Volumetric changes of the graft after maxillary sinus floor augmentation with Bio-Oss and autogenous...

Volumetric changes of the graft aftermaxillary sinus floor augmentation withBio-Oss and autogenous bone in differentratios: a radiographic study in minipigs

Thomas JensenS�ren SchouPatricia A. SvendsenJulie Lyng FormanHans J�rgen G. GundersenHendrik TerheydenPalle Holmstrup

Authors’ affiliations:Thomas Jensen, Department of Oral and MaxillofacialSurgery, Aalborg Hospital, Aarhus University Hospital,Aarhus, DenmarkS�ren Schou, Department of Oral and MaxillofacialSurgery and Oral Pathology, School of Dentistry,Aarhus University, Aarhus, DenmarkPatricia A. Svendsen, Department of Radiology,Aalborg Hospital, Aarhus University Hospital, Aarhus,DenmarkJulie Lyng Forman, Department of Biostatistics,University of Copenhagen, Copenhagen, DenmarkHans J�rgen G. Gundersen, Stereology and ElectronMicroscopy Research Laboratory, Aarhus Hospital,Aarhus University Hospital, Aarhus, DenmarkHendrik Terheyden, Department of Oral andMaxillofacial Surgery, Red Cross Hospital Kassel,Kassel, GermanyPalle Holmstrup, Department of Periodontology,School of Dentistry, University of Copenhagen,Copenhagen, Denmark.

Corresponding author:Dr. Thomas JensenDepartment of Oral and Maxillofacial SurgeryAalborg HospitalAarhus University Hospital18-22 HobrovejDK-9000 AalborgDenmarkTel.: þ 45 99 32 28 00Fax: þ 45 99 32 28 05e-mail: [email protected]

Key words: alveolar ridge augmentation, bone substitutes, bone transplantation, maxillary

sinus, oral implants, X-ray computed tomography

Abstract

Objective: The objective of the present study was to learn about the volumetric changes of the graft

after maxillary sinus floor augmentation with Bio-Oss and autogenous bone from the iliac crest or the

mandible in different ratios in minipigs.

Material and methods: Bilateral maxillary sinus floor augmentation was performed in 40 minipigs

with: (A) 100% autogenous bone, (B) 75% autogenous bone and 25% Bio-Oss, (C) 50% autogenous

bone and 50% Bio-Oss, (D) 25% autogenous bone and 75% Bio-Oss, and (E) 100% Bio-Oss. The

autogenous bone graft was harvested from the iliac crest or the mandible and the graft composition

was selected at random and placed concomitant with implant placement. Computed tomographies of

the maxillary sinuses were obtained preoperatively, immediately postoperatively, and at euthanasia

after 12 weeks. The volumetric changes of the graft were estimated using the Cavalieri principle and

expressed as mean percentage with a 95% confidence interval (CI).

Results: The mean volume of the graft was reduced by (A) 65% (95% CI: 60–70%), (B) 38% (95% CI:

35–41%), (C) 23% (95% CI: 21–25%), (D) 16% (95% CI: 12–21%), and (E) 6% (95% CI: 4–8%). The

volumetric reduction was significantly influenced by the ratio of Bio-Oss and autogenous bone

(Po0.001), but not by the origin of the autogenous bone graft (P¼ 0.2).

Conclusions: The volume of autogenous bone grafts from the iliac crest and the mandible is reduced

significantly after maxillary sinus floor augmentation in minipigs. The graft volume is better preserved

after the addition of Bio-Oss and the volumetric reduction is significantly influenced by the ratio of

Bio-Oss and autogenous bone. However, further studies are needed addressing the amount of new

bone formation and bone-to-implant contact before the final conclusion can be made about the

optimal ratio of Bio-Oss and autogenous bone.

Maxillary sinus floor augmentation applying the

lateral window technique is the most frequently

used method to increase the alveolar bone height

in the posterior part of the maxilla before or in

conjunction with implant placement, and the

treatment outcome has been reported in several

reviews (Tong et al. 1998; Wallace & Froum

2003; Del Fabbro et al. 2004; Esposito et al.

