bone healing in critical-size defects treated with bioactive glass/calcium sulfate: a histologic and...

Bone healing in critical-size defectstreated with bioactive glass/calciumsulfate: a histologic and histometricstudy in rat calvaria

Flavia A. C. FurlanetoMaria J. H. NagataStephen E. FuciniTatiana M. DeliberadorTetuo OkamotoMichel R. Messora

Authors’ affiliations:Flavia A. C. Furlaneto, Maria J. H. Nagata, TatianaM. Deliberador, Tetuo Okamoto, Michel R.Messora, Division of Periodontics, Department ofSurgery and Integrated Clinic, Dental School ofAracatuba, University of the State of Sao Paulo‘‘Julio de Mesquita Filho’’ – UNESP, BrazilStephen E. Fucini, Private Practice, Hanover,NH, USA.

Correspondence to:Maria Jose Hitomi NagataRua Afonso Pena, 325 – Apt. 51CEP: 16010-370 Aracatuba, SP, BrazilTel.: þ55 18 3621 7932Fax: þ55 18 3621 7932e-mail: [email protected]

Key words: bioactive, bone regeneration, bone substitutes, calcium sulfate, glass

Abstract

Objective: The purpose of this study was to analyze histologically the influence of bioactive

glass (BG) with or without a calcium sulfate (CS) barrier on bone healing in surgically

created critical-size defects (CSD) in rat calvaria.

Material and methods: A CSD was made in each calvarium of 48 rats. They were divided

into three groups: C (control): blood clot only; BG: defect filled with BG; and BG/CS: defect

filled with BG covered by a CS barrier. Animals were euthanized at 4 or 12 weeks. Formation

of new bone was evaluated histomorphometrically.

Results: No defect completely regenerated with bone. BG particles were observed in

Groups BG and BG/CS at both periods of analysis. The thickness throughout the healing area

in Groups BG and BG/CS was similar to the original calvarium, while Group C presented a

thin connective tissue in the center of the defect in both periods of analysis. At 4 weeks,

Groups C and BG/CS presented significantly more bone formation than Group BG. No

significant differences were found between Groups C and BG/CS. At 12 weeks, no

significant differences in the amount of bone formation were observed among the three

groups. When comparing 4 and 12 weeks, there was a significant increase in new bone

formation within groups BG and BG/CS, but not C.

Conclusion: BG particles, used with or without a CS barrier, maintained the volume and

contour of the area grafted in CSD. However, they did not lead to a significant difference in

bone formation when compared with control at 12 weeks post-operative.

A variety of graft materials and bone sub-

stitutes have been placed in bony defects in

order to facilitate and/or promote bone

regeneration (Lang et al. 1999). The syn-

thetic materials, or alloplasts, have been

largely used for their unlimited availability

and because they do not require additional

surgery of a donor site (Topazian et al.

1971; Norton & Wilson 2002).

Among the group of synthetic materials

used as bone substitutes, particulate bioac-

tive glasses (BG) have been studied consid-

erably. They act by forming a chemical

union with the surrounded tissues (Hench

et al. 1971; Hench & Paschall 1973). The

bonding between BG and the surrounding

bone tissue is the result of a series of

interfacial reactions that lead to the forma-

tion of a Si-rich layer covered by a Ca–P

rich layer. It has been suggested that osteo-

blasts deposit the organic matrix of bone on

this Ca–P layer, and that the bonding

results from cross-linking between ionic

sites on the collagen and the mucopolysac-

charides with those of the Ca–P rich layer

(Hench & Paschall 1974). Besides being

osteoconductive (Schepers et al. 1991;

Furusawa et al. 1998; Froum et al. 2002),

Date:Accepted 15 May 2006

To cite this article:Furlaneto FAC, Nagata MJH, Fucini SE, DeliberadorTM, Okamoto T, Messora MR. Bone healing in critical-size defects treated with bioactive glass/calcium sulfate.A histologic and histometric study in rat calvaria.Clin. Oral Impl. Res. 18, 2007; 311–318doi: 10.1111/j.1600-0501.2006.01331.x

c� 2007 Blackwell Munksgaard 311

BG particles have demonstrated an osteos-

timulatory effect (Schepers et al. 1991;

Schepers & Ducheyne 1997; Furusawa

et al. 1998; Froum et al. 2002; Norton

& Wilson 2002). Their use with barriers,

such as calcium sulfate (CS; Camargo

et al. 2000; Sottosanti & Anson 2003;

Melo et al. 2005), has been suggested.

