RESEARCH

EXPERIMENTAL FORMATION OF PERIODONTAL

STRUCTURE AROUND TITANIUM IMPLANTS
UTILIZING BONE MARROW MESENCHYMAL STEM
CELLS: A PILOT STUDY
Mona K. Marei, MScD; Manal M. Saad, PhD; Adham M. El-Ashwah, PhD; Rania M. El-Backly, MSc;
Mohammed A. Al-Khodary, MSc

Tissue engineering in the head and neck area, presents numerous advantages. One of the most
remarkable advantages is that regeneration of only a small amount of tissue can be highly beneficial
to the patient, particularly in the field of periodontal tissue regeneration. For decades, successful
osseointegration has provided thousands of restorations that maintain normal function. With the
increasing need to utilize dental implants for growing patients and enhance their function to
simulate normal tooth physiology and proprioception, there appears to be an urgent need for the
concept of periodontal tissue regeneration around dental implants. In the present work, 5 goats were
used for immediate implant placement post canine teeth extraction. Each goat received 2 implant
fixtures; the control side received a porous hollow root-form poly (DL-Lactide-co-Glycolide) scaffold
around the titanium fixture, and the experimental side received the same scaffold but seeded with
autogenous bone marrow–derived mesenchymal stem cells. One animal was killed 10 days
postoperatively, and the others were killed after 1 month. The results showed that on the
experimental side, periodontal-like tissue with newly formed bone was demonstrated both at 10
days and after 1 month, while the control specimens showed early signs of connective tissue
regeneration around the titanium fixture at 10 days, but was not shown in the 1 month specimens. It
can be concluded that undifferentiated mesenchymal stem cells were capable of differentiating to
provide the 3 critical tissues required for periodontal tissue regeneration: cementum, bone, and
periodontal ligament. This work may provide a new approach for periodontal tissue regeneration.

Key Words: adult stem cells, periodontal tissue regeneration, immediate implants, tissue
engineering

Mona K. Marei, MScD, is professor of prosthodontics and head,

Manal M. Saad, PhD, is lecturer of oral biology and researcher, INTRODUCTION
Adham M. El-Ashwah, PhD, is lecturer of oral surgery and

A
researcher, Rania M. El-Backly, MSc, is assistant lecturer of
restorative dentistry and lecturer, and Mohammed A. Al-Khodary,
MSc, is assistant lecturer of prosthodontics and researcher at the
Tissue Engineering Laboratories, Faculty of Dentistry-Alexandria
University, Egypt. Address correspondence to Dr Saad at Tissue
Engineering Laboratories, Faculty of Dentistry-Alexandria Univer-
sity, 122 Port-Said St. #12, Ibrahimeya, Alexandria, Egypt. (e-mail:
saad_manal@hotmail.com)
n osseointegrated implant may closely
resemble a natural tooth; however, the
absence of periodontal ligament and
connective tissue via cementum results
in fundamental differences in the adap-
tation of the implant to occlusal forces.

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The character of the tissues around the titanium
implant and the nature of the tissue attachment to the
implant surface influence the biomechanical respons-
es of this integrated system. Because of this structural
difference, functional differences also exist between
teeth and osseointegrated implants.1
In function, the mobility of an osseointegrated
implant is different from that of teeth, which may
result in biomechanical problems when teeth and
implants are combined for the support of a rigid
prosthesis. Although many investigators have recom-
mended using shock-absorbing elements to simulate
the natural resilience of the periodontal ligament in
the implant or its suprastructure, others have pre-
ferred to have anchorage of dental implants with the
same functional mobility as natural teeth.2–4
In addition, physiologic migration of natural teeth
is a functional requirement of adaptation to maintain
interdental and interocclusal relationships—a charac-
teristic that pertains to healthy periodontal ligament
and that it is impossible to achieve with dental
implants.5
Additional differences include the fact that the
potent cellular defense mechanism of the periodontal
ligament protects the tooth in case of inflammation, a
phenomenon that does not exist around dental
implants because of differences in the wound healing
mechanism around a natural tooth with a periodontal
ligament vs an osseointegrated implant with direct
bone-to-implant surface contact.6
In addition, the periodontal ligament has a
sensitive proprioceptive mechanism, which can detect
minute changes in forces applied to the teeth. Forces
applied to the teeth are dissipated through compres-
sion and redistribution of the fluid elements, as well as
through the fiber system. Forces transmitted to the
periodontal ligament can result in remodeling of tooth
movement as seen in orthodontics, or in widening of
the ligament space and an increase in tooth mobility
in response to excessive forces (eg, occlusal trau-
ma).7–9
In osseointegration, a greater level of bone contact
occurs in cortical bone than in cancellous bone, where
marrow spaces are adjacent to the implant surface,
which allows an initial period of healing after the
surgical procedures have been completed. This results
in bone resorption and is followed by bone deposition
over time. Although this is a dynamic process in which
bone turnover occurs, it is not an adaptive process, in
that the same happens with the natural tooth
surrounded by a periodontal ligament.10
Thus, the qualities of the periodontal ligament
(PDL) from the anatomic and functional stand points
Mona K. Marei et al
provide a number of potential advantages derived
from its presence on a dental implant (eg, for the use
of implants in growing patients). This can offer the
potential application of dental implants for orthodon-
tic tooth movement; hence, an implant with a PDL
could be moved orthodontically to optimized posi-
tions so as to achieve more favorable esthetics and
function.11–13
Recent studies have shown the possibility of
formation of periodontal ligament around titanium
implants when a special model of application is used;
this occurs when tooth-to-implant contact results
from orthodontic movement or movement within a
novel dentin chamber model. However, the methods
used were far from clinical applications.5,14
In the present study, we investigated the possibil-
ity of engineering a periodontal structure around a
titanium dental implant placed immediately in a fresh
extraction socket in a goat experimental animal
model. The tissue engineering principles utilized in
the present work included seeding of bone marrow
mesenchymal stem cells onto biodegradable porous
scaffolds to be placed around the titanium implant
fixture.
MATERIALS AND METHODS
Three-dimensional (3D) hollow porous root-form
scaffolds were prepared from 50:50 poly DL-lactide-
co-glycolide (PLG) (Absorbable Polymers International,
Pelham, Ala) with the use of the solvent casting/
compression molding/particulate leaching technique,
as described in our previous work.15,16 PLG scaffolds
were prepared from 50/50 PLG with 1 g of salt of
particle size 180–300 lm. The hollow porous PLG
scaffold was fabricated to fit around the fixture of the
implant, that is, a hexed-head, threaded, 3.75 mm,
hydroxyapatite-coated fixture with mount universal
head diameter, and measuring 10 mm in length
(IMTEC Corporation, Ardmore, Okla).
A mold to serve as the initial iteration was
designed and produced out of Teflon. The dimensions
of the mold were estimated based upon the previous
dimensions of the titanium dental implant screw. The
mold consisted of an insert, a hollow tube, and a solid
cylinder (Figure 1a and b).
In forming the scaffold, the PLG with the salt
particles was placed in the tube and was pressed, and
the flat end of the solid cylinder was used to press the
PLG between the end of the insert and the flat end of
the solid cylinder (Figure 1c). This procedure would
confirm a standardized size, length, and width of the
PLG scaffold each time (Figure 1d and e).
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FIGURE 1. Prototype of designed Teflon mold used to produce hollow porous scaffold around the dental implant screw. (a) The dimensions of
the insert and the tube. (b) The insert, the hollow tube, and the solid cylinder with a flat surface. (c) The three parts as they are used (left
side of photo) and the hollow Teflon tube, with insert inside showing the space to be occupied later by the DL-Lactide-co-Glycolide (PLG)
scaffold (right side of photo). (d and e) The PLG scaffold at the end of the insert as it comes out after molding.

