Human mandible bone defect repair by the grafting of dental pulp stem / progenitor cells and collagen sponge

Article (PDF Available)inEuropean cells & materials 18(7):75-83 · July 2009with80 Reads
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Abstract
In this study we used a biocomplex constructed from dental pulp stem/progenitor cells (DPCs) and a collagen sponge scaffold for oro-maxillo-facial (OMF) bone tissue repair in patients requiring extraction of their third molars. The experiments were carried out according to our Internal Ethical Committee Guidelines and written informed consent was obtained from the patients. The patients presented with bilateral bone reabsorption of the alveolar ridge distal to the second molar secondary to impaction of the third molar on the cortical alveolar lamina, producing a defect without walls, of at least 1.5 cm in height. This clinical condition does not permit spontaneous bone repair after extraction of the third molar, and eventually leads to loss also of the adjacent second molar. Maxillary third molars were extracted first for DPC isolation and expansion. The cells were then seeded onto a collagen sponge scaffold and the obtained biocomplex was used to fill in the injury site left by extraction of the mandibular third molars. Three months after autologous DPC grafting, alveolar bone of patients had optimal vertical repair and complete restoration of periodontal tissue back to the second molars, as assessed by clinical probing and X-rays. Histological observations clearly demonstrated the complete regeneration of bone at the injury site. Optimal bone regeneration was evident one year after grafting. This clinical study demonstrates that a DPC/collagen sponge biocomplex can completely restore human mandible bone defects and indicates that this cell population could be used for the repair and/or regeneration of tissues and organs.

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R d’Aquino et al. DPCs repair human mandible defects
European Cells and Materials Vol. 18 2009 (pages 75-83) ISSN 1473-2262
Abstract
In this study we used a biocomplex constructed from dental
pulp stem/progenitor cells (DPCs) and a collagen sponge
scaffold for oro-maxillo-facial (OMF) bone tissue repair
in patients requiring extraction of their third molars. The
experiments were carried out according to our Internal
Ethical Committee Guidelines and written informed consent
was obtained from the patients. The patients presented with
bilateral bone reabsorption of the alveolar ridge distal to
the second molar secondary to impaction of the third molar
on the cortical alveolar lamina, producing a defect without
walls, of at least 1.5 cm in height. This clinical condition
does not permit spontaneous bone repair after extraction
of the third molar, and eventually leads to loss also of the
adjacent second molar. Maxillary third molars were
extracted first for DPC isolation and expansion. The cells
were then seeded onto a collagen sponge scaffold and the
obtained biocomplex was used to fill in the injury site left
by extraction of the mandibular third molars. Three months
after autologous DPC grafting, alveolar bone of patients
had optimal vertical repair and complete restoration of
periodontal tissue back to the second molars, as assessed
by clinical probing and X-rays. Histological observations
clearly demonstrated the complete regeneration of bone at
the injury site. Optimal bone regeneration was evident one
year after grafting. This clinical study demonstrates that a
DPC/collagen sponge biocomplex can completely restore
human mandible bone defects and indicates that this cell
population could be used for the repair and/or regeneration
of tissues and organs.
Keywords: Dental pulp stem/progenitor cells (DPCs), bone,
human mandible, stem/progenitor cell graft, bioscaffold,
regenerative medicine, clinical study.
Address for correspondence:
Gianpaolo Papaccio
Department of Experimental Medicine
Section of Histology and Embryology, TERM Division
2
nd
University of Naples, via L. Armanni, 5,
80138 Naples (Italy)
Telephone Number: +39 081-5666014
FAX Number: +39 081-5666015
Skype: Stemface
E-mail : gianpaolo.papaccio@unina2.it
Introduction
The aim of tissue engineering (TE) is the regeneration of
tissues through the combined use of biomaterials and
biologic mediators in order to provide new tools for
regenerative medicine (RM). Over the last years, TE has
made significant progress, moving from being merely a
biomaterial science towards being a genuinely
multidisciplinary field, through the integration of biology,
medicine and various engineering sciences. Importantly,
future procedures will make increasing use of autologous
transplants (i.e., material obtained from the same
individual to whom they will be reimplanted); thus, the
need for immunotherapy will be avoided. Ideally, these
transplants will possess predictable patterns of
vascularisation and nerve supply, which are both important
aspects for a return to optimal functionality.
