Is there a connection between synthetic bone grafts and sisters chromatide exchange?

doi:10.4236/ojst.2013.38074

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Open Journal of Stomatology, 2013, 3, 447-451 OJST

http://dx.doi.org/10.4236/ojst.2013.38074 Published Online November 2013 (http://www.scirp.org/journal/ojst/)

Is there a connection between synthetic bone grafts and

sisters chromatide exchange?

Banu Gürkan Köseoğlu1, Amila Brkić2*, Mehmet Ali Erdem1, Şükrü Öztürk3, Şükrü Palanduz3,

Kivanç Çefle3

1Department of Oral and Maxillofacial Surgery, School of Dentistry, Istanbul University, Istanbul, Turkey

2Department of Oral Surgery, School of Dentistry, Sarajevo University, Sarajevo, Bosnia and Herzegovina

3Division of Medical Genetics, Department of Internal Medicine, School of Medicine, Istanbul University,Istanbul, Turkey

Email: *[email protected]

Received 24 September 2013; revised 25 October 2013; accepted 7 November 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Background: In oral and maxillofacial surgery, syn-

thetic bone grafts are most widely used as bone sub-

stitutes, due to the limited sources of autologous bone.

The aim of this study was to examine the influence of

three different synthetic bone grafts (Cerasorb, For-

toss and Perioglass) on sisters chromatide exchanges

(SCEs) in peripheral lymphocytes. Materials and

Methods: Peripheral blood samples taken from 68

patients (45 females and 23 males), who underwent

oral surgery procedures, such as apical resection, cyst

enucleation or periodontal curretage, were obtained

for SCE a day before and two months after the sur-

geries. A control group included 30 patients, while the

study group was made of the patients who underwent

bone grafting with Cerasorb® (11 patients), Fortoss®

VITAL (10 patients) or Perioglass® (17 patients). Re-

sults: Comparing with the results of the study group

before and after the treatment, it was concluded that

the results were statistically significant (p = 0.001). In

the Perioglass® subgroup, a greater statistical signifi-

cance (p = 0.003) was noted, than that in either the

Cerasorb® (p = 0.620) or Fortoss® (p = 0.210) sub-

groups, in which there was no statistical significance.

Conclusions: Although further investigations may be

necessary, our results suggest that the synthetic bone

grafts might have an influence on SCE in peripheral

lymphocytes.

Keywords: Bone Graft; Sisters Chromatide Exchange;

Genotoxic; Bone Defect

1. INTRODUCTION

Bone grafts present different spectrum of organic and

syntethic materials that are necessary to provide struc-

tural stability and linkage by stimulating osteogenesis,

which leads to biological repair of sceletal defects.

In oral and maxillofacial surgery, the use of bone

grafts is limited to reconstruction of bone defects after

trauma, cyst or tumor removals in implant and periodon-

tal surgery. The gold standard of bone grafts is consid-

ered to be an autologous bone (bone harvested from the

patient’s own body), due to the retaining of cell viability

[1,2]. However, limited autograft quantities and biologic

performances, extra donor site surgery including morbid-

ity, as well as infection risk and other complaints, have

brought some other materials in use such as alloplasts

[1,3-5]. These synthetic materials are biocompatible, re-

sorbable, easily and rapidly replaceable by authentic

bone tissue [1,6,7]. The resorption rate is synchronous

with bone remodelling [1]. Some of these materials are

bioactive glasses and calcium phosphates which can be

effective for reparation of bone defects in orthopedic and

maxillofacial surgery.

Bioactive glasses are composed of SIO2, CaO, Na2O,

P2O5 and bond to bone through the development of a

surface layer of carbonated hydrohylapatit (HA) [3,8,9].

Some other authors suggest that the mechanism of activ-

ity of Bioactive glasses have an effect of cortical con-

centrations of soluble Si, Ca, P and Na iones, inducing

favorable intracellular and extracellular responses, lead-

ing to rapid bone formation [8]. One of representatives of

these materials is PerioGlass® or Bioglass, which has

proven to be an effective graft material owing to the apa-

tit layer on the surface of the particles, which attracts

osteoprogenitor cells and osteoblasts, thus stimulating

bone formation [3,10]. This material is not only a osteo-

conductive scaffold for new bone formation, it also may

act as a biological membrane retarding epithelial down-

growth [11].

*Corresponding author.