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FIGURE 2. Isolation of bone marrow from goat. (a and b) An 8 mm skin incision was made in the thigh region to expose the bone, then a hole
was drilled in the bone of the femur using a micromotor. (c and d) The marrow was collected from the femur with the use of a 20 mL
syringe with a 14 gauge cannula.
Because the scaffold has to fit accurately around
the endosseous implant in vivo after being seeded, it
was noticed that the PLG scaffold might expand
during the leaching procedure. To avoid such
discrepancies, 8 samples were reproduced using the
previous mold. Multiple measurements were made on
each sample, and if any discrepancies in diameter
measurements were noted, the numbers were aver-
aged and the standard deviation was recorded.
Characterization of the 3D PLG scaffold
Structural Analysis
Imaging of the scaffold was done with a scanning
electron microscope (SEM) (JEOL 5510, JEOL Ltd,
Tokyo, Japan) under low and high vacuum. Samples
for SEM were prepared by fixation in osmium tetroxide
Mona K. Marei et al
at 48C overnight; then the samples were subjected to
dehydration in a graded series of alcohol solutions. For
the low vacuum imaging, samples were viewed
without coating, and then they were gold sputter
coated for high vacuum imaging.
Porosity and Interconnectivity
A total of 6 hollow root-form polymer scaffolds that
were prepared from biodegradable PLG composite
were exposed to porosity measurement by mercury
intrusion porosimetry (Poresizer 9320 V2.08) (ASTM:
D2873, Micrometrics, Norcross, Ga).
Degradation
In all, 36 hollow porous 3D PLG scaffolds were
incubated at 378C in phosphate-buffered saline (PBS)
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FIGURE 3. Implantation of endosseous titanium dental fixture and DL-Lactide-co-Glycolide (PLG) scaffold/cells in fresh extraction socket of
the goat canine tooth. (a) Schematic drawing of goat mandible indicating the area of the experiment. (b and c) The extraction procedure.
(d and e) The procedure of fixture implantation. (f ) Insertion of PLG scaffold/cells construct around the fixture.

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 Mona K. Marei et al

for 6 weeks. At the end of each week, 6 samples were

frozen then freeze dried and weighed for mass loss.
The percentages of mass loss were recorded at the
end of the second week, and the degraded samples
were exposed to mechanical testing under dynamic
loading. After the second week, the samples were too
fragile to be tested.

Animal study
Five goats, ranging in age from 6 months to 3.5 years,
were used in the study. For each one, the following
procedures were carried out.

Isolation of Bone Marrow

Each goat was anesthetized with Xylazine HCl 2% in a
dose of 3 mg/kg (body weight) intramuscular, and
ketamine/HCl 2 mg/kg body weight (b.w.) intrave-
nous. The bone marrow was collected from the femur
with a 20 mL syringe with a 14 gauge cannula
(Figure 2).

Culturing of Bone Marrow–Derived Mesenchymal

Stem Cells
The aspirated bone marrow was cultured in supple-
mented media (DMEM) with 10% FBS, 1% Pen-strep
(100.000 IU penicillin/10.000 lg streptomycin), 1% L-
glutamine, and 2% N-2-hydroxyethylpiperazine-N 0 -2-
ethanesulfonic acid (HEPES buffer). Cells were plated
in 75 cm2 tissue culture flasks and were placed in a
CO2 incubator (5% CO2, 378C, 95% humidity). On day
18, cells were trypsinized and subcultured at 90%
confluence.