2006; Browaeys et al. 2007; Pjetursson et al.

2008; Chiapasco et al. 2009; Jensen & Terheyden

2009; Nkenke & Stelzle 2009). In general, grafts

of autogenous bone from the mandible or the

anterior iliac crest are preferred (Burchardt 1983),

but bone harvesting implies a risk of donor site

morbidity (Clavero & Lundgren 2003; Cricchio

& Lundgren 2003). Moreover, substantial resorp-

tion of autogenous bone grafts occurs during

healing. Two-dimensional quantitation after

maxillary sinus floor augmentation has shown

that the graft reduction was as much as 40%

using mandibular bone (Schlegel et al. 2003) and

29–50% when iliac bone was used (Johansson et

al. 2001; Wiltfang et al. 2005; Zizelmann et al.

2007).

A bone substitute of bovine origin is frequently

used alone or in combination with autogenous

bone for the maxillary sinus floor augmentation

(Wallace & Froum 2003; Del Fabbro et al. 2004;

Esposito et al. 2006; Browaeys et al. 2007;

Pjetursson et al. 2008; Chiapasco et al. 2009;

Jensen & Terheyden 2009; Nkenke & Stelzle

2009). The anorganic bone matrix of Bio-Oss

appears to have a microscopic structure similar

to human cancellous bone (Weibrich et al. 2000),

and human and animal studies have indicated no

or limited resorption of the Bio-Oss particles after

Date:Accepted 23 Apr 2011

To cite this article:Jensen T, Schou S, Svendsen PA, Forman JL, GundersenHJG, Terheyden H, Holmstrup P. Volumetric changes of thegraft after maxillary sinus floor augmentation with Bio-Ossand autogenous bone in different ratios: a radiographic studyin minipigs.Clin. Oral Impl. Res. xx, 2011; 000–000.doi: 10.1111/j.1600-0501.2011.02245.x

c� 2011 John Wiley & Sons A/S 1

mailto:[email protected]

maxillary sinus floor augmentation with the

presence of non-resorbed Bio-Oss particles even

after 11 years (Piattelli et al. 1999; Maiorana

et al. 2000; Yildirim et al. 2000, 2001; Hallman

et al. 2001a, 2002a; Orsini et al. 2005; Degidi

et al. 2006; Lee et al. 2006; Busenlechner et al.

2009; Ferreira et al. 2009; Mordenfeld et al. 2010;

de Vicente et al. 2010).

Although Bio-Oss and autogenous bone have

been combined previously for maxillary sinus

floor augmentation (Maiorana et al. 2000; Hall-

man et al. 2001a, 2001b, 2002a, 2002b, 2005;

Yildirim et al. 2001; Hallman & Zetterqvist

2004; Hatano et al. 2004; John & Wenz 2004;

Marchetti et al. 2007; Simunek et al. 2008;

Cannizzaro et al. 2009; Kim et al. 2009; de

Vicente et al. 2010; Galindo-Moreno et al.

2010a, 2010b), different ratios of Bio-Oss and

autogenous bone have never been compared

within the same study. Therefore, the purpose

of the present study was to learn about the

volumetric changes of the graft after maxillary

sinus floor augmentation with Bio-Oss and auto-

genous bone from the iliac crest or the mandible

in different ratios in minipigs.

Material and methods

A license to perform the study was obtained from

the Ethical Committee for Animal Research,

Department of Justice, Copenhagen, Denmark

(Approval no. 2004/561-800).

Animals

The study included 40 adult female Gottingen

minipigs (Ellegaard Gottingen Minipigs A/S,

Dalmose, Denmark) with a mean age of 18

months (range: 17–19 months) and a mean

weight of 31 kg (range: 22–45 kg). During the

study, the minipigs kept in single cages were

fed with a standardized laboratory diet (Altromin

9023, Altromin International Gmbh, Lage, Ger-

many) and water ad libitum.

