JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

Generation of New Bone Around Titanium Implants

Using a Membrane Technique: An Experimental Study in
Rabbits
Christer Dahlin, DDS/Lars Sennerby, DDS/Ulf Lekholm, DDS, Odont Dr/Anders
Linde, MSc, DDS, Odont Dr/Sture Nyman, DDS, Odont Dr

Insufficient bone volume may be a significant problem in connection with

dental implants. In this study, a technique based on the principle of guided
tissue regeneration was tested for its ability to generate bone tissue around
titanium implants. Implants were inserted in tibiae of rabbits. To create a
secluded space for osteogenesis and to prevent soft-tissue ingrowth, a porous
Teflon membrane was placed around exposed parts of the implant. Where a
membrane had been used, the threads of the implant were completely covered
with significant amounts of new bone. This study indicates that the membrane
technique is a reconstructive surgical method that may be applicable to create
new bone around exposed parts of titanium implants in a clinical setting. (INT J
ORAL MAXILLOFAC IMPLANTS 1989;4:19-25.)
Key words: bone regeneration, osteogenesis, Teflon membrane, titanium implant

For more than 20 years, the osseointegration procedure ad modum Brånemark has
been successfully used in the treatment of edentulism.1 One crucial factor
influencing the outcome of the treatment is the anatomy of the jaw bone at the site of
fixture installation. Since the technique requires complete bone coverage of the
threaded part of the titanium implant at installation, a minimum of 7 x 4 mm of bone
tissue is needed when the smallest type of implant for oral application is used.2 In the
case of insufficient bone volume, either a bone-grafting procedure could be used to
increase the amount of bone tissue3,4 or exposed threads of the implant must be
accepted if treatment is still to be performed. The first alternative requires
hospitalization and is resource-demanding. Furthermore, that technique is still under
clinical evaluation.4 With regard to the second alternative, exposed threads of the
fixture head may create mucosal disorders, particularly if the threads are facing
movable oral mucosa.5 In addition, when a few small implants have to be installed,
the treatment is limited to support overdenture constructions only.6
With this in mind, it would be of interest to find an alternative to bone grafting
in order to increase the jaw bone volume. A method for healing of bone defects and
generation of new bone, using a membrane technique, has recently been described
by Dahlin et al.7 This technique implies that a mcmbrane is placed in such a way that
a secluded space is created into which cell populations from surrounding bone tissue
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

virtually exclusively can proliferate. Thus, ingrowth of connective tissue is

prevented. Similar techniques have been used for regeneration of other tissues, eg,
the attachment apparatus around teeth with advanced periodontal tissue loss.8
The purpose of the present investigation was to evaluate the feasibility of using
the principle of guided tissue regeneration to generate bone at exposed parts of
titanium implants. The tibial sites in the rabbit were chosen to mimic the clinical
situation, since the dimensions of this bone correspond well with human alveolar
bones.
Materials and Methods
Animals and Anesthesia. Fifteen adult New Zealand rabbits of both sexes (body
weight 3.1 to 3.7 kg) were used. General anesthesia was induced by intramuscular
injection of fluanison (Hypnorm®, Leo, Helsingborg, Sweden) at a dose of 7 mg/kg
body weight and intraperitoneal injection of diazepam (Stesolid®, Dumex,
Copenhagen) at a dose of 1.5 mg/kg body weight. Additional fluanison was provided
intramuscularly when needed. Local anesthesia at the site of surgery was given using
1 ml of 2% lidocain/adrenaline solution (Astra, Södertälje, Sweden).
Implants and Membrane. Thirty sterile, "commercially pure" titanium
implants (10-mlll fixtures, Nobelpharma, Gothenburg, Sweden), most of the without
cover screws, were installed. Polytetrafluoroethylene (PTFE) membranes
(Gore-Tex®, WL Gorc & Assoc, Flagstaff, Arizona) with a pore size of 0.45 µm
were cut into pieces approximately 2 cm2, sterilized, and used to cover the implants.
Surgical Procedure. The medial border of the proximal tibial metaphysis was
chosen as the site of implantation. The hind legs of each animal were shaved and
washed with 0.1% Jodopax® (Ferrosan, Malmö, Sweden), and the animal was
covered with a clinidrape perforated for the hind legs. The bone surface was exposed
via a skin incision and careful subperiosteal dissection. A fixture was installed at the
medial border of both tibiae using the standardized technique described by Adell et
al.9 Briefly, a series of stainless steel drills of successively increasing dimensions
were used at a speed of 2,000 rpm under vigorous cooling with saline. A titanium
screw tap was used to thread the bone site before the fixture was installed. Both of
these latter procedures were performed at a low speed (15 to 20 rpm) and under
extensive saline irrigation. The fixture was inserted into the bone in such a way that
three to four of the coronal threads were left exposed on one side of each implant (
Figs 1a and 1b). After installation, the fixture with surrounding bone tissue was
photographed using a Carl Zeiss dissection microscope ( x 9) Photometric
assessments were made to evaluate the amount of newly formed bone (described
later).
In each animal, a PTFE membrane was placed over one of the fixtures (test side)
in such a way that it also covered 5 to 8 mm of the surrounding bone (Figs 2a and 2b
). The muscle/periosteal flap was adapted on the outer surface of the membrane to
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