The need to develop tissue replacement and
implementation strategies is particularly felt in the oro-
maxillo-facial (OMF) field. Replacement of OMF
structures is tricky and peculiar because orofacial
functions – such as facial expression, articulation of
speech, chewing and swallowing – are exquisitely delicate,
being based on complex three-dimensional anatomical
structures formed from soft (skin, mucosa and muscle)
and hard (craniofacial skeleton and teeth) tissues (Bluteau
et al., 2008).
The repair and regeneration of bone is a major issue
in the OMF field and for the whole human body in general.
Bone loss is caused by many diseases (congenital or
degenerative), traumas and surgical procedures; it is a
problem for functionality and is having an ever-increasing
social impact, especially in elderly subjects.
Bone is formed by extracellular matrix (ECM) rich in
collagen and elastic fibres adherent to hydroxyapatite
crystals. Adult bone is continuously remodelled through
specific osteoblast/osteoclast interaction. Stem/progenitor
cells residing in the periosteum and endosteum of bone
possess a limited regenerative potential (Salgado et al.,
2006). For this reason, surgical intervention using
biocompatible fillers or bone-grafting techniques is
indispensable when significant bone loss occurs. To avoid
side effects produced by the use of biocompatible
materials and/or bone withdrawal, new biotechnological
approaches for repair must be envisaged.
Although stem/progenitor cells have been isolated
from different tissues and extensively studied in vitro and
in vivo in the past years, there is no information yet on the
application of human stem/progenitor cells for the repair
of OMF structures at a clinical level. Unfortunately, there
HUMAN MANDIBLE BONE DEFECT REPAIR BY THE GRAFTING OF DENTAL
PULP STEM/PROGENITOR CELLS AND COLLAGEN SPONGE BIOCOMPLEXES
Riccardo d’Aquino
1,2
, Alfredo De Rosa
1
, Vladimiro Lanza
1
, Virginia Tirino
2
, Luigi Laino
1
, Antonio Graziano
1
,
Vincenzo Desiderio
2
, Gregorio Laino
1
and Gianpaolo Papaccio
2*
1
Dipartimento di Discipline Odontostomatologiche, Ortodontiche e Chirurgiche,
2
Dipartimento di Medicina
Sperimentale, Sezione di Istologia ed Embriologia, Tissue Engineering and Regenerative Medicine (TERM)
Division, Secondo Ateneo di Napoli, Naples, Italy
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R d’Aquino et al. DPCs repair human mandible defects
are limits on the use of stem/progenitor cells in therapy,
such as the low number of stem/progenitor cells that can
be collected, morbidity at the site of collection and the
difficulties in reaching the site of repair.
Dental pulp is a niche housing neural-crest-derived
stem cells. This niche is easily accessible and there is
limited morbidity after collection (Jo et al., 2007; Lensch
et al., 2006; Mitsiadis et al., 2007). Previous studies have
shown that dental pulp stem cells (DPSCs) are capable of
differentiating into osteoblasts (Laino et al., 2005; Laino
et al., 2006b) that secrete abundant extracellular matrix
(ECM) and that can build a woven bone in vitro (Laino et
al., 2006a). Furthermore, DPSCs are capable of forming a
complete and well-vascularised lamellar bone after grafting
into immunosuppressed rats (d’Aquino et al., 2007;
Graziano et al., 2008). The quality and quantity of
regenerated bone formed by DPSCs was demonstrated in
in vitro and in vivo experiments using stem cells and
biomaterials (d’Aquino et al., 2008; d’Aquino et al., 2007;
Graziano et al., 2008; Laino et al., 2005). Thus, dental
pulp could be considered as an interesting and potentially
important source of autologous stem/progenitor cells that
are ready for use for therapeutic purposes, such as the
repair/regeneration of craniofacial bones.
The aim of this study, therefore, was to demonstrate
that DPCs could be used to repair bone defects in humans.
Here we give evidence that DPCs seeded onto collagen
sponge bioscaffolds repair alveolar defects of the mandible
produced after extraction of impacted third molars. The
autografts produced a fast regeneration of bone, which was
of optimal quality and quantity when compared to standard
techniques commonly used for guided bone regeneration
and bone grafts of various origins (Jensen et al., 2004).