Anaesthesia and drug administration

Anaesthesia was induced by an intramuscular

injection in the neck region with a mixture of

zoletil (0.125 ml/kg, Virbac SA, Carros CEDX,

France), xylazine (1.6 mg/kg, Rompun, Bayer

HealthCare AG, Leverkusen, Germany), keta-

mine (1.6 mg/kg, Ketaminol, Intervet Interna-

tional B.V., Boxmeer, the Netherlands), and

butorfanol (0.3 mg/kg, Torbugesic, Fort Dodge

Veterinaria S.A., Girona, Spain). For anaesthe-

sia, a standard straight 5.5 mm orotracheal

tube with a cuff (Portex, Kent, UK) was placed

and anaesthesia was maintained by inhalation

anaesthesia with 0.5–1.5% isoflurane (Forene,

Abott Gmbh, Wiesbaden, Germany). During

the surgical procedure, a continuous intrave-

nous infusion through an ear vein of a physiologic

saline solution containing rocuronium bromide

(50 mg/l, Esmeron, Orifarm AS, Denmark) and

fentanyl (250mg/l, Haldid, Janssen-Cilag, Den-

mark) was utilized. An intramuscular injection of

benzylpenicillin (1.2 g, Panpharma, Klampenborg,

Denmark) was given 1 h before surgery and once a

day for 3 days postoperatively. For pain control,

ketoprofen (100 mg, Orudis, Aventis Pharma A/S,

H�rsholm, Denmark) was administered intra-

muscularly once a day for 3 days after surgery.

Surgical procedure

The 40 animals were randomly divided into two

groups of 20 minipigs. The treatment sequence of

the animals was selected at random by drawing a

number between one and 20. Maxillary sinus

floor augmentation was performed bilaterally and

autogenous bone was harvested from the iliac

crest (Group 1) or the mandible (Group 2). The

allocation of graft and number of sinuses is out-

lined in Tables 1 and 2.

Bone graft harvesting from the iliac crest

A skin incision was made above the iliac crest

and the tissues were dissected to expose the

lateral surface of the posterior iliac crest (Fig.

1a, b). A cortico-cancellous bone graft involving

the entire posterior iliac crest was harvested with

a fissure bur during continuous cooling with a

sterile saline solution and chisel (Fig. 1c). The

periosteum and skin were readapted and sutured

in layers with resorbable sutures (Vicryl 2-0,

Ethicon, Norderstedt, Germany).

Bone graft harvesting from mandible

The lateral and inferior mandibular border was

exposed through a submandibular skin incision

and the tissues were dissected to expose the

mandible (Fig. 2a, b). The mental foramen in-

cluding the neurovascular bundle was identified

and protected. A 6 � 1.5 cm cortical bone graft

involving the lateral and inferior cortex was

harvested with a fissure bur during continuous

cooling with a sterile saline solution and chisel.

The periosteum and skin were readapted and

sutured in layers.

Bone graft

The harvested bone was particulated by a bone-

mill (Roswitha Quetin DentalProdukte, Leimen,

Germany) with 3 mm perforations to obtain bone

graft particles with a size of 0.5–2 mm3. The graft

composition was selected at random according to

Table 2. To achieve a standardized total graft

volume of 5 cm3 in each maxillary sinus, four

stainless-steel measuring cups with volumes of 5,

3.75, 2.5, and 1.25 cm3, respectively, were used

(Fig. 3). The selected ratio of autogenous bone

and Bio-Oss (Geistlich Pharma AG, Wolhusen,

Switzerland) (particle size: 1–2 mm) was mixed

and soaked in autogenous blood from an ear vein

until use.

Maxillary sinus floor augmentation and implantplacement

Maxillary sinus floor augmentation and implant

placement were performed according to the pro-

cedure described previously (Terheyden et al.

