Sinus augmentation analysis revised:the gradient of graft consolidation

Dieter BusenlechnerChristian D. HuberChristoph VasakAntonia DobsakReinhard GruberGeorg Watzek

Authors’ affiliations:Dieter Busenlechner, Christian D. Huber,Christoph Vasak, Reinhard Gruber, Georg Watzek,Department of Oral Surgery, Medical University ofVienna, Vienna, AustriaDieter Busenlechner, Christian D. Huber,Christoph Vasak, Antonia Dobsak, ReinhardGruber, Georg Watzek, Austrian Cluster for TissueRegeneration, Vienna, AustriaAntonia Dobsak, Ludwig Boltzmann Institute forClinical and Experimental Traumatology, Vienna,Austria

Correspondence to:Reinhard GruberDepartment of Oral SurgeryMedical University of ViennaWahringerstrasse 25aA-1090 ViennaAustriaTel.: þ 43 4277 67011Fax: þ 43 4277 67019e-mail: reinhard.gruber@meduniwien.ac.at

Key words: animal model, bone graft, bone regeneration, degradation, hydroxyapatite

osteoconduction, sinus augmentation

Abstract

Objective: Graft consolidation follows a gradient that reflects the properties of bone

substitutes at sites of sinus augmentation. Here we present an analytical method to

investigate the process of graft consolidation taking the distance from the maxillary host

bone into account.

Material and methods: We therefore evaluated histological specimens, 6 and 12 weeks

after the sinus of minipigs was augmented with Bio-Osss

, a deproteinized bovine bone

mineral, and Ostims

, an aqueous paste of synthetic nanoparticular hydroxyapatite. A curve

was drawn that represents the changes in histomorphometric parameters within a given

distance from the maxillary host bone.

Results: Based on this curve, three regions of interest were defined: R1 (0–1 mm) the

bridging distance where new bone is laid onto the host bone, R2 (2–3 mm) a region of

osteoconduction where new bone exclusively grows on the biomaterial, R3 (4–5 mm) and a

region of osteoconduction where bone formation has reached its maximal extension.

Qualitative and quantitative analysis of the three regions can reveal differences in graft

consolidation, depending on the bone substitutes and the observation period [Bone

volume (BV) per tissue volume after 6 weeks: R1: 19 � 8.4% for Bio-Osss

and 42.9 � 13.2%

for Ostims

(P¼0.03), R2: 3 � 2.4% for Bio-Osss

and 14.7 � 9.5% for Ostims

(P¼0.03), R3:

5 � 4.1% for Bio-Osss

and 5.3 � 5.3% for Ostims

(P¼0.86). BV per tissue volume after 12

weeks: R1: 38.0 � 13.3% for Bio-Osss

and 53.3 � 6.6 for Ostims

(P¼0.04), R2: 14 � 12.2

for Bio-Osss

and 26.4 � 11 for Ostims

(P¼0.18), R3: 6.6 � 7 for Bio-Osss

and 10.7 � 5.8 for

Ostims

(P¼0.32) after 12 weeks].

Conclusion: Based on the graft consolidation gradient, the impact of bone substitutes to

modulate the process of bone formation and the kinetic of degradation within a distinct

region of the augmented sinus can be investigated.

Bone substitutes are frequently used for

sinus augmentation (Esposito et al. 2006;

Browaeys et al. 2007; McAllister &

Haghighat 2007) and other areas requiring

reconstructive therapy (Giannoudis et al.

2005; Hak 2007). Bone substitutes can be

characterized by their physiochemical

properties (Kokubo et al. 2003; Rezwan

et al. 2006) and their impact on cells

in vitro (Buma et al. 2004; Liebschner

2004). The process of graft consolidation

is, however complex and involves the in-

teraction of bone substitutes with cells,

extracellular matrix and signaling mole-

cules provided by the host (Khan et al.

2005). Thus, the microenvironment of

Date:Accepted 1 April 2009

To cite this article:Busenlechner D, Huber CD, Vasak C, Dobsak A, GruberR, Watzek G. Sinus augmentation analysis revised: thegradient of graft consolidation.Clin. Oral Impl. Res. 20, 2009; 1078–1083.doi: 10.1111/j.1600-0501.2009.01733.x

1078 c� 2009 John Wiley & Sons A/S

the maxillary bone can influence the beha-

vior of bone substitutes, and the microen-

vironment of bone substitute can influence

the osteogenic response of the maxillary

bone. To gain insight into the complex

process of graft consolidation, we depend

on reliable preclinical models, understand-

ing the biological principles of bone regen-

eration, and sophisticated methods for

analysis.