Materials and Methods
Ethics
All procedures described here comply with Internal Ethical
Committee guidelines, approved on June 12
th
, 2005
(Second University of Naples Internal Registry:
Experimentation #914-Bone repair using stem cells).
Patients were invited, before being enrolled for the
study, to carefully read and sign an informed consent form,
drafted by us following instructions from our internal
Ethical Committee.
Objectives
The objective of this clinical study was to repair an alveolar
bone defect secondary to routine wisdom tooth extraction.
Usually, after extraction of impacted unerupted or partially
erupted wisdom (third molar) teeth, a proportion of patients
risk reabsorption of the alveolar ridge distal to the second
molar roots. In these patients, destruction of the tooth
socket produces a pocket formed from 2 walls, which are
represented by the root of the second molar and the distal
ridge, with the lingual wall forming a third wall when
present. This clinical condition (post-extractive alveolar
bone loss), in which vertical loss has a probing depth of at
least 7 mm, jeopardizes the second molar in an average of
five years or less and does not allow bone repair with
normal techniques (Dodson, 2007). Thus, it represents a
complication associated with the removal of the third
molars that should not be underestimated. For this reason,
we identified patients at risk of post-extractive alveolar
bone loss as candidates that could benefit for a bone
regeneration therapy.
Participants: patient selection and preparation
Eligibility criteria for participants and settings were the
following: extraction needed for all wisdom teeth, with
closely comparable conditions for the two lower impacted
teeth; no systemic disease; no pregnancy (for females); no
routine drug use. Patients with two similar lower molars
were needed for the study so that we could use one as a
test (T) site and the other one as a control (C) site.
Seventeen out of the 100 patients initially contacted for
this study consented to surgery. Of these 17 patients, 7 (6
females and 1 male) returned for the one-year follow-up.
The template for the enrolment of patients was set within
the limits of the approved clinical trial. All the procedures
were performed at the Department of Odontostomatology
of the Second University of Naples.
Patients were subjected to professional oral hygiene
one week before surgery. They were then instructed to
perform domiciliary hygiene of the oral cavity correctly,
which consisted in washing the mouth with 0.2%
Chlorhexidin (CHX) after tooth brushing, twice a day until
surgery was performed.
Pre-surgery evaluation of dental pulp stem/
progenitor cells
Before embarking on regeneration surgery, we needed to
obtain stem/progenitor cells from the pulps of the patients.
Patients were therefore subjected to the extraction of the
upper (maxillary) molars and the pulps harvested as
previously described (d’Aquino et al., 2007). Briefly, teeth
were washed in 0.2% CHX solution, the pulp chamber
opened using a surgical drill and the pulp collected. Then,
the pulp was rinsed in 1.5 ml saline solution and
mechanically dissociated; using previously described
procedures (Graziano et al., 2008). After dissociation, cells
were filtered through a 70μm strainer and cultured in α-
minimal essential medium (MEM) (Cambrex, Charles City,
IA, USA) with 20% FBS (Invitrogen, San Giuliano
Milanese, Italy) and the medium changed twice a week.
At day 21 cells were detached and analysed at the
Fluorescence Activated Cell Sorter (FACS Vantage,
Beckton Dickinson, Franklin Lakes, NJ, USA) for stem/
progenitor antigen expression in good manufacturing
practice (GMP) conditions. Cells were detached using
0.02% EDTA solution, centrifuged and incubated with 1μl
of antibody in 100 μl of phosphate buffered saline (PBS)
solution for 1h at 4°C. Antibodies were: anti-CD34 (clone
AC136, Miltenyi Biotech, Calderara sul Reno, Bologna,
Italy) and anti-flk-1 (c. sc-57135, Santa Cruz, CA, USA).
Extraction of wisdom teeth
Patients were prepared for surgery by decontamination of
the oral cavity with CHX. Then lower (mandible) impacted
third molars were extracted following a standard
procedure: after making a horizontal incision in the gum,
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the muco-periosteal flap is reflected and the bone covering
the tooth is removed using a round bur. The area is irrigated
with a steady stream of saline solution until the crown is
entirely exposed.