1999). Briefly, the lateral maxillary sinus wall

was exposed through a skin incision below the

lower eyelid (Fig. 4a). A 1 � 1 cm window to the

maxillary sinus was created with a burr (Fig. 4b),

and the Schneiderian membrane was carefully

elevated with blunt dissectors (Fig. 4c). The

maxillary sinus wall just posterior to the window

created was reduced to a thickness of 5 mm and

an implant bed was successively prepared by 2

Table 1 Allocation of graft and no. of sinuses

No. of sinuses (n) Iliac bone (%) Bio-Oss (%)

Autogenous bone from the iliac crest (Group 1)8 100 08 75 258 50 508 25 758 0 100

No. of sinuses(n)

Mandibular bone(%)

Bio-Oss(%)

Autogenous bone from the mandible (Group 2)8 100 08 75 258 50 508 25 758 0 100

Table 2 Randomized selection of graft

Animalno.

Right maxillarysinus

Left maxillarysinus

1 1 22 1 33 1 44 1 55 2 16 3 17 4 18 5 19 2 3

10 2 411 2 512 3 213 4 214 5 215 3 416 3 517 4 318 5 319 4 520 5 4

1, 100% autogenous bone; 2, 75% autogenous bone

and 25% Bio-Oss; 3, 50% autogenous bone and 50%

Bio-Oss; 4, 25% autogenous bone and 75% Bio-Oss; 5,

100% Bio-Oss.

Jensen et al �Maxillary sinus floor augmentation

2 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02245.x c� 2011 John Wiley & Sons A/S

and 3 mm twist drills at 800 rpm. An oral im-

plant (4 � 15 mm, Branemark, RP, TiUnite,

Nobel Biocare AB, Goteborg, Sweden) was in-

serted with primary stability and a cover screw

(Fig. 4d). The graft selected was packed around

the implant (Fig. 4e) and the window created was

covered by a membrane (25 � 25 mm, Bio-Gide,

Geistlich Pharma AG). Thereafter, the perios-

teum and skin were readapted and sutured in

layers.

Euthanasia

The animals were euthanized after 12 weeks.

The animals were deeply anaesthetized and a

midsternal incision, followed by a sternal split

was performed to perfuse the animals during the

left cardiac ventricle with a neutral-buffered

Ringer solution followed by a neutral-buffered

formaldehyde solution.

Radiography

Axial spiral single slice computed tomography

(CT) scans (Sensation 10, Siemens Medical So-

lutions, Erlangen, Germany) were performed

with 1 mm slice thickness and 1 mm distance

between the slices of the maxillary sinuses with

the animals in a supine position and with the

occlusal plane horizontal. Three sets of CT-scans

were obtained, i.e. preoperatively, immediately

after surgery, and after euthanasia.

To provide blinding of the radiographic evalua-

tion, the CT-scans were coded, and for the

assessment of the volumetric changes of the graft,

the border of the maxillary sinus was transferred

from the preoperative images to the images taken

postoperatively as well as at euthanasia. Initially,

the three sets of CT-scans were uniformly or-

iented in all dimensions using Syngo software

package (version A70A, Siemens Medical Solu-

tions). To obtain an equivalent starting point for

the systematic uniform random sampling of the

three sets of CT-scan images involving the max-

illary sinuses, all images from the posterior bor-

der of the infraorbital foramen to the posterior

part of the hard palate were selected. These two

well-defined anatomic structures were located

close to the anterior and posterior outline of the

maxillary sinus. A total of 20–45 images in-

volved the entire maxillary sinuses. The first

CT-scan image after the infraorbital foramen

was sampled randomly using a random number

table for each maxillary sinus (Gundersen et al.

1999). Every third CT-scan image was selected

from the three sets of CT-scans, thereby ensuring

an equal mutual distance between the selected 7–

15 images.

The selected images were transferred to a desk-

top and the outline of the maxillary sinus on the

preoperative CT-scans was determined using a

virtual marking instrument in Adobe Photoshop

(version CS2, 9.0, Adobe Systems, San Jose, CA,

USA) (Fig. 5a). The recorded outline was super-

imposed on the corresponding images taken post-

operatively and at euthanasia using well-defined

anatomic landmarks to outline the original max-

illary sinus boundary (Fig. 5b, c). Because of

growth, anatomic dissimilarity of the maxillary

sinus boundary was always present in images

taken at euthanasia. Therefore, the adjacent

CT-slice images taken at euthanasia were as-

Fig. 1. Surgical procedure for iliac bone harvesting. (a) Skin incision (arrow) above the posterior iliac crest. (b) The iliac crest

exposed. (c) The cortico-cancellous bone graft involving the entire posterior iliac crest.