complete coverage and was secured with a 4-0 Dexon Plus suture (Nobel Medica,
Sundbyberg, Sweden). The fixture implanted in the contralateral tibia in the same
animal (control side) served as a reference, as no membrane was placed. The skin
was secured with a 3-0 Novafil suture (Nobel Medica). The animals were divided
into three groups with five animals in each, and they were allowed healing periods of
6, 9, and 15 weeks, respectively. The animals were kept in separate steel cages and
given food and water ad libitum.
Tissue Processing. The animals in each group were sacrificed by an overdose of
Hypnorm. The fixtures with surrounding tissue were removed en bloc and fixed by
immersion in 4% buffered formalin. The membrane and its soft-tissue covering at
each test fixture and the soft tissue at each control fixture were carefully removed.
The exposed areas were photographed in the same manner as upon installation, and
the amount of newly formed bone was photometrically assessed.
Photometric Evaluation. The difference in surface area of exposed titanium
between the photos taken at installation and after healing represented the amount of
newly formed bone. The gain was expressed as percentage of the initial defect area.
A Kontron IBAS II interactive image analysis system was used for the assessment.10
The light transmittance of the image was detected by a television camera and the
video signal analog-to-digital converted to a gray scale of 512 x 512 pixels. By
means of the gray scale from the exposed area of the implant, a numeric value of the
amount of newly formed bone was obtained.
Histologic Examination. To obtain tissue sections for histologic analysis, a
sawing and grinding technique was used (Donath and Breuner11). In short, the
specimens were dehydrated in a graded series of ethanol and preinfiltrated for 6
hours in vacuum with a 1:1 solution of ethanol and 2-hydroxyethyl methacrylate
(Merck-Schuchardt, Hohenbrunn, West Germany). After this, the specimens were
infiltrated for 7 days at 4° C with LR white resin (Polaron, Johnson Instrument,
Stockholm, Sweden). Thereafter, vacuum infiltration and embedding were
performed using fresh LR resin. After polymerization, the tissue samples were
sawed under water irrigation (Exact, Cutting Grinding System, Copenhagen) to
approximately 200 ,µm thickness and subjected to grinding (Struers Scientific
Instruments, Copenhagen). The thick sections produced after polishing, mounting,
and sawing were ground to about 10 µm and stained with toluidine blue/pyronin G.
The sections were examined in a Zeiss light microscope.
The newly formed bone was analyzed with respect to structure, quality, and
quantity. Histologic measurements were also performed along the midaxis of the
implant in the exposed area. Since this region would represent the longest distance
for the bone-forming cells to migrate, the least favorable location for new bone
formation was thus used for assessment of the amount of new bone. Using the crest
of the original bone (OB) as a starting point, the distance between OB and a
reference point (RP) on the fixture, as well as the distance between OB and the crest
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