When CS was used as a barrier in

conjunction with other bone graft materi-

als, such as demineralized freeze-dried

bone allograft and autogenous bone graft,

it resulted in favorable bone and perio-

dontal responses (Sottosanti 1992; Kim

et al. 1998; MacNeill et al. 1999).

Camargo et al. (2000) clinically evalu-

ated the use of BG particles with a CS

barrier in extraction sites. According to

the authors, the combination of the two

materials was beneficial for preserving the

dimensions of the alveolar ridge follow-

ing extraction of teeth. Melo et al. (2005)

conducted a histomorphometric study in

rat tibias to evaluate the combination of

materials used by Camargo et al. (2000).

Surgically created bone defects were treated

with either BG, a CS barrier over the blood

clot or a combination of BG with a CS

barrier. The defects of the control group,

filled only with blood clot, presented sig-

nificantly more bone formation than the

defects of the experimental groups. These

results were attributed to the non-critical-

size nature of the surgically created defects.

The purpose of this study was to analyze

histologically the influence of BG with

or without a CS barrier on bone healing

in surgically created critical-size defects

(CSD) in rat calvaria.

Material and methods

Experimental model

Forty-eight 3–4-month-old, male rats (Rat-

tus norvegicus, albinus, Wistar) weighing

350–400 g (University of State of Sao Paulo

– UNESP, Dental School of Aracatuba,

Animal Care Unit) were used. The rats

were kept in a room with a 12 h light/dark

cycle and temperature between 22 and

241C. The experimental protocol was

approved by the UNESP – Dental School

of Aracatuba Institutional Animal Care

and Use Committee. They were randomly

assigned to one of three experimental

groups: Group C (control), Group BG

(BG implant; Biograns

, 3i Implant Innova-

tions Inc., Palm Beach Gardens, FL, USA)

and Group BG/CS (BG implant and CS

barrier; Calcigent Oral, Biomet Orthope-

dics Inc., Warsaw, IN, USA/3i Implant

Innovations Inc.).

Surgical procedure

Animals were anesthetized by an intra-

muscular injection of xylazine (6 mg/kg

body weight) and ketamine (70 mg/kg

body weight). After aseptic preparation, a

semilunar incision was made in the scalp

in the anterior region of the calvarium

allowing reflection of a full-thickness

flap in a posterior direction. A 5 mm in

diameter CSD was made with a trephine

(3i Implant Innovations Inc.) used in a

low-speed handpiece under continuous

sterile saline irrigation. The defect included

a portion of the sagittal suture. Reference

marks were made 2 mm anterior and 2 mm

posterior to the margins of the CSD, both

of which were located on a longitudinal

axis bisecting the surgical defect. The

marks were made using a small tapered

carbide fissure bur and then filled with

amalgam (Bosch et al. 1998; Fig. 1). Their

purpose was to allow identification of the

center line of the original defect during

laboratory processing and also to be used

as references to locate the original bone

margins of the surgical defect during histo-

metric analysis.

In Group C, the surgical defect was filled

with a blood clot only. In Group BG, the

surgical defect was filled with BG particles

(Biograns

). In Group BG/CS, it was filled

with BG particles (Biograns

) and covered

with a CS barrier (Calcigent Oral). The

BG particles were composed of 45% SiO2,

24.5% Na2O, 24.5% CaO and 6% P2O5

by weight percentages, and had a size range

limited to 300–355mm.

The soft tissues were then repositioned

and sutured to achieve primary closure

(Silk 4.0, Ethicon, Sao Paulo, SP, Brazil).