During graft consolidation, new bone ori-

ginates from the maxillary bone and pro-

gresses towards the augmented area (Roldan

et al. 2004; Busenlechner et al. 2008). The

gradient of graft consolidation that develops

is characteristic for each biomaterial, as

reflected by the osteogenic response of

the host bone and the degradation profile of

the bone substitutes. The inhomogeneous

distribution of bone and biomaterials within

the augmented sinus was recently consid-

ered in the analysis by focusing on selected

regions (Fuerst et al. 2004; Roldan et al.

2004). However, the full spectrum of

changes that occur within the augmented

sinus has not been considered so far.

Here we performed a preclinical study

aiming to obtain a curve that reflects the

histomorphometric changes with increas-

ing distance from the maxillary bone.

Based on this curve, three characteristic

regions of graft consolidation could be

distinguished; (i) the area where the host

bone provides the surface for the new

bone, commonly termed ‘bridging dis-

tance’ (R1; 0–1 mm), (ii) the area where

the osteoconductive biomaterial provides

the surface for the new bone (R2;

2–3 mm), and (iii) the area of the maximal

progression of bone formation (R3;

4–5 mm) (Roldan et al. 2004). This precli-

nical model provides a one-wall defect

situation where new bone grows into the

augmented area between the host bone and

the elevated sinus mucosa (Schenk &

Buser 1998; Fuerst et al. 2004). Thus,

all three regions can be defined in the

augmentation maxillary sinus of minipigs.

Based on this preclinical model and cor-

responding clinical studies, the consolida-

tion of Bio-Osss

and Ostims

have been

studied (Browaeys et al. 2007; McAllister

& Haghighat 2007). Moreover, the two

bone substitutes show a different degra-

dation profile, suggesting that also the

gradient of graft consolidation is different.

Bio-Osss

is a slow resorbing deproteinized

bovine bone mineral. Ostims

is a synthetic

nanocrystalline hydroxyapatite paste, con-

sidered to be rapidly degraded (Huber et al.

2006). However, in neither in these

studies, the impact of the biomaterials on

the osteogenic response of the host and its

own degradation profile has been investi-

gated throughout the augmented sinus.

The purpose of the study was to develop

a method that allows histological analysis

of three characteristic regions of graft

consolidation, based on the minipig model

with Bio-Osss

and Ostims

serving as bone

substitutes.

Material and methods

Animals and anesthesia

Ten adult minipigs (Ellegaard Gottinger

Minipig, Solo Landevij, Denmark) with a

weight of 41.5� 9.5 kg were fed a high-

calorie diet and allowed to drink water ad

libitum. The study was approved by the

local Committee for Animal Experiments

of the Ethikkommission der Stadt Wien

(M58/02213/2005/6). General anesthesia

was induced by application of 6.25 ml

xylyzin (Rompuns

, Bayer Austria, Vienna,

Austria), 1.25 ml ketamin (Ketasols

,

Schoeller Chemie Produkte GmbH,

Vienna, Austria) and 2.5 ml butorphanol

(Butomidors

, Richter Pharma AG, Wels,

Austria), together with zolazepam HCl (Zo-

letils

, Virbac, Vienna, Austria) at 1 ml/kg

body weight by intramuscular (i.m.) injec-

tion. General anesthesia was maintained by

inhalation of O2, N2O, and Isoflurane (For-

anes

, Abbott GmbH, Vienna, Austria) un-

der controlled respiration frequency and

electrocardiogram monitoring.