of the newly formed bone (NB), were measured. The thickness of the newly formed
bone was estimated in the midportion of the previous defect along the same axis.
Estimation of Errors. To estimate the inherent error in the photometric
assessments and the histologic examinations from a total of 30 fixture surfaces with
different amounts of new bone formation, all fixtures were re-examined after 2
months. For the photometric measurements, the mean difference between the first
and second examination was 1.4 ± 4.3% (mean value ± SD), and the standard
deviation of one single measurement was 3.1. For the histologic measurements, it
was 1.6 ± 3.9% (mean value ± SD). The differences in both cases were not
statistically significant.
Results
Healing in all surgical sites occurred uneventfully, and all 15 test and 15 control
fixtures with surrounding tissue were available for photometric evaluation and
histologic examination. When the test areas were exposed, the membranes were
found to be retained in proper position under the soft tissue and tightly adapted to the
surface of the newly formed bone; considerable force was required to separate the
membrane from the underlying bone. No clinical signs of inflammatory reactions
were seen around the membranes.
In general, it was found that new bone, covering the threads of the titanium
implants, already had formed after a 6-week observation period (Figs 3a and 3b).
Because of this, all of the 15 animals (ie, the 6-, 9-, and 15-week groups) were
evaluated as one group. No statistical differences were seen between the groups.
The results of the photometric measurements of the newly formed bone covering
the titanium implants are given in Fig 4. A larger amount of new bone (highly
statistically significant; P < 0.0001) was formed at the test sites (B = 99. 5%; range
95.6% to 100%) compared with the controls (B = 66.4%; range 38.9% to 92.4%)
(Student's t test).
In histologic sections it could be seen that the newly formed bone in both test
and control sites was highly trabecular and stained darker than the original tibial
bone (Figs 5a to 5c). Osteoblasts were found lining marrow spaces containing a
loose connective tissue (Figs 6a and 6b). Osteocytes were seen embedded in the
mineralized bone. Direct contact between the new bone tissue and the titanium
surface was evident at all sites, on the test as well as control sides. Various amounts
of new bone were also seen in a few specimens on the hexagonal head of the test
fixtures above the reference point.
The results of the linear measurements along the midaxis of the exposed implant
areas are presented in Fig 7. In the test areas, the new bone reached a significantly (P
< 0.0001) higher coronal level (B = 3.8 mm; range 3.1 to 4.6 mm) compared with
the controls (B = 2.2 mm; range 0.8 to 3.8 mm) (Student's t test). In the test
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

specimens, the newly formed bone had a uniform thickness all along the previously
exposed surface of the implant (average of 0.7 mm) compared with the controls,
where the bone became clearly thinner in the coronal direction. There were no
statistically significant differences between the healing periods.
Discussion
The purpose of this investigation was to study the potential of a new membrane
technique, based on the principle of guided tissue regeneration for bone healing,7
used in connection with exposed parts of titanium implants. The results showed that
all of the exposed surfaces on the test side became filled with newly formed bone
(see Fig 4) of a uniform thickness (see Figs 5a to 5c). The bone grew predictably all
the way up to the reference point on the test side but, with large variations, on
average only half the way on the control side (see Fig 7).
The results of the present study thus clearly demonstrate that a complete
selective formation of bone within a defined bone defect can be accomplished by
preventing cells not derived from the surrounding bone from repopulating the defect
area. The regeneration already had occurred after 6 weeks. These results are in
agreement with the authors' earlier findings,7 in which complete bone regeneration of
large perforating bone defects in the mandibles of rats was obtained using a similar
surgical technique.
By placing a biologically inert membrane with a pore size that will not allow
penetration of cells, and by suturing the soft tissue flap (including the periosteum) on
the outer surface of the membrane, an empty space was created into which only cells
capable of inducing bone formation were allowed to proliferate. This, in turn,
resulted in the successful closure of the space with bone tissue. Since the newly
formed bone completely filled the space between the membrane and the exposed
parts of the fixtures, the thickness of the new bone is obviously determined by the
width of the space. This means that the new bone may be designed to a required
thickness by varying the contour of the membrane, which can be achieved by
varying the stiffness of the membrane material.
New bone formation was also seen in control areas of the present study, but to a
much lesser extent than in the test areas. While on the test side only bone-forming
cells were allowed to migrate into the wound, granulation tissue derived from the
soft-tissue cover flap could also invade the wound area on the control side, thereby
competing with the bone-forming cells. The amount of newly formed bone in this
situation may be highly dependent on the differences in proliferation rate of the
different types of cells and the size of the available space. This may explain the
pronounced variation in new bone formation at the control fixtures and also the
finding that only two out of 15 control implants showed a bone coverage reasonably
close to that obtained on the membrane side. All test implants presented practically
complete bone coverage of the exposed threads.
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