Seeding of 50/50 PLG tubes with goat
bone marrow–derived mesenchymal stem cells
Second passage goat bone marrow derived mesen-
chymal stem cells were used for the seeding
procedures. Prepared polymer scaffolds were sterilized
by soaking in 95% ethyl alcohol for 30 minutes. They
were then transferred to 24 well tissue culture plates,
one scaffold in each well. The scaffold was washed
with PBS for 1 hour, and the PBS was changed every
15 to 20 minutes. All PBS was aspirated and scaffolds
were prewet with 2 mL supplemented media. Each
animal received 2 scaffolds: a control scaffold, which
was unseeded, and an experimental scaffold, which
was seeded with bone marrow–derived mesenchymal
stem cells.
Media were added to the control group. For the
test group, after 30 minutes, the media were aspirated
and 500 lL of media containing cells (9.4 3 106 cells/
FIGURES 4–6. FIGURE 4. DL-Lactide-co-Glycolide scaffold expansion data.
% increase ¼ 30%; averaged (65) produced scaffold ¼ 7.1 mm;
averaged (65) mold dimensions ¼ 5.5 mm. FIGURE 5. Percentage of
weight loss of scaffolds after degradation. FIGURE 6. Complex
modulus for samples up to 2 weeks after degradation.
mL) was added to each well (1 scaffold per well). The
following day, the media and unattached cells were
aspirated, then 500 lL of fresh supplemented media
was added to each well. The number of unattached
cells was calculated. The seeded scaffolds were
monitored daily with an inverted light microscope.
On day 2, the scaffolds were used for the animal
experiment. For each experimental animal used,
before the surgery was begun, 2 scaffolds (1 control
and 1 seeded) were utilized for SEM examination using
low and high vacuums.

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FIGURE 7. Characterized DL-Lactide-co-Glycolide (PLG) degraded samples under dynamic loading. (a) PLG scaffold dimensions, which include
5 mm outer diameters, a 4 mm inner diameter, and a 5 mm length. (b) Scanning electron microscopy (SEM) of PLG scaffold shows porosity
and voids in the polymer scaffold. SEM 335. (c) PLG scaffold 1 week after degradation. SEM 335. (d) One week degraded scaffold after
being exposed to dynamic compression load up to 4000 Pa. SEM 3200.

Surgical procedure: implantation of titanium fixture Radiographic and histologic evaluation

and the seeded scaffold in fresh extraction socket
in the goat model
Location: For each animal, lower right and left canine
teeth were used.
The control was the right side, and the experi-
mental side was the left side. On the control side, the
canine tooth was extracted and the titanium fixture
was inserted, then the hollow porous PLG scaffold was
fitted around the fixture. On the experimental side, the
canine was extracted and the fixture was inserted,
then the PLG scaffold/seeded with bone marrow–
derived mesenchymal stem cells (BMDSCs) was placed
and fitted around the titanium fixture, as shown in
Figure 3a through f.
Four animals were killed 4 weeks postoperatively, and
1 animal was killed at l0 days, the mandible of which
was retrieved; occlusal and periapical radiographs
were taken. The 10 day specimens for both control
and experimental sides were radiographed and
processed for non-decalcified sectioning with existing
implant fixtures, then hard sections were obtained and
stained with Van Geison picro-fuchsin and Stevenel’s
blue for light microscopy examination.17 For the 4
week animals, periapical radiographs were taken to
determine the exact place of the implant fixtures. The
mandibles were retrieved and sectioned on the right
and left sides of the implant fixtures. The implant post
was removed from the healed socket to allow study of
the bone/implant interface and comparison of the

112 Vol. XXXV /No. Three /2009


FIGURE 8. Culturing goat bone marrow derived mesenchymal stem cells. (a) Day 6 320. (b) Day 10 320. (c) Day 12 320. (d) Day 18 320.
healing mechanism in bony sockets that received cell/
polymer construct around the implant vs control
specimens, and to examine all surfaces of the fixtures
by SEM.
Four-week specimens were processed and pre-
pared for plastic resin embedding and non-decalcified
sectioning. Hard sections were stained with Van
Geison picro-fuchsin and Stevenel’s blue to be
examined under light microscopy (Olympus, Center
Valley, Pa), and the implant fixtures were prepared for
SEM examination, then for histologic evaluation.
Histophotometric Analysis
Histologic sections also were analyzed with static
cytophotometer microscopy (Leitz, Wetzlar, Germany)
to measure the optical density of newly formed bone
tissue in the peri-implant area. This was done to
evaluate the quality of bone growth at the extraction
Mona K. Marei et al
socket lining wall following the implantation of
different implant constructs.18 Control and experimen-
tal specimens were compared at different levels
around the implant along its vertical axis, from the
top coronal level down to its apical part, corresponding
to the fundus of the extraction socket. At the
horizontal level, measurements were taken to compare
2 different bone layers at the implant/socket wall
interface: the innermost bony layer directly at the
interface level (‘‘the closest to the implant surface’’) and
the layer just next to it (‘‘toward the outer surface of
the jaw bone’’). Collected data were analyzed by means
of 3-factor analysis, with 3-factor interaction occurring
as an error. The analysis of variance (ANOVA) proce-
dure was performed with the Statistical Analysis
System (SAS, SAS Institute, Inc, Cary, NC) program,19,20
and comparisons among means were done to least
significant difference .05 values.
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FIGURE 9. Seeding goat bone marrow derived mesenchymal stem cells onto porous DL-Lactide-co-Glycolide (PLG) scaffold. (a) PLG seeded
scaffold after 2 days under inverted light microscope. (b) Photograph for the seeded scaffold before implantation. (c) Scanning electron
microscopy (SEM) view for the porous surface of unseeded scaffold. SEM 32000. (d) SEM view for the seeded porous surface scaffold. SEM
32000.