Bone substitutes and sinus augmentationprocedure

Through an extraoral aperture, the sinus

membrane was detached from the maxillary

floor. The cavity created was randomly

filled with 2 cm3 of Bio-Osss

(0.25–1 mm

particle size, Geistlich, Wolhusen, Switzer-

land) on one side and with Ostims

(Heraeus

Kulzer GmbH, Hanau, Germany) on the

contra lateral side, according to the instruc-

tions of the manufacturer. Subsequently,

wounds were closed with an absorbable

suture (Vicryl 4-0 and Vicryl 3-0, Ethicon,

Johnson & Johnson Medical, Vienna,

Austria). For pain relief, animals received a

single i.m. dose of amoxicillin (Duphamaxs

,

Pfizer, Vienna, Austria) and carprofen

(Rimadyls

, Pfizer) at 4 mg/kg. After 6 and

12 weeks, animals were sacrificed by a

lethal anesthetic dose of barbiturate.

Preparation of histological samples

The relevant part of the skull was removed

and fixed in neutral buffered 4% formalin.

Block sections were prepared by cutting the

maxillary sinus with a diamond saw (Exakt

Apparatebau, Norderstedt, Germany). Block

sections were dehydrated in ascending

grades of alcohol and embedded in light-

curing resin (Technovit 7200 VLCþBPO,

Kulzer & Co, Hanau, Germany). Further

processing was done with the exact cutting

and grinding equipment (Exakt Apparate-

bau) according to the sawing–grinding

method of Donath (1988). Undecalcified

ground sections with a thickness of appro-

ximately 30mm were stained with the

Levai–Laczko dye (Fig. 1).

Histomorphometric analyses

Digital pictures at a resolution of 1 pixel

equal to 4.37mm were obtained with a light

microscope (Nikon Microphot-FXA,

Nikon, Tokyo, Japan) and a digital camera

(Nikon DXM 1200) coupled with a

motorized stage (Marzhauser Wetzlar

GmbH&Co KG, Wetzlar Steindorf, Ger-

many). Single pictures were assembled to

generate large overview images (Lucia G

4.71, Laboratory Imaging Ltd, Brno, Czech

Republic). Based on interactively drawn

false colour images (Adobe Photoshops

,

Adobe, San Jose, CA, USA), bone tissue

and bone substitutes were measured with

the histomorphometry software Definiens

Developer 7 (Definiens, Munich, Germany).

Subsequently, the boundary of the host bone

tissue was gradually enlarged by 20 steps of

250mm, resulting in 20 segments with in-

creasing distances to the host bone (Fig. 3).

In all of the 20 segments, bone volume (BV)

and bone substitute volume (BSV) was

determined. Calculations were based on the

ratio of BV per tissue volume (BV/TV)

and BSV/TV, evaluated for each of the 20

segments (Fig. 2).

Assembly of segments into curvesrepresenting the gradient of graftconsolidation

To visualize the change of BV/TVand BSV/

TV with increasing distance to the host

Busenlechner et al . Sinus augmentation analysis revised

c� 2009 John Wiley & Sons A/S 1079 | Clin. Oral Impl. Res. 20, 2009 / 1078–1083

bone, a Bezier spline was fitted to the

succession of the 20 measurements for

every histological sample (Kim et al.

1999); (Wolfram Research Inc., Mathema-

tica, Version 5.0, Champaign, IL, USA).

Residual plots were used to verify the

adequacy of the fitted splines. Curves in

Fig. 3 show the means and standard devia-

tions of the single Bezier splines, represent-

ing the change of the mean BV/TV and

BSV/TV with increasing distance to the

host bone. Based on the curve, three char-

acteristic regions of graft consolidation

were defined, each consisting of four seg-

ments. The regional characteristic was

confirmed by qualitative histological ana-

lysis. Histomorphometric data gained

from region 1 (R1; 0–1 mm), region 2 (R2;

2–3 mm), and region 3 (R3; 4–5 mm) were

subjected to statistical analysis (Fig. 3).

Statistical analysis

Paired t-test was used to compare the means

of histomorphometric data of the three re-

gions, after having tested for normal distri-

bution. Po0.05 was considered significant.

Results

Definition of three characteristic regions ofgraft consolidation

Here we defined the impact of different

biomaterials on bone formation within char-

acteristic region in an augmented sinus. A

curve that represents the changes of histo-

morphometric parameter in relation to the

distance from the host bone was established.

Based on the curve characteristics, the area

including the ‘bridging distance’ (R1;

0–1 mm), the area of exclusive osteoconduc-

tion (R2; 2–3 mm) and the most distant area

showing bone formation (R3; 4–5 mm)

could be distinguished (Fig. 3), but any

further graduation is possible.