A certain amount of new bone formation also occurred in a few specimens on

the hexagonal head of the test fixtures. This was most probably caused by variation
of the tightness in adaption between the membrane and the surface of the hexagonal
head. Hence, if the membrane is located in close contact with the surface of the
hexagonal head, no bone can form in this area; however, if a small space exists,
bone-forming cells can invade the space and new bone will form. In this context, it
should be realized, however, that if the most coronal parts of the fixture, ie, the
hexagonal heads (no cover screws were used), are covered with new bone, this bone
must be removed in a second procedure to connect an abutment cylinder to the
fixture.
The histologic examination of the material showed that the newly formed bone
stained darker than the original tibial bone, presumably representing a more active
phase of osteogenesis. The implant surface indeed had a partly direct contact with
the newly formed bone, so-called osseointegration12 (see Figs 6a and 6b). The partial
contact between bone and the implant surface normally appears at this stage of
healing.13 The surface in direct contact will gradually increase during prolonged
time of healing.
Conclusion
The present study has documented that guided tissue regeneration provides a
biologic principle for the development of reconstructive surgical techniques aimed at
generating bone tissue of sufficient volume for installation of titanium implants.
Although this study was performed in the rabbit, it may be surmised that the
technique is also applicable to humans in a clinical setting.
Acknowledgments
The authors gratefully acknowledge the technical assistance given by Ms Kerstin
Bjurstam and Ms Anneli Sagebring. This study was supported by the Faculty of
Odontology at the University of Gothenburg, the Public Dental Health Service of the
City of Gothenburg, the Swedish Medical Research Council, and the Swedish
National Board for Technical Development. The titanium implants were kindly
supplied by Nobelpharma Implant System, Gothenburg, Sweden.
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