Each animal received an intramus-

cular injection of 24,000 IU penicillin

G-benzathine (Pentabioticon Veterinario

Pequeno Porte, Fort Dodges

Saude Animal

Ltd., Campinas, SP, Brazil) post-surgically.

Tissue processing

Each group of animals was divided into

two sub-groups for euthanasia at either

4 or 12 weeks post-operative. The area of

the original surgical defect and the sur-

rounding tissues were removed en bloc.

The blocks were fixed in 10% neutral

formalin, rinsed with water and then dec-

alcified in 16% ethylediaminetetraacetic

acid solution. After an initial decalcifica-

tion, each specimen was divided longitud-

inally into two blocks exactly along the

center line of the original surgical defect

using the amalgam reference marks. Trans-

verse cuts were made perpendicular to the

longitudinal axis at the medial edge of each

amalgam reference mark. Each specimen

then measured 9 mm in length along

the longitudinal axis running through the

center of the defect, allowing for identifica-

tion of the original surgical defect margins

during both histologic and histometric

evaluations (Fig. 2). After additional

decalcification, they were processed and

embedded in paraffin. Serial sections 6mm

thick were cut in a longitudinal direction

starting at the center of the original surgical

defect. The sections were stained with

either hematoxylin and eosin or Masson’s

Trichrome for analysis by light microscopy.

Histomorphometric analysis

Before the analysis, criteria were estab-

lished in order to conduct a more object-

ive evaluation of the acute (neutrophils)

and chronic (macrophages, lymphocytes

and plasma cells) inflammatory infiltrates.

The following criteria were used to describe

the inflammatory infiltrate in each field

using a light microscope with a � 40

objective: (a) light: 1–100 cells; (b) moder-

ate: 100–250 cells; and (c) intense: more

than 250 cells.

Four histologic sections, representing the

center of the original surgical defect, were

selected for the histologic and histomor-

phometric analyses in order to increase the

reliability of the data used in the statistical

analysis. The images of the histologic

Fig. 1. Critical-size defect (5 mm diameter) and the

two reference marks created on the calvarium.

Furlaneto et al . Bone healing with bioactive glass/calcium sulfate

312 | Clin. Oral Impl. Res. 18, 2007 / 311–318 c� 2007 Blackwell Munksgaard

sections were captured by a digital camera

connected to a light microscope with an

original magnification of � 32. The digital

images were saved on a computer and then

copied to ‘ImageLab 2000’ software (Dir-

acon Bio Informatica Ltd., Vargem Grande

do Sul, SP, Brazil), which was used for

histomorphometric analysis.

The following criteria, based in part on

the work of Melo et al. (2005), were used to

standardize the histomorphometric analy-

sis of the digital images:

(1) The total area (TA) to be analyzed

corresponded to the entire area of the

original surgical defect. This area

was determined by first identifying

the external and internal surfaces of

the original calvarium at the right and

left margins of the surgical defect, and

then connecting them with lines

drawn following their respective curva-

tures. The center of the histologic

section (considering its total length)

was localized and 2.5 mm were mea-

sured to the right and to the left of this

center point in order to determine the

limits of the original surgical defect

(Fig. 3). The newly formed bone area

(NFBA) and the areas of the remnants

of the implanted materials, named

bioactive glass area (BGA) (Fig. 3) and

calcium sulfate area (CSA), were deli-

neated within the confines of the TA.

(2) The TA was measured in mm2

and was considered 100% of the

area to be analyzed. The NFBA, the

BGA and the CSA were also measured

in mm2 and calculated as a percentage

of TA.

Fig. 2. (a) Longitudinal cut along the center line of critical-size defects indicated in blue; (b) transverse cuts indicated in green; (c) histologic specimen ready to be

embedded in paraffin.

Fig. 3. Captured image of a histological section. The total area (TA) is delineated by the pink line and

corresponds to the area of the calvarium where the surgical defect was originally created. The height of the TA

(X) corresponds to the thickness of the original calvarium (Y). The newly formed bone area (NFBA) is delineated

by the blue line and the bioactive glass area (BGA) is delineated by the green line.