Fig. 2. Surgical procedure for mandibular bone harvesting (a) Submandibular skin incision (arrow). (b) The cortical bone graft

involving the lateral and inferior mandibular cortex.

Fig. 3. Stainless-steel cups with a volume of 5, 3.75, 2.5, and 1.25 cm3, respectively, to obtain different ratios of Bio-Oss and

autogenous bone.


c� 2011 John Wiley & Sons A/S 3 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02245.x

sessed. If the comparison revealed improved ana-

tomic uniformity, this CT-image was selected for

the stereologic evaluation. Moreover, minor

transversal growth was seen in all animals.

Thus, the recorded frame was slightly adjusted

to fit the margins of the maxillary sinus on the

CT-scans taken at euthanasia. These procedures

were included to compensate for the growth of

the maxillary sinus during the study period.

A point grid test system was superimposed at

random on all images taken after surgery and

after euthanasia, allowing 100–200 points to hit

the graft of each maxillary sinus (Fig. 5d) (Weibel

1979; Gundersen et al. 1981). The numbers of

intersections (i.e. upper right corner of the

crosses) over the graft were counted on each

selected image. The Cavalieri volume estimation

principle was used to estimate the total volume of

the graft:

V ¼Xn¼7�15

i¼1

P� ða=pÞ � t

where V is the volume, t is the distance between

the sampled images, (a/p) is the area associated

with each test point corrected for magnification,

andP

P is the total number of points hitting the

graft (Gundersen et al. 1988a). Finally, the volu-

metric changes (%) of the graft during the 12-

week healing period were estimated.

Evaluation of the stereologic procedure

To evaluate the sampling design, the coefficient

of variation (CV) (SD/mean) was calculated based

on the original estimates. Moreover, all proce-

dures on eight randomly selected animals, in-

cluding CT-scanning and transfer of outline of

the maxillary sinus, were repeated and the coeffi-

cient of error (CE) (SEM/mean) based on the

differences between the first and the second set

of estimates was calculated. Moreover, the differ-

ences between the repeated estimates were also

examined against there corresponding means by a

scatter diagram (Bland & Altman 1986). The

same investigator (T. J.) performed all recordings.

Data management and statistical analysis

Data management and analysis including the

calculation of descriptive statistics were carried

out using the statistical software R version 2.8.0

(R Development Core Team 2008), in particular

the nlme package (Pinheiro et al. 2008). The

outcome was the relative change of the graft

volume (i.e. the ratio of measurement after eu-

thanasia to postoperative measurement). The data

were analysed in a random-effect ANOVA model

for normally distributed data. As increased volu-

metric changes tend to be characterized by in-

creased variability, a logarithmic transformation

was applied before the analysis to achieve normal

distributions within the groups. Estimated mean

log-reductions were back-transformed to yield

estimates of median percentage reduction of graft

with 95% confidence intervals (95% CI) (Dou-

glas 1991). The effects of the following explana-

tory variables were tested: (1) origin of the

autogenous bone graft (mandible, iliac crest), (2)

ratio of Bio-Oss and autogenous bone, and (3) left

or right maxillary sinus. Further, minipigs (a

categorical variable with 40 levels, represented

by animal number) was included in the model as

a random effect in order to account for the two

repeated measurements made on each minipig.

Results

Minor perforation of the Schneiderian membrane

occurred in six sinuses. The perforation was

Fig. 4. Surgical procedure for maxillary sinus floor augmentation. (a) Skin incision (arrow) below the lower eyelid. (b) Created

window to maxillary sinus. (c) Schneiderian membrane elevated. (d) Implant placed. (e) Graft packed around implant.



covered with a resorbable membrane (Bio-Gide)

and the augmentation procedure was completed.