1. Adell R, Eriksson B, Lekholm U, Brånemark P-I, Jemt T: A long-term follow-up

study of osseointegrated implants in the treatment of the totally edentulous jaw.
In manuscript, 1988.
2. Lekholm U, Zarb GA: Patient selection and preparation, in Brånemark P-I, Zarb
GA, Albrektsson T (eds): Osseointegration in Clinical Dentistry. Chicago,
Quintessence Publ Co, 1985, pp 199-204.
3. Breine U, Brånemark P-I: Reconstruction of alveolar jaw bone. An experimental
and clinical study of immediate and preformed autologous bone grafts in
combination with osseointegrated implants. Scand J Plast Reconstr Surg
1980;4:23-48.
4. Adell R, Lekholm U, Gröndahl K, Brånemark P-I, Lindström J, Jacobsson M: Fresh
autogenous bone grafts combined with fixtures in the treatment of severely
resorbed edentulous upper jaws. In manuscript, 1988.
5. Lekholm U, Adell R, Brånemark P-I: Complications, in Brånemark P-I, Zarb GA,
Albrektsson T (eds): Osseointegration in Clinical Dentistry. Chicago,
Quintessence Publ Co, 1985, pp 233-240.
6. Zarb GA, Jansson T: Prosthodontic procedures, in Brånemark P-I, Zarb GA,
Albrektsson T (eds) Osseointegration in Clinical Dentistry. Chicago,
Quintessence Publ Co, 1985, pp 241-282.
7. Dahlin C, Linde A, Gottlow J, Nyman S: Healing of bone defects by guided tissue
regeneration. Plast Reconstr Surg 1988,81:672-676.
8. Nyman S, Lindhe J, Karring T: Reattachment—New Attachment, in Lindhe J (ed):
Textbook of Clinical Periodontology. Copenhagen, Munksgaard, 1988, pp
409-429.
9. Adell R, Lekholm U, Brånemark P-I: Surgical procedures, in Brånemark P-I, Zarb
GA, Albrektsson T (eds): Osseointegration in Clinical Dentistry. Chicago,
Quintessence Publ Co, 1985, pp 211-232.
10. Kälebo P, Jacobsson M, Albrektsson T, Turesson I: Bone healing following
irradiation during tourniquet ischaemia. Acta Oncol 1987;26:63-68.
11. Donath K, Breuner G-A: A method for the study of undecalcified bones and teeth
with attached soh tissue. The Saege-Schliff technique. J Oral Pathol 1982;
11:318-325.
12. Brånemark P-I, Hansson B-O, Adell R, Breine U, Lindström J, Hallén O, Öhman
A: Osseointegrated Implants in the Treatment of Edentulous Jaw. Experience
from a 1O-year Period. Stockholm, Almqvist & Wiksell Int, 1977.
13. Johansson C, Albrektsson T: Integration of screw implants in the rabbit: A l-yr
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

follow-up of removal torque of titanium implants. Int J Oral Maxillofac Implants

1987;2:69-75.
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

Figs. 1a and 1b Insertion

of fixtures into the medial border of the tibia. Fig 1a (left) shows the installation of the
fixture at the bone site under vigorous cooling with saline. In Fig 1b (right), the fixture has
been installed in such a way that three to four of the coronal threads were left exposed
on one side of each implant.

Figs.
2a and 2b Covering of the implant with membrane. In each animal, one fixture (test) was
covered with a PTFE membrane extending 5 to 8 mm around the fixture (left). The
periosteal flap was adapted on the outer surface of the membrane, aiming at full
coverage and stabilization of the membrane (right).

Figs.
3a and 3b Macroscopic appearance of titanium implants after 6 weeks of healing. On
left
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

the test side (left), where a membrane had been placed, all threads were covered with
bone, whereas on the control side (right) with no membrane, several exposed threads
were still seen.

Fig. 4 Area of new bone

formed on test (membrane) and control (no membrane) side. Results of photometric
assessments in each experimental animal. Healing periods were 6, 9 and 15 weeks
respectively, with five animals in each group. Results are expressed as bone gain in
percent of the original defect area.

Figs
thick) represent the midportion of the fixtures. The sections were stained with toluidine
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

blue/pyronin G. Histologic measurements expressed the distance (mm) from the original
bone crest after preparation of the implant site (OB) to the reference point on the fixture
(RP), as well as the distance from the original bone crest (OB) to the crest of the newly
formed bone (NB). The results were expressed as percent gain of bone cover. Fig 5a
(left) shows the situation at the time of fixture installation (original magnification x40).
Figs 5b (center) and 5c (right) demonstrate bone healing on the control side (5b, no
membrane, x40) and the test side (5c, membrane, x80) after 6 weeks.

Figs
side. Ground section, stained with toluidine blue/pyronin G. Original magnifications x125
(left) and x500 (right). Deposition of bone (B) into the threads of the implant (IM) is
evident. Numerous osteoblasts can be seen. Osseointegration is evident, as indicated by
light microscopy.

Fig. 7 New bone formed on

test (membrane) and control (no membrane) side. Results of histologic measurements in
longitudinal sections from each experimental animal. Healing periods were 6, 9, and 15
JOMI on CD-ROM, 1989 Jan (19-25 ): Generation of New Bone Around Titanium Implant… Copyrights © 1997 Quint…

weeks, respectively, with five animals in each group. Results are expressed as
longitudinal bone gain in percent of the previously exposed area.