When the oral surgeon is not able to extract the whole
tooth in one go, a groove is created vertically (along the
long axis of the tooth) at the cervical line of the tooth,
using a fissure bur, in order to separate the crown from the
root. The groove created by the bur must not be deep, since
the mandibular canal is often found in close proximity to
the tooth and the risk of injuring or severing the inferior
alveolar nerve must be avoided. After being placed in the
groove, a straight elevator is used to separate the crown
from the root, with a rotary movement. The crown is
removed separately, using the same elevator, with a rotary
movement upwards, and the root is then easily removed,
using a straight or angled elevator, the blade end of which
is placed in a purchase point created on the buccal side of
the root.
In the case of a tooth with multiple roots, the crown
must be sectioned and removed, as above described.
Afterwards, if the roots of the impacted tooth are separated
during crown sectioning, they can be easily removed one
in succession, starting with the distal root and then the
mesial root.
After smoothing the bone, the area is irrigated with
saline solution and the distal root of the second molar is
planned with a Gracey curette, and all the necrotic tissue
is taken away.
Stem/progenitor cells, obtained as above described,
were gently endorsed with a syringe onto a collagen sponge
scaffold (Gingistat, Vebas, San Giuliano Milanese, Italy).
The sponge-cell implant was used to fill the space left by
the extraction procedure (test (T) site). A sponge without
cells was used to fill the control (C) site.
A flap of gum was then sutured as a tendon in order to
avoid any contact with the oral cavity. A suture was then
placed at the distal portion of the second molar and the
others were placed at the interdental papillae and at the
posterior end of the incision. For both sites, a replacement
jig was placed to ensure correct localization for sample
withdrawal.
Post-surgery patient evaluations
Clinical and radiological controls were performed. The first
control was scheduled at day 7 after surgery, when X-ray
(for each patient the Ethical Committee permitted to
perform a maximum of 4 X-ray Orthopantomographies
(OPTs) per year and a maximum of 8 endo-oral X-rays
per year), clinical observation and suture removal were
performed. Oedema, presence of inflammation and
functionality were clinically evaluated.
Patients were controlled once a month thereafter, up
to the third month. During these controls, clinical
observations and X-rays were performed. During the
fourth control, at three months after surgery and before
bone sampling, probing depth was performed to evaluate
the retrieved clinical attachment.
A sample was then collected from the T and C sites of
each patient for histological and immunofluorescence (IF)
analyses. Each bone sample was collected using a drill
with a replacement jig. Bone specimens were used for
histological observations. For this purpose, each sample
was decalcified in 10% EDTA in distilled water for 2
months. Then, each specimen was sectioned and stained.
Other than haematoxylin-eosin staining, samples were used
for IF analyses, using the following antibodies: anti-
osteocalcin (OC), anti-osteonectin (ON), anti-bone alkaline
phosphatase (BAP), anti-bone morphogenetic protein
(BMP)-2 and anti-vascular endothelial growth factor
(VEGF) (all purchased from Beckton Dickinson). One year
from surgery, further analyses were performed.
Statistical analysis
Student t-test (two-tailed) was used for statistical
evaluation. The level of significance was set at p<0.05.
Results
The dental pulp cells of the third maxillary molars collected
for pre-surgery evaluation were strongly positive to both
CD34 and Flk-1 (Fig. 1A, B) and clearly comprised stem/
progenitor cells in sufficient quantity to perform the in
vivo experiments. All the selected patients were therefore
subjected to surgery to extract third mandible molars and
regenerate bone defects at the level of the lower, impacted
third molars (Fig. 2 A, B, C, D, E, F, G, H, I).
Seven days after implantation of the biocomplex,
clinical and radiological controls revealed that T and C
sites did not display differences (Fig. 3A, B, C). There
was slight oedema and inflammation at the sites of surgery,
Fig. 1. Fluorescence activated cell sorting before surgery. (A) Cells collected from maxillary (upper) third molars
were challenged for CD34 and flk-1. Representative 2D plot image showing high positivity levels for both flk-1
and CD34 antigens (77,3%). (B) Isotype control.