RESULTS Figure 5 shows the results of degradation indicating

Scaffold characterization
Results of the expansion of the scaffolds are shown in
Figure 4. This represents 1 iteration, which was used
primarily to calculate the expansion coefficient; then a
precise mold could be designed to produce the
desired size polymer scaffold cylinder. With the
optimized mold, the scaffolds were produced success-
fully to fit around the titanium screw satisfactorily.
The scaffold characteristics were 80% porosity,
measured by mercury intrusion porosimetry that
reflected open-celled structures, with good intercon-
nection between the pores shown by SEM. The
geometry of the 3D hollow root-form scaffold allowed
distribution of masticatory load along the viscoelastic
nature of the scaffold wall and interconnected pores.
that weight loss increased over up to 6 weeks in vitro.
Samples at the second interval demonstrated marked
loss of their mechanical properties under dynamic
compression loading, larger pores were obliterated,
and the smaller-diameter pores were obliterated and
disappeared as shown in Figures 6 and 7.
Cell seeding and infiltration
Cultured cells proliferated and reached confluence in
18 days (Figure 8). Figure 9 shows the cell/scaffold
construct examination by inverted light microscope as
well as SEM, which shows the adherence of cells to the
scaffold with marked proliferation onto the porous
surface.

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 Mona K. Marei et al

Radiographic and histologic evaluation

Radiographic views for the retrieved goat mandible at
10 days are shown in Figure 10. Both sides; the control
and experimental ones, demonstrated similar healing
results radiographically (Figure 10A). The tissue
surrounding the implant fixture resembled the tissue
surrounding the natural tooth of the goat and
distances previously occupied by the periodontal
tissue had the same width of the natural periodontal
tissue surrounding the tooth (Figure 10a through c).
The 10 day histologic specimen of the control side,
where the implant fixture was surrounded for 10 days
by the polymer scaffold without BMDSCs, showed
some areas with spaces resembling periodontal tissue
width (Figure 11a). At larger magnification, connective
tissue fibers seemed to be extending from the original
socket wall through an opening in the newly formed
bone toward the surface of the titanium fixture (Figure
11b). In addition, fibers from these previously ob-
served bundles extended to the titanium surface of
the serration, while other bundles of fibers extended
toward each other, forming a network and filling the
space between the fixture and the newly formed bone
(Figure 11c and d).
In Figure 12, we observed at the control side newly
formed woven bone extending onto the titanium
surface in a way similar to osseointegration; this
phenomenon was noticed in some areas throughout
the whole titanium fixture (Figure 12a through d). In
all control specimens, no polymer scaffold remained
or was noticed histologically.
On the experimental side of the 10 day specimen FIGURE 10. Radiographic views of the 10 days retrieved bony
specimen of goat mandible. (a) Overall radiographic view of the
(Figure 13a), bone regenerated along almost the whole mandible, showing both control side (right side) and
whole length of the implant fixture, maintaining the experimental side (left side) (original magnification 31). (b and c)
space between the titanium screw surface and the Radiographic views of the goat mandible (side view) showing
control side at (b) and experimental side at (c) (original
bone. The width of the space appeared the same all magnification 31). Note the similarities of the interfaces: Tooth/
around the implant and resembled the space occu- surrounding tissue and implant/surrounding tissue.
pied by PDL around the natural teeth. Upon exami-
nation of the specimens, a heavy bundle of connective
tissue fibers was seen extending from the original (Figure 14a), similar to the direction of the natural
socket wall through an opening in the newly formed gingival/periodontal fibers. Toward the middle part of
bone while tightly adhering to the bone and the fixture, we noticed the presence of circular
extending to meet other circular fibers running toward collagen fibers that adhered well to the serration of
and parallel to the titanium surface of the fixture the implant screw, then started to fill the space
(Figure 13a through c). Signs of remodeling were between the implant screws extending toward the
noticed with the presence of osteoclasts away from newly formed bone (Figure 14b through d). These new
the implant fixture, while bone-forming areas were collagen fibers were highly cellular and vascular. No
seen on the newly forming bone surface toward the remaining degraded polymer scaffold was seen in any
implant fixture (Figure 13d). area. Figure 15 demonstrates the dense collagen
Figure 14 shows obvious preservation of the width bundles around the coronal part of the fixture,
of the periodontal ligament space, and the newly extending from the implant surface and running to
formed connective tissue fiber bundles appeared to be inserted into the surface of the newly formed
run parallel to the titanium fixture in the coronal part woven bone (Figure 15a through d).

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FIGURE 11. Histologic evaluation for the 10 day specimen at the control side. (a) Overall implant fixture with the surrounding peri-implant
tissue showing the surface of the implant fixture, newly formed bone around it, and part of the bony socket wall (original magnification
310). (b) Connective tissue fibers extending from the socket wall through the openings in the newly formed bone toward the implant
serration (original magnification 340). (c and d) Larger magnification of the area at (b), showing the bundles of connective tissue fibers
extending with intimate contact to the new bone surface (arrows) (original magnification 3100).