The accidental membrane perforation occurred

once in each group and appeared not to influence

the volumetric changes of the graft. All implants

achieved good primary stability. Uneventful heal-

ing occurred in 34 animals, and one animal died 2

months after surgery due to unknown reasons. In

addition, unilateral postoperative infection devel-

oped in six animals involving six sinuses: one

with 100% iliac bone, two with 75% mandibular

bone and 25% Bio-Oss, and three with 50%

mandibular bone and 50% Bio-Oss. Two im-

plants were almost entirely surrounded by soft

tissue at the time of euthanasia, probably due to a

chronic infection. The graft used was 100% Bio-

Oss in one sinus, while 50% iliac bone and 50%

Bio-Oss were used in the other sinus. Finally, it

was not possible to place the entire amount of the

graft around the implant in one sinus due to a

very small sinus cavity. Consequently, these

sinuses were excluded from the study. To obtain

equal numbers of observations within each group,

six additional animals were included in the study.

Volumetric changes of graft

The volumetric changes of the graft are presented

in Fig. 6. The mean volumetric changes of the

graft involving iliac as well as mandibular bone

were: (A) 100% autogenous bone: 65% (95% CI:

60–70%), (B) 75% autogenous bone and 25%

Bio-Oss: 38% (95% CI: 35–41%), (C) 50%

autogenous bone and 50% Bio-Oss: 23% (95%

CI: 21–25%), and (D) 25% autogenous bone and

75% Bio-Oss: 16% (95% CI: 12–21%), and

100% Bio-Oss: 6% (95% CI: 4–8%).

The volumetric reduction of the graft was

significantly influenced by the ratio of Bio-Oss

and autogenous bone (Po0.001), but not by the

origin of the autogenous bone (P¼0.2), i.e. iliac

and mandibular bone, and not by the left or the

right maxillary sinus (P¼0.1). Examples are

presented in Figs 7–9.

Evaluation of the stereologic procedure

The mean CVs of the estimated total volume of

the graft varied between 12% and 21% for the

postoperative CT-scans and 16% and 41% for

the CT-scans after euthanasia. The correspond-

ing mean CEs varied between 4% and 8%. A

scatter diagram showed no relation between the

differences of the repeated estimates against the

corresponding means (Bland & Altman 1986).

The analysis of the repeated measurement on the

CT-scans showed that the total number of points

hitting the augmented region on the second CT-

scan was within �15.3% to þ9.2% of the first

CT-scan, with an average 3.8% lower than the

first CT-scan (P¼0.01) (Fig. 10). However, this

Fig. 5. Evaluation of the volumetric changes of the graft. (a) The right maxillary sinus was outlined on the preoperative

computed tomography (CT)-scan image. (b) CT-scan image taken after sinus floor augmentation and implant placement. The

outline of the original sinus was transferred from the preoperative CT-scan. (c) CT-scan image obtained after euthanasia with

the original outline of the maxillary sinus transferred. (d) A point-counting grid was superimposed at random to estimate the

volumetric changes of the graft using the Cavalieri volume principle.

Vol

umet

ric r

educ

tion

of g

raft

(%)

0

20

40

60

80

Fig. 6. Volumetric reduction of graft (%). (�, iliac bone; ., mandibular bone; –, mean).



bias cannot explain the differences between the

groups.

Discussion

The volumetric changes of the graft after maxillary

sinus floor augmentation with Bio-Oss and auto-

genous bone in different ratios were evaluated on

CT-scans in the present study. Significant volu-

metric reduction of grafts composed of autogenous

bone from the iliac crest and the mandible was

revealed. Increased graft preservation occurred

after the addition of Bio-Oss and the volumetric

reduction was significantly influenced by the ratio

of Bio-Oss and autogenous bone.