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but patients did not present with morbidity or infections
after intervention. In all cases, the post-surgery time for
recovery was normal. Patients did not complain about
particular post-operative pain, so no analgesic medication
was given. Functionality was normal in all cases except
for one patient, who suffered a little distortion in mouth
opening and an increased level of oedema at both sites.
Post-operative clinical observation revealed normal healing
without scar tissue formation or functional disturbances;
no bleeding, no swelling or other side effects were
observed.
Thirty days after surgery, clinical parameters were well
balanced in all the patients. X-ray controls showed
significant differences between the C and T sites: the latter
presented with a high rate of mineralisation (Fig. 4A,B).
Clinical control performed two months after surgery
did not reveal the presence of any alteration. X-ray analyses
clearly revealed different levels of cortical bone at the T
(Fig. 4C) and C sites (Fig. 4D): whereas at the T site the
cortical margin reached the cementum-enamel junction
(CEJ) level of the second molar, demonstrating vertical
regeneration, this was not seen at the C site in any of the
patients.
Three months after the surgery, X-ray analyses
confirmed that the T sites were completely regenerated
and that the cortical level was much higher than at the C
sites (Fig. 5A,B). From day 7 up to the third month, patients
did not show signs of local or general infections or diseases.
All the parameters (oral and general) were within normal
ranges. The functionality (dental functions, chewing in
particular) and quality of life were optimal in all cases.
Samples of bone were collected for histology and IF
analyses (Fig. 5C,D).
The probing depth analyses revealed an increase of
clinical attachment that was quantitatively higher at the T
site than at the C site: whereas the C sites presented with a
gain of 4.4±1.2 mm, the gain at the T sites was 6.2±2.3
mm. In addition, we collected a bone sample for each site,
using mini-invasive surgery. The bur was positioned in
the right place using a replacement jig placed before the
Fig. 2. Surgical procedure. (A) Pre operatory X-ray (T: test site; C: control site). In this representative X-ray it is
possible to see how the lower right third molar is in close contact with the second molar root. (B) Control site tooth
extraction. (C) Pulp withdrawal from the extracted tooth. (D) In the control site, a collagen sponge without DPCs is
put in the gap left by the surgery and is then sutured. (E) Evaluation of the depth of the defect produced by tooth
extraction carried out with a parodontal probe at the test site. (F) Test surgical site. After extraction of the third molar,
a gap is left in the mandible. (G) Construction of the collagen-cell biocomplex. Stem/progenitor cells obtained from
pulp being seeded onto the collagen sponge. (H) Grafting of the biocomplex at the test site. (I) Surgery ends with the
placing of sutures.
Fig. 3. X-ray and clinical control 7 days after surgical intervention. (A) Control X-ray (T: test site; C: control site) of
patient N. 3. At the T site it is possible to observe the wide gap behind the distal roots of the right second molar. The
yellow double-headed arrow evidences the vertical gap and the white double-headed arrow indicates the horizontal
gap. (B) Test site. (C) Control site.
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surgery. In this way we were able to collect bone from the
regenerated sites, avoiding any potential loss of periodontal
tissue. The samples belonging to the T sites were very
different when compared with those withdrawn from the
C sites: T site samples were made up of well organized
and well vascularised bone with a lamellar architecture
surrounding the Haversian channels (Fig. 6A); bone from
C sites was immature, with fibrous bone entrapped among
new lamellae, incomplete and large Haversian channels
and evidence of bone reabsorption (Fig. 6B). In all cases
the collagen sponge was completely reabsorbed.
IF analyses were performed to assess the expression
of bone proteins (ON, OC and BAP), as well as BMP-2
and VEGF, which are both important molecular signals
during bone regeneration. ON, OC and BAP expression
was slightly different between T and C sites: they were
expressed at both sites, but with a different distribution
(Fig. 6 C,D,E,F,G,H). Significant differences were
observed for BMP-2 and VEGF expression: these two
molecular signals were expressed at much higher levels
(p<0.001) in the T samples with respect to the C ones (Fig.
6 I, J, K, L, M).
One year after surgery, patients presented with a normal
oral cavity without signs of alterations. The mucosae were
normal at both sites. X-ray analysis confirmed that the bone
regeneration at the T site was complete and stable (Fig. 7).