The deposited cellular cementum-like layer on the
titanium surface is demonstrated in Figure 16, which
reveals an evenly thick layer of cementum. In Figure
16a and b, the layer appears to be adherent to the
titanium surface, while the periodontal-like fiber
bundles are inserted into this layer (Figure 16c). The
connective tissue fibers appeared to be attached to
this cementum-like layer. Higher magnification in
Figure 16d shows the cementum-like layer.
In the 1 month specimens, the phenomenon of
periodontal tissue regeneration around the titanium
implant was observed clearly. The titanium screws
were retrieved from the sockets so their surfaces could
be examined via SEM and light microscope. In Figure
17, where the titanium screw was surrounded by PLG
scaffold alone in the control specimen, very few bony
spicules were regenerated in the space surrounding
the retrieved screw, and no epithelium was seen
growing in the space at any time. Healing at the
socket area did not reveal any periodontal tissue–like
fibers, yet new bone projections were seen to extend
toward the space where the screw had existed (Figure
17a through c).
On the experimental side, where the implant
fixture was surrounded for 4 weeks by PLG scaffold
seeded with BMDSCs, peri-implant wound healing was
similar to that seen in the 10 day specimens (Figure
18). Newly regenerated bone trabeculae were ob-
served surrounding the titanium fixture, so the socket
appeared to have double walls of bone: the outside
layer, representing the original socket wall, and the
inside layer, representing the newly formed wall

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 Mona K. Marei et al

FIGURE 12. Histologic evaluation for the 10 day specimen at the control side. (a) Overall implant fixture with the surrounding peri-implant
tissue shows the surface of the implant fixture, newly formed bone around it, and part of the inner bony socket wall (toward midline of the
jaw) (original magnification 310). (b) Newly formed bone adapted directly to the metal surface of the implant (osseointegrated) (original
magnification 340). (c and d) Newly formed bone extending toward implant serration comprised of woven bone and parallel fibers, while
the center of the bone shows varying amounts of lamellar bone (original magnification 340 at [c] and 3100 at [d]).

(Figure 18b), as well as new bone trabeculae that have
been extensively distributed between these two walls
(Figure 18c). In addition, finer bony spicules appeared
to start from the inside bony wall, extending in a
horizontal direction toward the space of the relieved
fixture (Figure 18c). These horizontally arranged
spicules seemed very active and were lined by
osteoblasts (Figure 18d), showing no remnants of
the degraded scaffold (Figure 19a).
At larger magnification, dense bundles of connec-
tive tissue fibers were seen between the active bone
trabeculae also directed horizontally. These bundles
appeared highly cellular and vascular and extended
toward the implant fixture space from all around the
inside bony wall (Figure 19c through f).
Upon examination, the retrieved fixture surface by
SEM showed that some areas in the serrations had
bone segments adhering to them, as shown in Figure
20a and b, while the entire fixture surface was covered
with a cementum-like layer of even thickness. In other
areas, particularly in the middle (third to sixth
serrations), we observed periodontal-like tissue with
obvious bundles; although many of them were cut
during fixture retrieval, the remaining ones demon-
strated the characteristic appearance of connective
tissue fiber bundles anchored to the calcified tissue
surface, resembling Sharpey’s-like fibers (Figure 20c
and d). Although the fibers of new cementum were
running parallel to the fixture surface, the periodontal-
like tissue fibers were seen to originate from the
cementum layer, running away from the fixture
surface toward the new bone spicules and confirming

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FIGURE 13. Histologic evaluation of the 10 day specimen at the experimental side. (a) Overall implant fixture with the surrounding peri-
implant tissue shows the newly formed, almost continuous, bony trabeculae all around the implant fixture (original magnification 310). (b)
The bony wall of the extraction socket and the remaining periodontal tissue extending from the socket wall through an opening between
the newly formed trabeculae toward the implant fixture (original magnification 340). (c) Larger magnification for the top part of the
previous photo shows the remaining periodontal tissue extending from the socket wall to completely surround the newly formed woven
bone and the connective tissue fibers extending toward the implant fixture (original magnification 340). (d) Larger magnification for the
bottom part of the photo at (b) shows the periodontal tissue that extends from the socket wall to completely surround the newly formed
bone. Bone remodeling is obvious, the arrows indicate the presence of osteoclasts at the outer surface of the newly formed bone away
from the implant fixture, and the arrowheads indicate the newly formed woven bone. The same photo shows connective tissue fiber
bundles tightly attached to the surface of the newly formed bone. The surface of the titanium fixture exhibits attachment of the circular
fibers that extend toward the newly woven bone (original magnification 3100).

what was seen in the previous figure (Figure 19). The
histology section of the fixture demonstrated the
tightly adhered cementum-like tissue to the implant
serration surface that was stained with Van Gieson and
Stevenel’s blue (Figure 21a and b).
Histophotometric results
According to the analysis of variance (ANOVA) test for
bone density evaluation at the implant/socket wall
interface, no significant differences in bone density
were found at any level, along the vertical axis of the
implant nor over the bone layers at the horizontal
plane, between the experimental socket wall interface
and the control one in 10 day specimens (Figure 22).
Although the newly formed bone on the experimental
side regenerated in the form of a continuous thin
bony plate along the whole length of the implant, a
tiny consistent distance at the interface area was
found to be filled with connective tissue fibers that
had been growing and extending between the
growing bone and the implant surfaces.

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FIGURE 14. Histologic evaluation of the 10 day specimen at the experimental side implant/tissue interface. (a and c) Preservation of the
original width of the periodontal ligament between the titanium fixture and the newly formed bone. Although the direction of the fibers
was not organized in the coronal part, they seem parallel to the titanium surface, then change direction to horizontal toward the middle of
the fixture, to run from the surface of the fixture to be inserted into the newly formed bone (original magnification 340). (b and d) Higher
magnification of the previous photos demonstrating newly formed bone and bundles of collagen fibers that are inserted into the titanium
surface (original magnification 3100).