In R1, the region within 1 mm distance

from the host bone represented a continu-

ous broad seam of newly formed bone. The

presence of secondary osteons after 12

weeks indicated that the maturation of

the bone took place. Bio-Osss

particles

were loosely distributed, while only a few

areas of Ostims

were visible. In R2, graft

consolidation occurred at an almost con-

stant rate. Histological analysis of R2 con-

firmed the osteoconductive properties

of both bone substitutes based on their

Fig. 1. Histological specimens of the augmented maxillary sinus of minipig. The upper pictures shown

histological specimens stained according to Levai–Laczko. In the corresponding lower pictures, bone tissue is

depicted in red while the biomaterial is depicted in yellow. Note the broad seam of new bone that lies down on

the host bone and the area of bone formation that depends on the osteoconductive properties of the biomaterial.

Fig. 2. Regions of interest in the augmented maxillary sinus. Quantitative and qualitative histological analysis

of bone and biomaterial volume was performed in the three indicated regions of graft consolidation. The regions

represent the indicated distances to the host bone. (R1: 0–1 mm, R2: 2–3 mm, and R3: 4–5 mm).

Busenlechner et al . Sinus augmentation analysis revised

1080 | Clin. Oral Impl. Res. 20, 2009 / 1078–1083 c� 2009 John Wiley & Sons A/S

intimate contact with the newly formed

bone. The remaining surface of both bone

substitutes showed light zones occupied by

multinucleated cells. With increasing dis-

tance from the host bone, the relative

portion of newly formed bone declined to

almost zero. R3 was defined as the area

with the least significant amount of detect-

able bone formation. What further became

obvious was the size distribution of the two

bone substitutes. While the islands of Os-

tims

measured approximately 50 mm in

areas close to the host bone, up to 3 mm

were measured in R3. The size distribution

of the single Bio-Osss

particles was con-

stant. Thus, the curve provides insight into

the behavior of biomaterial in three defied

regions of graft consolidation (Figs 2 and 3).

Histomorphometric analysis of the threeregions of graft consolidation

Based on these evaluations, the progress on

graft consolidation can be quantified by

taking the distance from the host bone

into account. In agreement with the histo-

logical analysis, bone area was significantly

higher within the ‘bridging distance’ (R1;

38� 13.3 for Bio-Osss

and 53.3� 6.6 for

Ostims

) than in the region of graft conso-

lidation (R2; 14� 12.2 for Bio-Osss

and

26.4� 11 for Ostims

) and than in the

region where bone formation ceases (R3;

6.6� 7 for Bio-Osss

and 10.7� 5.8 for

Ostims

) at 12 weeks (Figs 3 and 4). A

similar trend was observed for the earlier

observation period, and the volume of Bio-

Osss

was apparently less in R1 (15.6� 2.1)

than in the two other regions (28.7� 6.1

and 26.1� 4.4 in R2 and R3, respectively).

In contrast, substantially more Ostims

was

detectable with increasing distance from

host bone (6.2� 2.8 in R1, 24.5� 6.5 in

R2, and 41.2� 10.2 in R3) as indicated by

the 12-week data (Fig. 4).

Consistent with the qualitative impres-

sion gained by the curve characteristics, R1

showed significant differences between

Bio-Osss

and Ostims

after 6 weeks (16.1�6.2 for Bio-Oss

s

and 3.9� 2.8 for Ostims

),

as well as after 12 weeks (15.6� 2.1 for

Bio-Osss

and 6.2� 2.8 for Ostims

). In R2,

the only significant difference was observed

when comparing BV/TV after 6 weeks

(Po0.05). No difference was found between

the two bone substitutes in BV/TVand BSV/

TV. In R3, more BSV/TV in the Ostims

group (34.7� 29.2 after 6 weeks and

41.2� 10.2 after 12 weeks) than in the

Bio-Osss

group (25.9� 6.9 after 6 weeks

and 26.1� 4.4 after 12 weeks) was ob-

served (Po0.01) (Fig. 4).