Final scores for the bone regeneration per patient are given
in Table 1. Quality of life, chewing, oral cavity and relative
functions remained optimal in all the cases.
Discussion
Approximately 1,500,000 subjects undergo craniofacial
reconstruction each year in Europe, and about 20% of these
patients experience a loss of function despite
reconstruction. 30,000 of these subjects suffer from donor
site morbidity relating to flap reconstructions after oral
and maxillofacial surgery. The missing parts that are
involved in the defect of a given tissue or organ have
specific functions, and replacement is often quite difficult.
For example, the closure of a bone defect is commonly
associated with the transfer of tissue (e.g., a flap), which
may not fully restore the unique function of the lost part.
Furthermore, tissue transfer is associated with donor site
morbidity, accompanied by scarring, infection and loss of
function. Craniofacial reconstruction aims mainly to repair
gross aesthetic and functional disfigurement in cases of
Fig. 4. X-ray control 30 days after graft (T: test site; C:
control site). The image shows that significant bone
regeneration has started at the T site of patient N. 3
(compare with Fig. 3A).
Fig. 5. X-ray, withdrawal and bone sampling 3 months after graft. (A) X-ray control 3 months after grafting (patient
N. 3) (T: test site; C: control site). (B) Enlargement of the T site X-ray. The red arrow indicates the cementum-
enamel-junction; the white double-headed arrow indicates the minimal exposure of the second molar root due to
significant bone regeneration. (C) Enlargement of the C site X-ray. The red arrow indicates the cementum-enamel-
junction; the white double-headed arrow indicates the considerable exposure of the second molar root due to a lack
of bone regeneration. (D) Bone sample collected 3 months after grafting.
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Differentiation of stem cells into osteoblasts in vitro
was assessed by osteocalcin expression, a cell membrane
protein that identifies osteoblasts. In addition, the
extracellular matrix that is secreted by these cells was
measured by the expression of ON, a marker of bone
matrix, and BAP, a tetrameric glycoprotein found on the
surface of osteoblast and an indicator of bone turnover.
Morphologically, bone biopsies collected from the treated
sites and used for histological analysis revealed the
presence of well differentiated lamellar bone, with typical
Haversian channels and lamellae and spider-like
osteocytes. Interestingly, the significantly higher amount
of BMP-2 and VEGF observed at the treated sites, when
compared to the control sites, explains and confirms the
different levels of bone maturation. In fact, these two
molecules are responsible for fine-tuning the balance of
secreted bone ECM and neo-angiogenesis during bone
formation. For bone development and healing, vigorous
vessel sprouting is extremely important to sustain
mineralisation and maturation.
Regarding the most appropriate scaffold for bone
regeneration, we have previously demonstrated that
complete bone regeneration can be obtained in vivo with
DPSCs seeded on reabsorbable collagen sponges
(Gingistat). These gave optimal results, allowing cells to
proliferate and differentiate into osteoblasts (Graziano et
al., 2008). Therefore, we used the same scaffold in this
clinical study. The results that we have obtained and shown
above allow us to assume that this scaffold is an optimal
support for stem/progenitor cells in cell-guided
regeneration.
The present study is the first that accomplishes
autologous bone regeneration in humans. This was
obtained with the use of a biocomplex constructed from
dental pulp stem/progenitor cells seeded onto a collagen-
based scaffold. We have given evidence here that the
procedure described results in optimal bone repair, with
the restoration of complete periodontal tissue behind the
congenital malformations, tumour resections and post-
traumatic deformities.
Recent TE products are based on novel biomaterials
integrating stem/progenitor cells that are capable either of
self-renewal or of differentiating into several specific cell
types. The use of adult stem/progenitor cells can be
extensive, since stem/progenitor cells can be harvested
from various tissues such as adipose tissue, bone marrow,
dental pulp and periodontal ligament. Dental stem/
progenitor cells collected from dental pulp can be
differentiated in vitro and then transplanted with
biomaterial scaffolds into the host without immunologic
rejection (d’Aquino et al., 2007; Graziano et al., 2008).
The use of appropriate biomaterial scaffolds combined with
selected growth factors can significantly improve the
survival and differentiation of the transplanted stem/
progenitor cells.