Fig. 4. Group C (control): 4 weeks. Surgical defect with a fibrous connective tissue that is thinner than the

original calvarium. Hematoxylin and eosin stained; original magnification � 25.


c� 2007 Blackwell Munksgaard 313 | Clin. Oral Impl. Res. 18, 2007 / 311–318

Statistical analysis

The values of NFBA for each animal were

represented by the mean percentage of the

four histologic sections. These percentage

data were transformed into arccosine

for the statistical analysis. The significance

of differences between groups in relation

to NFBA was determined by an analysis of

variance, followed by a post hoc Tukey’s

test when the analysis of variance sug-

gested a significant difference between

groups (Po0.05). The values of BGA for

each animal in Groups BG and BG/CS and

of CSA in Group BG/CS were represented

by the mean percentage of the four histo-

logic sections. The proportion test at the

5% significance level was used to compare

the values of BGA between 4 and 12 weeks

post-operative in Group BG and in Group

BG/CS (Po0.05). The values of CSA

in Group BG/CS at both 4 and 12 weeks

post-operative were practically zero. There-

fore, statistical analysis was not performed

on this parameter.

Results

One specimen of Group BG/CS at 4 weeks

post-operative and one specimen of Group

BG/CS at 12 weeks post-operative were

lost because of problems encountered

during lab processing.

Qualitative histologic analysis

No surgical defect in any of the groups

completely regenerated with bone.

Group C (Control)

At 4 weeks, new bone formation was

restricted to areas close to the borders of

the surgical defect in most specimens. At

12 weeks, well-developed newly formed

bone surrounded by an osteoid matrix rich

in osteoblasts was observed in those areas.

In all specimens, at both 4 and 12 weeks,

the connective tissue in the central part

of the defect was thinner than the origi-

nal calvarium (Figs 4 and 5). It was

well vascularized and rich in fibroblasts

with oriented collagen fibers. The presence

of osteoid matrix was observed in some

areas. Light acute and chronic inflamma-

tory infiltrates were present throughout the

surgical defect. In a few specimens, areas

with an intense, predominantly chronic

inflammatory infiltrate were noted.

Group BG and Group BG/CS

Well-developed newly formed bone sur-

rounded by an osteoid matrix was observed

restricted to the borders of the surgical

defect in both periods of analyses. At

12 weeks, it extended toward the center

of the defect in a few specimens in Group

BG and in most specimens in Group BG/

CS. At 4 weeks, a large amount of BG

particles was distributed throughout the

defect and at 12 weeks, they were smaller

than those at 4 weeks. Most BG particles

presented with cracks, many of which had

an ingrowth of connective tissue within the

cracks and excavated centers, in both per-

iods of analysis (Fig. 6). Other particles

showed amorphous material in their exca-

vated centers instead (Fig. 7). In some

areas, BG particles were surrounded by a

fibroblast-rich connective tissue that

was organized and dense with the presence

of osteoblasts and osteoid matrix (Figs 8

and 9). In other areas, the surrounding

connective tissue was loose and not orga-

nized. In all specimens, BG particles and

the surrounding connective tissue formed

a strip with a thickness similar to that of

the original calvarium (Figs 10 and 11). In

Group BG/CS, minimal remnants of CS

were observed, sometimes surrounded by

Fig. 5. Group C (control): 12 weeks. Surgical defect with a fibrous connective tissue that is thinner than the

original calvarium. Masson’s Trichrome; original magnification � 25.

Fig. 6. Group BG/CS (bioactive glass/calcium sulfate): 4 weeks. Bioactive glass particles surrounded by

connective tissue that is also seen within cracks and excavated centers. Hematoxylin and eosin stained;

original magnification � 160.



newly formed bone. In Group BG/CS, in

both period of analyses and in Group BG at

4 weeks, light acute and chronic inflam-

matory infiltrates were observed in most

specimens, while a few demonstrated an

intense chronic inflammatory infiltrate

composed of lymphocytes, plasma cells

and histiocytes between the BG particles.