On the other hand, the 4 week specimen analysis DISCUSSION

revealed differences in the bone density measurement
at the bony socket wall between the side that received
the implant/polymer construct with BMDSCs and that
of the control socket; this difference was found to be
highly significant (P , .01). These differences were
position dependent, in that the experimental group
showed less bone density at the apical half around the
implant than did controls (P , .05), while no
significant differences have been found between
groups at the implant coronal half. Furthermore, in
both groups, the innermost bone layer (L1) at the
interface showed significantly less density than was
noted in the next layer (outward L2) (P , .05) (Table 1).
Recent advances in the mechanisms of tissue regen-
eration have laid the foundation for novel approaches
in clinical tissue engineering as a product of biotech-
nological advances in the field, and one of the most
significant discoveries in postnatal life.
In dentistry, over the past few decades, the use of
endosseous implants has increased as a means of
providing a foundation for intraoral prosthetic devices,
from single crowns to full arch dentures or other
devices, because of the increasingly convincing data
of long-term clinical success rates, which promoted
the use of implants in more challenging clinical
situations than were previously envisioned.21–23

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PERIODONTAL TISSUE ENGINEERING AROUND TITANIUM IMPLANTS

FIGURE 15. Histologic evaluation of collagen fiber bundles in the 10 day specimen of the experimental side implant/tissue interface, coronal
area. (a) Histologic section at the coronal part of the implant/tissue interface shows bundles of periodontal ligament fibers running parallel
to the implant surface, and newly formed bone inserted into both surfaces (original magnification 340). (b and c) Larger magnification
shows the highly cellular network of periodontal-like fibers filling the space between implant and bone while inserted into the surface of
the newly formed bone from one side and of the newly formed cementum matrix on the implant surface on the other side (original
magnification 3100). (d) Large magnification shows the periodontal-like fibers as they are inserted into the newly formed cementum
matrix on the titanium surface and active newly formed woven bone surface (original magnification 3400).

Although osseointegrated implants provide high
survival rates on a long-term clinical basis, their
supporting mode is not considered ideal. Several
investigators have shown evidence of periodontal
ligament formation around dental implants or other
artificial surfaces.3–5 It has been shown that healing
characteristics of the wound created from implant
placement are determined by the cells derived from
the bone compartment: bone cells, bone marrow cells,
and blood cells, all of which determine the pattern of
healing that results in direct bone apposition on the
titanium surface (osseointegration).24,25
Previous studies have demonstrated that only cells
residing in the periodontal ligament are capable of
forming new cementum by inserting collagen fibers
into exposed root surfaces. These studies focused on
the use of periodontal ligament cells after they had
been grown in culture in vitro. Results showed that
these cells were able to retain their viability and
phenotype, which led to new attachment formation
after seeding in vivo.5,14,26,27
The research model of the present work was
designed to be in the extraction sockets immediately
after tooth removal. The hypothesis was to benefit
from the remaining periodontal ligament with its cells.
Circumstantial evidence suggests that during wound

120 Vol. XXXV /No. Three /2009

 Mona K. Marei et al

FIGURE 16. Formation of cellular cementum-like layer at the surface of the titanium screw at the experimental side (10 days specimen). (a and
b) Continuity of cementum-like layer with even thickness deposited on the titanium surface (original magnification 3400). (c) Even
thickness of cementum-like layer with extrinsic fibers similar to Sharpey’s fibers (original magnification 3200). (d) Higher magnification for
the titanium surface shows the deposited cellular cementum and the extracellular matrix with almost even thickness (original
magnification 3400).

healing in the periodontal ligament, different cell
groups originating from the periodontal ligament
possess the potential to form new/reparative cemen-
tum while simultaneously inserting new attachment
ligament fibers into the newly formed tissue matrix.
However, these observations have been confined to
the areas closest to the source of progenitor cells at
the wound periphery.28
In the present work, we observed growth of
periodontal tissue–like bundles of connective tissue
fibers extending from the periphery of the wound
toward the surface of the titanium fixture (Figures 11
through 15). These observations were seen at both the
control and experimental sides in the 10 day
specimens; this confirms previous study findings,
indicating that progenitor cells from the remaining
periodontal ligament have the capacity to differentiate
into formative cells like cementoblasts and fibroblasts
as long as the peri-implant wound area is close to the
periphery of the extraction socket24,25 (Figures 11
through 15).
Because of the anatomic position of the canines in
the goat, which are tipped toward the orifice of the
oral cavity, and because of the fact that the endo-
sseous implant fixtures were placed in the same
position as the extracted tooth, 1 surface of the
implant fixture was closer than the other to the wound
periphery, and these were the same surfaces in which
we observed extension of new periodontal-like
ligament structure.29,30 Previously, cultured cells de-