To date, only few cases of translation from biological
studies and TE to patients have been reported (Gurtner et
al., 2008). The difficulties linked to cell manipulation and
the quantity and quality of stem/progenitor cells are the
main reasons for the slow progress. In addition, cell
differentiation is more a limitation than an implementation
for results. Stem cells are more capable of regeneration
than their differentiated daughter cells. For example,
mesenchymal stem cells exhibit properties that allow their
use in regenerative medicine, including their
immunosuppressive activity (Dazzi and Horwood, 2007;
Guo et al., 2006; Le Blanc and Ringden, 2007; Locatelli
et al., 2008; Samuelsson et al., 2009). It has been shown
that this property is also displayed by DPSCs
(Pierdomenico et al., 2005; Wada et al., 2009). Moreover,
it has been demonstrated that infusion of mesenchymal
stem cells (MSCs) expanded in vitro exert a therapeutic
effect in patients with steroid-resistant severe graft-versus-
host disease (Le Blanc et al., 2008), paving the way to
new clinical use of MSCs, even though they derive from
an allogenic source.
Table 1. Patient Characteristics and Final Clinical Score
Score Legend: 0, NO regeneration; 1, =30% regeneration; 2, =70% regeneration; 3, complete regeneration (80-
100%). F, female; M, male. p<0.01, T vs C for all patients except N. 7.
Patient N. Age (YEARS) SEX FINAL SCORES
1 27 F 0 2
2 25 F 1 2
3 29 M 2 3
4 31 F 1 2
5 40 F 2 3
6 36 F 1 2
7 24 F 2 2
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Fig. 6. Histological and
Immunofluorescence analyses on samples
collected after 3 months from surgery.
Haematoxylin-eosin staining reveals better
bone formation at the T site (A) than that
at the C site (B). IF analyses were
performed on bone samples to assess the
expression of bone proteins including
Osteonectin (C - Test site; D - Control site),
Osteocalcin (E - Test site; F - Control site),
BAP (Bone Alkaline Phosphatase) (G-
Test site; H- Control site) and growth
factors such as BMP-2 (I- Test site; J-
Control site) and VEGF (K- Test site; L-
Control site). M: isotype negative control
for fluorescein isothiocyanate (FITC).
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R d’Aquino et al. DPCs repair human mandible defects
second molars, as assessed by clinical and X-ray
evaluation.
The technique that we developed for this clinical study
can be easily applied to any other area of reconstructive
and orthopaedic surgery. Stem cells represent an easy and
natural alternative to repair/regenerate damaged tissues.
This is essential especially when bone loss subsequent to
degenerative or traumatic diseases cannot be amended
through conventional therapies.
Within the craniofacial region, maxillary and mandible
bones often undergo reabsorption following degenerative
diseases, including periodontal disease (the first cause of
tooth loss in the elderly), mandible necrosis or tumour
resections. Autologous DPCs are a new tool for bone tissue
engineering. The procedure is efficient, exhibits low
morbidity of the collection site, and is free from diseases
incurred by transmission of pathogens. The regeneration
process is fast and efficient.
Conclusions
This clinical study has demonstrated the following: (i)
dental pulp stem/progenitor cells can be used for OMF
bone repair; (ii) the use of DPCs on appropriate
reabsorbable scaffolds produces an efficient biocomplex;
(iii) collagen sponges can be considered an optimal support
for the stem/progenitor cells in cell-guided regeneration.
We have given evidence here that autologous DPCs
can be used in a low-risk and effective therapeutic strategy
for the repair of bone defects. The result we have obtained
is encouraging and prompts further clinical trials on a larger
scale of bone loss.
Limitations
Despite the optimal results, the flaws of this study reside
mainly in the small number of patients enrolled. Longer
patient follow-up would ascertain the lifespan of the
regenerated bone. In further studies, regeneration will be
ascertained, other than in bone, in other tissues of the OMF
area.
Acknowledgements
This research was funded by grants from the Italian MIUR
(FIRB 06 n. RBIP06FH7J_006 to GP (Strategic project:
tissue repair using bioscaffold and stem cells).