OPEN ACCESS

B. G. Köseoğlu et al. / Open Journal of Stomatology 3 (2013) 447-451

448

Calcium phosphates are synthetic materials in the form

of isolated alpha or beta-tri calcium phosphate (beta-TCP)

(Cerasorb®) or in combination with Hydroxyl sulphate

matrix (Fortoss® VITAL). The TCP mostly behaves as

osteoconductive material, which allows bone osteopro-

genitor cells to grow on its surface or in its pores, chan-

nels or pipes, and to differentiate into osteoblast, thus

bringing bone deposition [12]. This phenomenon might

also be called “osteostimulation”, which implies upregu-

lation of osteoblast precursors [13]. However, due to the

fact that this differentiation does not take place in an

ectopic site, the phenomenon does not meet the strict

standard of osteoinduction [14].

Synthetic bone grafts were a subject of various studies

in which their biocompatibility and osteoinductive and

osteoconductive qualities were examinated [1,3,8,10,15].

So far, potential genotoxic effects caused by synthetic

bone grafts are almost unknown.

Sisters chromatide exchange (SCE) is one of the me-

thods used for examination of chromosomes and ge-

netic damages, caused by exposure to potential environ-

mental mutagens and carcinogens [16-19]. It represents

the interchange of DNA replication at apparently ho-

mologous chromosomal loci, involving DNA breakage

and reunion [18].

The aim of this study was to compare in vivo geno-

toxic effects of three commercially available synthetic

bone graft materials: beta-tri calcium phosphate (Cera-

sorb®), beta-tricalcium phosphate in a hydroxyl sulphate

matrix (Fortoss® VITAL) and bioactive glass ceramic

(Perioglass®) by analyzing the frequency of sisters

chromatide exchange of peripheral lymphocytes.

2. MATERIAL AND METHODS

The study was approved by the Istanbul University Eth-

ics Committee for Scientific Research in Humans (NO:

2006/507). Written and oral consents were obtained from

all patients.

2.1. Patient Selection

Sixty eight patients (45 females and 23 males), age range

from 24 to 58 years, who were referred to the Istanbul

University, School of dentistry, Department of Oral and

Maxillofacial Surgery, for oral surgery procedures, such

as apical resection, cyst enucleation or periodiodontal

curettage were included into a study. All participans in

the study were medically healthy, were nonsmokers, and

were not taking any drugs for at least 2 months before

surgery. Work histories excluded relation to chemical

mutagens and/or exposure to ionizing radiation. The pa-

tients were randomly separated into a two groups. The

first group was control group and included 30 patients

(16 females and 14 males). The second group, subdi-

vided into three subgroups, included 38 patients who

underwent oral surgery procedures in combination with

one of the three synthetic bone grafts; Beta-tricalcium

phosphate (Cerasorb®), Beta-tricalcium phosphate in a

hydroxyl sulphate matrix (Fortoss® VITAL) or bioac-

tive glass ceramic (Perioglass®). The Cerasorb sub-

group included eleven patients (7 females and 4 males),

the Fortoss® subgroup comprised ten patients (7 fe-

males and 3 males), while 17 patients (15 females and 2

males) were included into the Perioglass® subgroup. The

quantity of the graft materials, used in all of the patients

from the second group was 2 gr.

Following surgical procedures, the patients were under

antibiotic (Amoxicillin 2000 mg/day for 7 days) and anal-

gesic (Naproxen sodium 1100 mg/day, as needed) therapy.

2.2. Sisters Chromatid Exchange (SCE) Analysis

Procedure

Sisters Chromatid Exchange (SCE) analysis was per-

formed in the cytogenetic laboratory of the Istanbul

University, School of Medicine, Department of Internal

Medicine, Division of Medical Genetics.

Heparinized blood samples (3 ml) taken from all of the

patients one day before and two months after the oral

surgery procedures, were cultured and examined for sis-

ters chromatid exchanges. The peripheral lymphocytes

were cultured in medium containing RPMI 1640 (Bio-

chrom KG, Berlin, Germany) supplemented with 1%

phytohaemagglutinin-M (Biological Industries, Kibbutz-

Beit Haemek, Israel), 20% fetal calf serum, 1% peni-

cilin-streptomycin and 1% L-glutamine (PAA Laborato-

ries GmbH, Pasching, Austria). At 24 hours 0.5 μg/ml

5’-bromodeoxyuridine (BrdU, Sigma Chemical Com-

pany, USA) was added to the medium and it was further

incubated in the dark. Later at the 70th hours a Colchi-

cine (0.2 μ/ml) (Colchicine powder, Sigma Chemical Co,

USA) was added. Two hours later the cells were col-

lected and treated 10 minutes with 0.075 mol/L KCl at

37˚C, then fixed with methanol-acetic acid (Merck,

Darmstadt, Germany) and standard harvesting procedure

was performed. Slides were stained by flourescein plus

Giemsa technique (FPG). The slides were examined by a

Leitz-Ortoplan microscope (100×) with respect to SCE

frequency per metaphases. 30 metaphases were analyzed

for each patient before and after treatment.