In Group BG, at 12 weeks, a light and

predominantly chronic inflammatory infil-

trate was dispersed throughout the defect.

Histometric and statistical analyses

The data normality and homogeneity of

variances were verified. Means and stan-

dard deviations of NFBA for each group, as

well as the comparison among the groups,

at 4 and 12 weeks post-operative are docu-

mented in Table 1. Table 2 demonstrates

the increased resorption of BG particles

from 4 to 12 weeks post-operative in

Groups BG and BG/CS.

Discussion

This study evaluated the influence of

BG particles (300–355mm) with or with-

out a CS barrier on the healing of CSD in

rat calvaria.

According to Bosch et al. (1998), the use

of large bone defects results in some incon-

veniences, such as inclusion of the sagittal

suture in the calvarial defect, thereby in-

troducing the connective tissue of the su-

ture in the evaluation of bone regeneration.

Because of the size of the calvaria of the

animals used in the present study, the

experimental defect could not be made

only in parietal bone. Therefore, a location

involving the sagittal suture was chosen in

order to standardize the experimental

model. Any possible effect from the inclu-

sion of connective tissue of the sagittal

suture would be present in all groups.

Similar experimental models that included

the sagittal suture have been reported in

other studies as well (Brunel et al. 1996;

Mardas et al. 2002, Kim et al. 2004).

The outcome of any type of regenerative

procedure is strongly dependent upon the

available space under the mucoperiosteal

flap (Wikesjo & Selvig 1999). In the pre-

sent study, the flap collapse seen in Group

C was probably prevented by the use of BG

particles in Groups BG and BG/CS, similar

to what was observed by Sculean et al.

(2002). One of the advantages of BG is

the in situ formation of a calcium-phos-

phate shell, which serves to maintain the

overall volume of the particle–bone matrix

(Furusawa et al. 1998). This is an extre-

mely advantageous property for a bone aug-

mentation material where predictable

modification of the defect or deficiency is

required.

In the present study, osteoid matrix rich

in osteoblasts and fibrous connective tissue

were observed surrounding most BG parti-

cles. These collagen fibers may indicate

matrix development for future mineraliza-

tion and bone formation (Wheeler et al.

1997). This mineralization and bone

formation might have been observed if

the present study had included a longer

period of healing. Cracks and excavated

centers were observed in the BG particles,

with ingrowth of cells, probably undiffer-

entiated mesenchymal cells, at both 4

and 12 weeks post-operative, as found

in other studies (Furusawa et al 1998;

Cordioli et al. 2001; Tadjoedin et al.

2002). However, the osteostimulatory

effect described in other histological

human and animal studies (Schepers et al.

1991; Schepers & Ducheyne 1997;

Furusawa et al. 1998; Froum et al. 2002;

Norton & Wilson 2002) was not seen in

any of the specimens of the present study

at either 4 or 12 weeks post-operative.

Fig. 7. Group BG (bioactive glass): 4 weeks. Bioactive glass particles showing cracks and excavated centers

with ingrowth of connective tissue. Amorphous material can also be observed in some excavated centers.

Masson’s Trichrome; original magnification � 160.

Fig. 8. Group BG (bioactive glass): 12 weeks. Bioactive glass particles surrounded by a fibrous connective

tissue and osteoid matrix with osteoblasts. Hematoxylin and eosin stained; original magnification � 160.



BG particles were observed in Groups

BG and BG/CS in both periods of analysis,

confirming the slow process of resorption

of this material observed in histologic stu-

dies in humans (Tadjoedin et al. 2000,

2002; Cordioli et al. 2001; Norton &

Wilson 2002) and in animals (Schepers &

Ducheyne 1997; Cancian et al. 1999; Stav-

ropoulos et al. 2003). Schepers & Duch-

eyne (1997) observed the presence of BG

particles up to 24 months after their

implantation in surgically created bony

defects in mandibles of dogs.

At 4 weeks post-operative, Groups C

and BG/CS showed significantly greater

bone formation than Group BG (Table 1).