Journal of Oral Implantology 121

PERIODONTAL TISSUE ENGINEERING AROUND TITANIUM IMPLANTS
FIGURE 17. Histologic evaluation for the 4 week specimen at the
control side. (a) Healed bony socket at the control side after
removal of titanium fixture (original magnification 310). (b) The
side wall of the bony socket where bone trabeculae extend from
the coronal part of original socket wall toward the implant fixture
space (original magnification 340). (c) Larger magnification for the
socket wall at the side with few bony spicules extending from
socket wall toward the implant fixture space (original magnification
3100).
122 Vol. XXXV /No. Three /2009
TABLE 1
Significance level for ANOVA for bone density evaluation of
studied specimens at different levels around the implant
Significance Level
30 Day Specimens 10 Day Specimens DF S.O.V.
** ns 1 G
* ns 9 P
* ns 1 L
* ns 9 G3P
ns ns 1 G3L
ns ns 9 P3L
0.0101 0.0291 9 Error
Statistical data of the 3 factors under study: (1) Control group and
experimental group (G); (2) position in the vertical axis from top
(P1) to bottom (P10) around the implant; and (3) bone layer at the
interface with the implant (L1) closest to the implant and L2 next to
it. ns, indicates nonsignificance; *, significance at .05 level of
probability; **, significance at .01 level of probability; DF, degree of
freedom; SOV, source of variance; G, group of specimen; P, position
at the socket wall; L, layer of bone.
rived from periodontal ligament and alveolar bone
were found to be basically capable of synthesizing
periodontal tissue after their in vivo reimplantation,
and these cells retained their capability to differentiate
and participate in the regeneration of periodontal
structure.31–34
In the present work, the ability of periodontal
tissue to regenerate on both control and experimental
sides at the periphery of the wound confirmed the
previous findings of these authors that progenitor
cells in the remaining periodontal tissue at the wound
periphery maintain their phenotype to be differenti-
ated under environmental factors such as specific
glycol proteins and under growth factors into
osteoblasts, cementoblasts and fibroblasts.
Histologic findings for the 10 day specimen at the
control side showed areas of osseointegration where
new woven bone was in direct contact with the fixture
serrations (Figure 12). All of these areas were away
from the wound periphery, and the fact that the PLG
scaffold degraded allowed the osteoblasts to reach
the surface of the titanium fixture. This direct bone
apposition or osteoconduction in areas away from the
wound periphery has been recognized by others in
peri-implant healing around endosseous implants.
During this cascade of wound healing, the differenti-
ated osteogenic cells would reach the bone/implant
interface to start the de novo bone formation.35,36
In the experimental sides that received seeded PLG
scaffolds with stem cells at the time of fixture
placement, we observed new continuous woven bone
all around the implant fixture; this was separated from
 Mona K. Marei et al

FIGURE 18. Histologic evaluation for the 4 week specimen at the experimental side. (a) Macroscopic view for the bony specimen before
implant fixture removal. (b) Longitudinal section at the place of the implant fixture shows the empty socket after implant retrieval with
double bony walls; the outside is the original socket wall and the inside is the new bony wall (original magnification 310). (c) Larger
magnification for the socket wall shows the newly formed bony trabeculae parallel to the socket wall and newly formed bony spicules
toward the center of the socket at larger magnification for the square area at (b). (d) De novo regenerated bone extends toward the center
of the socket. These fine spicules are lined by osteoblast cells (original magnification 3400).

the surface of the implant at the bone/implant
interface with a continuously even space that ap-
peared filled with periodontal-like tissue (Figures 13
through 15). These cells along with the progenitor
cells from remaining periodontal tissue in the wound
differentiated into cementoblasts, fibroblasts, and
osteoblasts which were all responsible for periodontal
tissue regeneration and the maintenance of periodon-
tal ligament width. The presence of newly formed
woven bone, the clear bundles of collagen fibers, and
the presence of the cementum-like layer observed on
the titanium surface were all obvious indications of
periodontal-like structure regeneration around the
titanium fixture in the 10 day results (Figures 13
through 16).
Previous investigators have used BMDSCs to
regenerate periodontal tissue in periodontal defects
around natural teeth. These investigators have shown
new cementum, greater cellularity, and sparse extrin-
sic Sharpey’s fibers upon implantation of BMDSCs into
periodontal defects.37–41
In this study of 10 day experimental specimens, we
observed the extension of periodontal tissue toward
the implant fixture in the area near the wound
periphery to separate new woven bone from the
titanium surface. Although progenitor cells from the
remaining tissue residing in the extraction socket

Journal of Oral Implantology 123

PERIODONTAL TISSUE ENGINEERING AROUND TITANIUM IMPLANTS

FIGURE 19. Histologic evaluation for the regenerated periodontal-like tissue in the space surrounded by the inside bony wall experimental
side at 4 weeks. (a) Overall view for the peri-implant tissue area surrounded by the inside bony wall of bone, showing epithelium coverage,
de novo trabecular bone lined by organized bony spicules, and a band of periodontal-like tissue (from outside toward implant) (original
magnification 310). (b) Section showing the superficial epithelium over the socket area that covers the titanium implant fixture (original
magnification 340). (c and d) Section (part of the side wall) showing bony spicules with bundles of connective tissue fibers extending
between the previously mentioned newly formed bony spicules toward the space where the implant fixture existed (original
magnification: [c] 3100, [d] 3400). (e) Section representing the bottom area in the peri-implant tissue located at the fundus of the socket;
de novo bony spicules are apparent, and collagen bundles are running from one side to the other in the socket area, and in between the
bony spicules. At larger magnification (f), highly cellular bundles of connective tissue fibers are very obvious to surround the bony spicules
(original magnification: [e] 3100, [f] 3400).

