. Computer-aided characterization for effective
mechanical properties of porous tissue scaffolds. Computer-Aided Design
2005;37:65–72.
[31] Kim BH, Kim YK, Ok JH. Development of liquid chromatographic method for
the analysis of kanamycin residues in varicella vaccine using phenyl-
isocyanate as a derivatization reagent. J Chromatogr B Biomed Sci Appl
2001;752:173–7.
[32] Leong KF, Cheah CM, Chua CK. Solid freeform fabrication of three-dimensional
scaffolds for engineering replacement tissues and organs. Biomaterials
2003;24:2363–78.
[33] Graziano A, d’Aquino R, Cusella-De Angelis MG, De Francesco F, Giordano A,
Laino G, et al. Scaffold’s surface geometry significantly affects human stem cell
bone tissue engineering. J Cell Physiol 2008;214:166–72.