No differences were found between Groups

C and BG/CS. According to MacNeill

et al. (1999), graft materials that require

extended time periods for complete resorp-

tion will reduce the total amount of newly

formed bone due to their continued pre-

sence. The slow resorption of the BG

particles probably accounted for the greater

amount of newly formed bone observed in

Group C when compared with Group BG,

as also observed in previous studies by

MacNeill et al. (1999) and Melo et al.

(2005). In this study, Group BG/CS re-

ceived less BG particles than Group BG

because of the space occupied by the CS

barrier. Thus, in a similar manner, the

presence of a greater amount of a slowly

resorbed material in Group BG may help to

explain why Group BG/CS presented sig-

nificantly more bone formation than Group

BG in this study. Furthermore, the rapid

resorption of CS observed in the present

study probably allowed early ingress of os-

teoprogenitor cells, thus accelerating bone

regeneration. An almost complete resorp-

tion of the CS barrier was observed at

4 weeks post-operative. According to the

results observed by Pecora et al. (1997) in a

histologic study conducted in rats, the pre-

sence of the CS barrier for 3 weeks was

enough to halt ingrowth of soft connective

tissue and promote osseous formation. It

should be remembered that it is not possible

to know exactly how long the CS main-

tained its function as a barrier in the present

study. Thus, additional histologic studies

are necessary to evaluate the resorptive

process of CS when used as a barrier.

At 12 weeks post-operative, Group BG/

CS presented more bone formation than

Groups C and BG, although there were no

significant differences among the three

groups (Table 1). A statistically significant

increase in the amount of newly formed

bone was observed in the groups treated

with BG (Groups BG and BG/CS) from 4 to

12 weeks post-operative, probably due to

the combination of new bone formation

with the material resorption process (Tad-

joedin et al. 2000). The decrease in the

percentage values of BGA in Groups BG

and BG/CS from 4 to 12 weeks indicates

a clear trend, although the difference

within each group was not statistically

significant.

The time necessary for complete resorp-

tion of BG particles remains unknown.

Norton & Wilson (2002) stated that this

material can be used for filling dentoalveolar

defects and/or ridge preservation at the site

of extraction sockets before placement of

dental implants. According to the authors,

even though the BG particles are slowly

replaced by bone, there was no negative

impact on the clinical success of dental

implants placed into the graft/tissue mass.

In this study, the finding that there was

an insignificant increase in new bone for-

mation in Group C from 4 to 12 weeks

post-operative corroborates the findings of

Takagi & Urist (1982). They surgically

Fig. 9. Group BG/CS (bioactive glass/calcium sulfate): 12 weeks. Bioactive glass particles surrounded by a

fibrous connective tissue and osteoid matrix with osteoblasts. Hematoxylin and eosin stained; original

magnification � 160.

Fig. 10. Group BG (bioactive glass): 12 weeks. Bioactive glass particles and surrounding connective tissue

forming a strip with a thickness similar to that of the original calvarium. Hematoxylin and eosin stained;

original magnification � 25.



created CSDs in rat calvaria and found that

bone formation had terminated at 4 weeks

post-operative.

Within the limits of this study, it can be

concluded that BG particles, used either

with or without a CS barrier, maintained

the volume and contour of the area grafted

in CSD. However, they did not lead to a

significant difference in bone formation

when compared with control at 12 weeks

post-operative.

Acknowledgements: This study was

partially supported by 3i Implant

Innovationss

. The authors thank

Johnson & Johnson (Sao Jose dos

Campos, SP, Brazil) for donating the

suture materials.

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Table 1. Mean percentage of newly formed bone area (NFBA) within the area of thesurgically created defect with comparison among the groups, 4 and 12 weeks post-operative

Group n Mean-NFBA(%) 4 weeks

Standarddeviation4 weeks

Mean-NFBA(%) 12 weeks


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Mean-BGA(%) 12 weeks


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BG, bioactive glass; BG/CS, bioactive glass/calcium sulfate.



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bone healing in critical-size defects treated with bioactive glass/calcium sulfate: a histologic and...

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