Stereological methods are precise tools for

obtaining quantitative information about three-

dimensional structures, based mainly on observa-

tions made on two-dimensional images (Gunder-

sen et al. 1988a, 1988b). These methods have not

been used previously for the assessment of volu-

metric changes of the graft after maxillary sinus

floor augmentation. The Cavalieri volume esti-

mation principle is an unbiased method for esti-

mating the volume of three-dimensional

structures based mainly on observations on

two-dimensional images of the object (Gunder-

sen et al. 1988a, 1988b). Systematic uniform

random sampling at all levels of the stereological

procedure is mandatory to obtain unbiased and

efficient estimates (Gundersen et al. 1999). The

sampling must be uniform random to obtain

unbiased estimates and systematic uniform ran-

dom sampling must be used to increase effi-

ciency. Systematic means that the distance

between the sampled images is identical, while

uniform random means that the position of the

first image is random by sampling the first image

at a uniform random position within the distance

between two consecutively sampled images.

The Cavalieri principle requires that the position

of the first image is random, the images are

parallel, and the distance between the images is

identical (Gundersen et al. 1988a). In the present

study, the assumptions for using the Cavalieri

principle were fulfilled. The first CT-scan image

was chosen at random using a random number

table (Gundersen et al. 1999), and the following

parallel CT-scan images were sampled using a

systematic uniform random sampling design in-

cluding a uniform image thickness of 1 mm and a

constant distance of 3 mm between the consecu-

tive images. Using such a sampling design at all

levels of the quantitation procedure, the quantita-

tion can be performed by counting 100–200 points

on 5–10 images from each specimen in most cases

with a CE below 5–10% (Gundersen et al. 1999).

To obtain an equivalent starting point for the

systematic uniform random sampling of the CT-

scan images, two titanium screws were inserted

as landmarks at the time of maxillary sinus floor

augmentation in the frontal and nasal bones.

However, a pilot study on two minipigs revealed

that the titanium screws were displaced due to

growth of the skull, which is why the screws

could not be used as landmarks for the selection

of the CT-scan images. Therefore, the infraorbi-

tal foramen was used as a landmark.

The demarcation of the original border be-

tween the graft and the maxillary sinus becomes

indistinct as the graft becomes integrated. Hence,

evaluation of graft volume changes without

superimposing the original border of the maxil-

lary sinus may not yield a reliable estimate of the

true changes of the graft volume. Therefore, the

original border of the maxillary sinus was trans-

ferred from the preoperative images to the images

taken postoperatively as well as at euthanasia.

Although the mean age of the minipigs was 18

months, minor adjustments of the transferred

frames were necessary to compensate for growth

of the skull during the study period.

The observed total variance of stereologic esti-

mates is a combination of an actual difference be-

tween the specimens (i.e. biologic variation) and the

variance added by the stereological procedure (i.e.

methodological variation). The sampling design of

the present study was assessed by calculating the

Fig. 7. Sinus floor augmentation with a 100% iliac bone graft. (a) Postoperative computed tomography (CT)-scan image. (b)

CT-scan image after euthanasia demonstrating a substantial volumetric reduction of the graft.

Fig. 8. Sinus floor augmentation with a 50% iliac bone graft and 50% Bio-Oss. (a) Postoperative computed tomography (CT)-

scan image. (b) CT-scan image after euthanasia showing limited volumetric reduction of the graft.

Fig. 9. Sinus floor augmentation with 100% Bio-Oss. (a) Postoperative computed tomography (CT)-scan image. (b) CT-scan

image after euthanasia displaying nearly total preservation of the graft.



CV (SD/mean) and the CE (SEM/mean). The CVs

were always much larger than the corresponding

CEs, which is why the variance of the estimates

was caused almost exclusively by an actual differ-

ence between the specimens (i.e. ‘‘biological varia-

tion’’) and not by the stereological methods.

The two-dimensional changes of the graft after

maxillary sinus floor augmentation with Bio-Oss

mixed with autogenous bone have been evaluated

in three human studies (Hallman et al. 2002b;

Hatano et al. 2004; Kim et al. 2009) and one

animal study (Schlegel et al. 2003). The dimen-

sional changes of the graft were evaluated on

tomograms when Bio-Oss (80%) was mixed

with chin bone (20%) (Hallman et al. 2002b).