124 Vol. XXXV /No. Three /2009


might be responsible for this previous finding at areas
near the wound periphery, we observed the dense
collagen bundles and the extrinsic Sharpey’s-like fibers
all around the fixture. The differentiation of BMDSCs
into cementoblasts, osteoblasts, and periodontal
ligament fibroblasts might be responsible for this
previous finding. In addition, a bioactive hydroxyap-
atite layer on the titanium surface of the fixture has
been identified to be a suitable layer for periodontal
regeneration and cementogenesis, because it provides
a low contact angle and superior wettability as well as
adhesion characteristics when compared with the
titanium surface alone.42,43
At 4 weeks, we could not detect any periodontal-
like tissue in the socket created by implant fixture
removal, or at the surface of the fixture on the control
side, which shows that the presence of progenitor
cells from remaining periodontal tissue was not
enough to maintain the newly regenerated collagen
fibers that appeared at the control side in the 10 day
specimen (Figure 17). Instead, bony spicules appeared
to surround the implant fixture, yet they did not fill the
entire area at the bone/implant interface. The
incomplete regeneration of new connective tissue is
believed to be due to the limited capacity of the
progenitor cells from the remaining periodontal
ligament to form and maintain the new attachment.
This fact has been shown by other investigators to be
caused by decelerated restricted migration and
premature differentiation of progenitor cells.29,30
In addition, statistical analysis of the histophoto-
metric measurements showed that the innermost
bone layer (L1) density of the socket wall on the
experimental side was significantly less than that of
the control side 1 month after implant insertion. This
confirms the histologic picture of the widely distrib-
uted newly forming and active bone spicules all
around the lining wall of the extraction socket. These
spicules are obviously of the immature woven bone
type, which is characterized by high cellularity and less
calcium content, compared with mature lamellated
bone, which eventually will be replacing the new
immature form. Such structure could explain the lower
bone density at the experimental socket/implant
interface than in the control group. This phenomenon
was obvious around the implant apical half that
showed the pattern of bone regeneration starting
from the socket fundus up toward the coronal level.
This reflects the role of seeded BMDSCs in new bone
formation. Furthermore, these cells have shown in the
present study their high potential and plasticity
characteristics as mesenchymal unspecialized cells
Mona K. Marei et al
capable of being differentiated to variable cell groups
and producing different tissue types of mesenchymal
origin. Consequently, at the implantation site where
the bony socket was originally formed of alveolar
bone lining containing anchored periodontal ligament
fibers at one extremity, while the other fiber
extremities were inserted into the root cementum of
the extracted tooth, it was found that implanted
BMDSCs demonstrated signs of differentiation and
regeneration of new tissues that had existed originally
at the same site. These tissues represented the main
elements of the tooth-supporting structures surround-
ing and attached to the implant fixture surface.44,45
In our previous work, we have shown that the
presence of BMDSCs seeded onto porous PLG
scaffolds influences the rate of degradation and
enhances bone formation.15,16 The 4 week specimens
at the experimental side confirmed our previous
observations.
All experimental specimens showed a space of
even thickness at the bone/implant interface that was
filled with new connective tissue fiber bundles all
around the implant fixture. These fibers extended
from the bone to the implant surfaces. The histologic
sections showed the connective tissue bundle ar-
rangement emerging between new bone spicules in
an organized direction toward the implant fixture.
Other investigators have shown regeneration of dense
collagen bundles on the root surface 1 month after
transplantation of BMDSCs into furcation defects.41
The surface of the titanium fixture seemed to be
covered with an even layer of cementum-like tissue
that appeared to be adhered well to the fixture
surface. Scanning electron micrographs showed the
cementum-like layer to consist of densely packed
collagen fibrils parallel to the fixture surface, while the
collagen bundles of the periodontal tissue–like struc-
ture appeared to be distinct and loose as they left the
cementum layer. The 1 month retrieved implant
fixtures appeared to be covered with cementum
matrix, which apparently was not fully mineralized,
with collagen bundles simulating periodontal liga-
ment fibers (Figures 20 and 21) being inserted into
and extending toward the newly formed bone
trabeculae.
The formation of periodontal-like structures as
noticed in the present study may open new prospects
in our understanding of wound healing in peri-implant
tissues and may provide a novel approach for
periodontal tissue regeneration. The results of this
work encourage more research in this promising area.
Journal of Oral Implantology 125
PERIODONTAL TISSUE ENGINEERING AROUND TITANIUM IMPLANTS

FIGURE 20. Scanning electron microscope for the titanium fixture retrieved from the 4 week specimen at the experimental side. (a) Overall
view for the retrieved titanium fixture (SEM 325). (b) Area between serrations 2 and 3 showing part of the peri-implant tissue attached to
the surface of the serration covered with densely packed collagen fibers similar to natural cementum; typical perforated-like bone
structure appears at the outer layer of the peri-implant tissue (SEM 3140). (c and d) Area between serrations 3 and 4 showing periodontal-
like tissue with numerous bundles of collagen fibers attached, while many were cut during fixture removal (SEM: [c] 3170, [d] with larger

126 Vol. XXXV /No. Three /2009

 Mona K. Marei et al

FIGURES 21–22. FIGURE 21. Longitudinal histologic section in the retrieved titanium fixture at experimental side. (a) Titanium serrations with
cementum-like tissue that appears structureless and homogenous. It is obvious that this cementum layer is well deposited on the titanium
surface (original magnification 340). (b) Larger magnification for the homogenous cementum-like tissue stained with Van Geison and
Stevenel’s blue (original magnification 3200). FIGURE 22. Diagram of a longitudinal section of goat extraction socket. L1 represents the
innermost layer of the bony socket wall at the interface with the implant, and L2 is the next bony layer. Across the vertical axis, the socket
wall is divided into 10 levels to be studied, starting from P1 down to the socket bottom (P10).

ACKNOWLEDGMENTS moud Hammad for their technical assistance in

We are grateful to Prof. Mamdouh El-Rouby for his manuscript preparation. This work was funded by
valuable contribution to the statistical analysis. We the Academy of Scientific Research and Technology-
also thank Rami Abdelaty, Ahmed Saad, and Mah- Egypt and the US-Egypt joint program.

magnification for 1 bundle; original magnification 31500). (e and f) Area between serrations 4 and 5 showing serrations covered with
cementum-like structure and periodontal-like tissue with obvious numerous collagen bundles of fibers attached to the cementum-like
structure covering the fixture serration. Although the densely packed collagen fibers of the cementum are running parallel to the implant,
the collagen bundles of periodontal tissue are attached to the cementum and are growing perpendicular (SEM: [e] 3250, [f] 31500).

Journal of Oral Implantology 127

PERIODONTAL TISSUE ENGINEERING AROUND TITANIUM IMPLANTS
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