Discussion

Graft consolidation in the augmented sinus

is a complex process where new bone

originating from the maxillary host bone

is guided by the osteoconductive properties

of biomaterials up to the Schneiderian

membrane. The gradient of graft consolida-

tion characteristically represents the inter-

action of new bone with the biological

properties of biomaterials within each re-

gion of the augmented sinus. To consider

these changes in the augmented maxi-

llary sinus, we performed a sequential

Fig. 3. Changes of histomorphometric parameters with increasing distance from the host bone. The selection

of the three regions of interest was performed based on the curve characteristics, bone volume (BV) and bone

substitute volume (BSV) was determined. BV per tissue volume (BV/TV) and BSV/TV were calculated. Solid

curves represent the means of five observations; interrupted curves represent the corresponding standard

deviation. Blue and red curves stand for Bio-Osss

and Ostims

, respectively.

Fig. 4. Histomorphometric parameters of the three region of interest. (Region 1) indicates the -bridging

distance-, where new bone is mainly laid onto the host bone, (region 2) represents a region of osteoconduction,

where new bone exclusively grows on the biomaterial and (region 3) indicates a region of limited bone

formation. Bone volume (BV) and bone substitute (BSV) was determined. BV per tissue volume- (BV/TV) and

BSV/TV were calculated. Blue curves represent Bio-Osss

(n¼ 5), red curves represent Ostims

(n¼ 5).

Busenlechner et al . Sinus augmentation analysis revised

c� 2009 John Wiley & Sons A/S 1081 | Clin. Oral Impl. Res. 20, 2009 / 1078–1083

histomorphometrical analysis that led to

the formulation of a curve. Based on the

curve characteristics, three regions of inter-

est were selected: (i) a ‘bridging distance’,

where new bone is mainly laid onto the

host bone, (ii) a region of osteoconduction,

where new bone exclusively grows on the

biomaterial, (iii) and a region of osteo-

conduction that represents the maximal

distance of the new bone. Based on these

data, the properties of bone substitutes in

the augmented sinus can be described –

first, histological changes with increasing

distance from the host bone – second,

histological changes over time within a

particular region. Thus, the sequential ana-

lysis of graft consolidation in the augmen-

ted sinus provides further insights into the

characteristics of bone substitutes, includ-

ing their capacity to modulate bone forma-

tion and their degradation.

Equivalent intervals representative for

each region were chosen for the analysis.

R1 stood for the histological picture

where host bone is covered by new bone.

This area of intense bone formation can be

termed ‘bridging distance’ because small

defects are rapidly bridged by this mechan-

ism. Also when the Schneiderian mem-

brane is kept up, bone grows spontaneously

in the sinus floor (Botticelli et al. 2003).

R1 also represented parts of osteoconduc-

tive bone formation, which has to be con-

sidered in the interpretation of the results.

The curve characteristic and the corre-

sponding statistical analysis indicated that

the two bone substitutes significantly

differ in R1. Bone formation was more

pronounced in the sinus augmented with

Ostims

than in the Bio-Osss

group. While

only a marginal amount of Ostims

was

present in R1, a substantial amount of

Bio-Osss

was detectable in R1. Both bio-

materials allowed the progression of bone

formation over time, with Bio-Osss

slightly kept up to Ostims

. The observed

statistical advantage for Ostims

in R1 is

apparent and in line to the histological

picture. One might get the impression

that growing bone originating from the

wall of the sinus has pushed away Ostims

strongly and Bio-Osss

weakly. Another

interpretation is that degradation of Ostims

proceeds more rapidly than Bio-Osss

in

regions close to the host bone than in

deeper layers of the augmented area. Even

though we currently lack evidence that

would support either of the two theories,

there is obviously more space for the newly

formed bone in the R1 Ostims

group than

in the R1 Bio-Osss

group.

The curve characteristics further revealed

a zone where the bone area only slightly

declined with increasing distance from the

host bone. R2 strictly showed graft consoli-

dation by means of osteoconductive bone

formation. At 12 weeks, the bone area in

the Ostims

group became comparable to

the bone area in the Bio-Osss

group. The

relative amount of the residual Ostims

and

Bio-Osss

in R2 remained constant over

time. In R3, the volume occupied by Os-

tims

was even higher than the volume

occupied by Bio-Osss

. Thus, the residual

volume of Ostims

but not of Bio-Osss

is

strongly influenced by the distance from the

host bone. These findings are consistent

with our recent observations from a precli-

nical model of calvaria augmentation,

where no attempts were made to define

particular regions (Busenlechner et al.