The height and width of the graft were not

significantly changed from 3 to 12 months after

surgery. In contrast, the height and width were

reduced significantly after 24 months. However,

the reduction was o10%.

The height reduction of the graft has also been

assessed after the application of 33% Bio-Oss

mixed with 66% mandibular bone (Hatano et

al. 2004). The grafted sinus floor was consis-

tently located above the implant tip just after

surgery, while the grafted sinus floor level was

located at or slightly below the implant tip after

2–3 years. Only minor changes occurred after 3

years.

The height changes of Bio-Oss mixed with

small volumes of autogenous bone from the

maxilla have also been assessed (Kim et al.

2009). The alveolar bone height was 19 mm

immediately after sinus floor augmentation and

17.2 mm 1 year after surgery. No statistical

comparison was made.

In conclusion, these human studies indicated

limited reduction of the graft when Bio-Oss was

mixed with autogenous bone.

The two-dimensional changes of the graft after

maxillary sinus floor augmentation with Bio-Oss

or autogenous bone have been addressed in beagle

dogs (Schlegel et al. 2003). The mandibular bone

graft was reduced by 4% and 40% after 90 and

180 days, respectively, while the corresponding

figures for Bio-Oss were 15% and 17%.

The results of the above-described studies are

all based on two-dimensional quantification. The

graft within the maxillary sinus is an inhomoge-

neous and three-dimensional anisotropic struc-

ture, which is why three-dimensional methods

should be applied for most studies assessing the

reduction of the graft. As discussed previously,

CT-scan images provide the opportunity to esti-

mate the volumetric reduction of the graft, pro-

vided that the outline of the original border of the

maxillary sinus can be determined. Volumetric

estimates of autogenous bone grafts using CT-

scans have been described previously (Johansson

et al. 2001; Smolka et al. 2006; Zizelmann et al.

2007). The outline of the graft was plotted on

each CT-scan image, and the area of the graft on

all images was calculated using computer soft-

ware. The graft volume was estimated by calcu-

lating the total number of points over the graft on

all images multiplied by the thickness of the CT-

images. However, the original border of the

maxillary sinus was not transferred from the

preoperative images, which is why precise de-

marcation of the original border between the graft

and the maxillary sinus may be compromised as

the graft integrates.

In conclusion, the present study has shown

that while the volume of autogenous bone grafts

from the iliac crest and the mandible is signifi-

cantly reduced after maxillary sinus floor aug-

mentation in minipigs, graft volume preservation

is improved after the addition of Bio-Oss. More-

over, the volumetric reduction is significantly

influenced by the ratio of Bio-Oss and autoge-

nous bone, although the optimal ratio for obtain-

ing the highest amount of new bone formation

and bone-to-implant contact still remains un-

known.

Acknowledgements The authors are

deeply indebted to Mr Jens S�rensen, Mr Ole

S�rensen, and Mr Torben Madsen for excellent

handling of the animals, and Ms Anni

Wehrmann Pedersen, Ms Jette Bruun, and Ms

Lene Bjerg Jensen for valuable assistance

during the surgical procedures. Finally, the

authors would like to thank Dr Graziella

Andersen for her help with the radiographic

procedures and her valuable input. Bio-Oss and

Bio-Gide membranes were kindly provided by

Geistlich Pharma, Switzerland, and implants

by Nobel Biocare AB, Sweden. The study was

supported by grants from FUT/Calcin-fondene,

KOF/Calcin-fondene, Det Obelske

Familiefond, Nordjyllands Amts

Forskningsrad, Direkt�r E. Danielsens og

Hustrus Fond, Familien Hede Nielsens Fond,

Gangstedfonden, Augustinus Fonden,

Speciallæge Heinrich Kopp’s Legat, Else og

Mogens Wedell-Wedellsborgs Fond, and

Peder Kristen T�fting samt Dagmar T�fting’s

Fond.

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