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    • "Among human stem cells, dental pulp stem cells (DPSC) have relatively easy accessibility, fast multiplication rate, and multi-potential capability, thus could be used in clinic applications [10,11]. It has been reported that DPSC have possibility to repair bone defect in humans [12,13]. Therefore, as undifferentiated mesenchymal stem cells, DPSC may be good candidates to study stem cell regenerative therapies. "
    [Show abstract] [Hide abstract] ABSTRACT: Advances in treatment of tooth injury have shown that tooth regeneration from the pulp was a viable alternative of root canal therapy. In this study, we demonstrated that Gutta-percha, nanocomposites primarily used for obturation of the canal, are not cytotoxic and can induce differentiation of dental pulp stem cells (DPSC) in the absence of soluble mediators. Flat scaffolds were obtained by spin coating Si wafers with three Gutta-percha compounds: GuttaCore™, ProTaper™, and Lexicon™. The images of annealed surfaces showed that the nanoparticles were encapsulated, forming surfaces with root mean square (RMS) roughness of 136-211 nm. Then, by culturing DPSC on these substrates we found that after some initial difficulty in adhesion, confluent tissues were formed after 21 days. Imaging of the polyisoprene (PI) surfaces showed that biomineral deposition only occurred when dexamethasone was present in the media. Spectra obtained from the minerals was consistent with that of hydroxyapatite (HA). In contrast, HA deposition was observed on all Gutta-percha scaffolds regardless of the presence or absence of dexamethasone, implying that surface roughness may be an enabling factor in the differentiation process. These results indicate that Gutta-percha nanocomposites may be good candidates for pulp regeneration therapy.
    Full-text · Article · May 2016
    • "The most well characterized source for MSCs is still the bone marrow, however in the past decade, populations of stem cells have been isolated from different dental tissues (Gronthos et al., 2000; Miura et al., 2003). A population of adult stem cells isolated from dental pulp (DP) tissues and designed as dental pulp stem cells (DPSCs), has been identified as a promising source of MSCs for tissue engineering (Gronthos et al., 2000; Daltoe et al., 2014; Mori et al., 2011; Mori et al., 2010; Spath et al., 2010; d'Aquino et al., 2009; Mangano et al., 2010; Galli et al., 2011; Mangano et al., 2011). http://dx.doi.org/10.1016/j.scr.2015.09.011 1873-5061/© 2015 The Authors. "
    Full-text · Dataset · May 2016 · BMC Oral Health
    • "This multipotency, in addition to their relative accessibility, made DPSCs an appealing source of cells for application in regenerative medicine15161718. In fact, several papers have proved their superiority in different aspects, including osteogenic differentiation [19, 20], which supported their use for regeneration of craniofacial defects [21, 22], as well as alveolar bone defects [23, 24] . Additionally, the similar embryonic origins of dental pulp cells and periodontal cells [25] and their presence within protective layers of tooth structure have encouraged their use for periodontal tissue regeneration [26, 27] . "
    [Show abstract] [Hide abstract] ABSTRACT: Background Regeneration of periodontal tissues is a major goal of periodontal therapy. Dental pulp stem cells (DPSCs) show mesenchymal cell properties with the potential for dental tissue engineering. Enamel matrix derivative (EMD) and platelet-derived growth factor (PDGF) are examples of materials that act as signaling molecules to enhance periodontal regeneration. Mineral trioxide aggregate (MTA) has been proven to be biocompatible and appears to have some osteoconductive properties. The objective of this study was to evaluate the effects of EMD, MTA, and PDGF on DPSC osteogenic differentiation. Methods Human DPSCs were cultured in medium containing EMD, MTA, or PDGF. Control groups were also established. Evaluation of the achieved osteogenesis was carried out by computer analysis of alkaline phosphatase (ALP)-stained chambers, and spectrophotometric analysis of alizarin red S-stained mineralized nodules. Results EMD significantly increased the amounts of ALP expression and mineralization compared with all other groups (P < 0.05). Meanwhile, MTA gave variable results with slight increases in certain differentiation parameters, and PDGF showed no significant increase in the achieved differentiation. Conclusions EMD showed a very strong osteogenic ability compared with PDGF and MTA, and the present results provide support for its use in periodontal regeneration.
    Full-text · Article · Dec 2015
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