2008). However, regions were defined in

preclinical studies with the maxillary sinus

such as territorial division based on the total

augmentation height (Fuerst et al. 2004), or

the coronal and apical parts of periimplant

bone (Liu et al. 2008). The novelty of the

present study is the systematic curve-based

strategy to define the regions, independent

of the augmentation height.

Our study was based on the histomor-

phometric determination of the volume of

bone and bone substitutes in the augmen-

ted sinus. However, this analytic method

represents only one possible application of

our curve based analysis. The biological

properties of bone substitutes can not

only relate to the amount of bone forma-

tion, but possibly also its quality e.g.

mineral distribution, formation and ma-

turation of the non-mineral matrix, and

the birth and death of bone cells (Manola-

gas 2000; Roschger et al. 2008). Curve

based analysis can also provide insights

into the function of biologicals such as

the recently approved bone morphogenetic

protein-2 for sinus augmentation (Boyne

et al. 2005). One might expect a different

gradient when osteogenic cells of the sinus

mucosa are forced to make bone (Gruber

et al. 2004). Our observations justify

a more general use of this analytical tool

in biomaterial research and related clini-

cal fields.

There are also limitations in our study.

The curves are based on a two-dimensional

analysis, while significant more structural

information can be achieved by the three-

dimensional mCTanalysis. However, radio-

density of the biomaterial must be signifi-

cantly higher than bone tissue. Another

aspect that might be included in future

study designs is the dimension of the host

bone, which can influence the osteogenic

response to a biomaterial e.g. the height of

the maxillary bone (Spray et al. 2000). The

observation period of 6 and 12 weeks

provides a snapshot of the early phase of

graft consolidation, whereas long-term

studies remain to be done.

Statistical power can be considered low.

However, according to the published

results (Huber et al. 2006; Busenlechner

et al. 2008), the expected difference be-

tween the fast- and slow-resorbing bioma-

terials is rather high. Thus, the number of

animals that were included in the study

was kept low. Moreover, we have a ‘paired’

situation, which adds to the power of the

analysis. Indeed, we obtained significant

differences with data from five animals.

The statistical power of the study is there-

fore sufficient to support the conclusions of

our method.

How do these results observed in the

minipig add to the overall concept of sinus

augmentation? In most two-stage sinus

augmentation studies, the earliest time

point of functional loading is at 3–6

months after augmentation, so that, ac-

cording to our data, both grafts would

have a similar performance (Wallace &

Froum 2003). If implant placement is per-

formed at an earlier time point, the relative

amount of bone at the basis but also at the

most apical distance of interconnecting

bone trabeculae is relevant. In theory,

best is to have a broad basis with a large

bone area that reaches beyond the tip of the

implant. While R1 and R2 are suitable

areas that reflect the bone area at the basis,

R3 represents the most apical distance of

bone. Considering all three regions at an

indicated time point is therefore of scien-

tific relevance. However, we have to be

careful to draw conclusions applied to the

clinic relevance from the observed results.

It must be clearly stated that our study

was designed to reveal the process of graft

consolidation and not the impact of bone

substitutes on osseointegration of dental

Busenlechner et al . Sinus augmentation analysis revised

1082 | Clin. Oral Impl. Res. 20, 2009 / 1078–1083 c� 2009 John Wiley & Sons A/S

implants. Moreover, bone substitutes have

to function under functional loading of the

implants. Our findings do not allow draw-

ing any conclusions about the clinical as-

pects of implant survival and function. We

also want to emphasis that this study was

not driven by a hypothesis. Aim of the

study was to report on a new method to

evaluate the process of graft consolidation,

exemplified by two well-described bioma-

terials. Based on our new method, hypoth-

esis-driven studies can be designed.

In summary this study supports further

attempts for sequential analysis of graft

consolidation in the augmented sinus. Se-

quential analysis can provide deeper insights

into the impact of bone substitutes on their

consolidation. These findings should offer a

base for future investigations of bone sub-

stitutes in graft consolidation models.

Acknowledgements: The authors

are grateful to Prof. Dr h.c. mult. Karl

Donath for histological evaluation,

to Prof. DDr Gabor Furst for

sharing his surgical experience, to

Dr Birgit Mair for her support during

surgical procedures. This study was

generously supported by

Heraeus Kulzer

Germany.

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