2.3. Statistical Analyses

Analyses in this study were performed by NCSS 2007

packet program.

Kruskal-Wallis test was used for group comparison,

while Dunn’s multiple comparison test was performed

for subgroups comparison. For repeated-measures Wil-

coxon test was used. The results were evaluated in a con-

B. G. Köseoğlu et al. / Open Journal of Stomatology 3 (2013) 447-451

449

OPEN ACCESS

fidence interval of 95% and p < 0.05 was considered

statistically significant.

3. RESULTS

A blood samples from 68 patients (separated into control

and experimental groups), who underwent one of oral

surgery procedures in combination with synthetic bone

grafts or without, were evaluated for sisters chromatid

exchange a day before and two months after the surgery.

Evaluation of results in control and working groups, be-

fore and after the surgeries have showed no statistical

significance (p = 0.501 and p = 0.209). The mean SCE

frequencies before the surgeries, in patients from control

group, Cerasorb®, Fortos® and Perioglass® subgroups

were 7.06 ± 1.54, 7.41 ± 0.62, 6.95 ± 0.87 and 7.47 ±

0.78 respectively, while these values after the surgery

were 7.06 ± 1.54, 7.32 ± 0.72, 7.08 ± 0.71 and 7.78 ±

0.83, respectively.

The results from the Cerasorb® (p = 0.620) and For-

toss® (p = 0.210) subgroups were not statistically sig-

nificant. However, the results from the Perioglass® sub-

group have shown a difference between blood samples

taken before and after the surgeries, which were statistic-

cally significant p = 0.003 (Table 1). The statistical sig-

nificance of p = 0.001 in SCE frequency before and after

the surgeries, between subgroups appears in Table 2.

While the comparision of the Cerasorb® versus Fortoss ®

and Fortoss® versus Perioglass® subgroups did not show

any statistical significance (p = 0.245 and p = 0.669 re-

spectively), the Cerasorb® versus Perioglass® subgroup

showed a meaningful low value of p = 0.018 (Table 3).

4. DISCUSSION

Sisters chromatid exchange (SCE) of peripheral lym-

phocytes presents one of cytogenetic methods for de-

tecting DNA damage [16]. It is well known that different

medications, chemicals, viral infections, malignant dis-

eases, ultraviolet light and smoking are the most com-

mon factors inducing SCE [17-19]. Because of the men-

tioned, in our study the selection of the patients was lim-

ited to those individuals, who’se medical histories ex-

cluded these factors.

Using the sisters chromatid exchange method, muta-

genetic and carcinogenic potentials of different sub-

stances were examinated [16,17,20-25]. Antibiotics and

non-steroidal anti-inflammatory drugs (NSAIDs) were

just some of the most investigated subjects. Various

studies in vitro and vivo deal with different results and

conclusions. Xie et al. [21] suggest that Tetracycline

induce significant increases in SCE even at the lowest

concentrations, while, according to Istifli & Topaktaş [20]

and Jaju et al. [22], Amoxicillin does not behave

genotoxic, with recommendation for safe usage in cases

of bacterial infections. Also the metabolites of Amoxicil-

lin do not have a significant effect on cell proliferation

[20]. Benzathine penicillin G (BPG) might also be con-

sidered as the “safe” antibiotic for a short-term ex posure

[23], although in cases of a long-time exposure, SCE

Table 1. Results in controle and working groups, before and after the surgeries.

Cerasorb®

subgroup

Fortoss®

ubgroup

Perioglass®

subgroup

Control

group KW P

SCE before procedure 7.41 ± 0.62 6.95 ± 0.87 7.47 ± 0.78 7.06 ± 1.54 2.36 0.501

SCE two months later 7.32 ± 0.72 7.08 ± 0.71 7.78 ± 0.83 7.06 ± 1.54 4.63 0.209

Z −0.48 −1.24 −3,01 -

p 0.620 0.210 0,003 -

Table 2. Results show statisticaly significance in SCE frequency before and after the surgeries.

Cerasorb® subgroup Fortoss® subgroup Perioglass® subgroup KW p

SCE Difference −0.09 ± 0.49 0.13 ± 0.32 0.31 ± 0.34 16.5 0.001

SCE % Changes −1.51 ± 6.56 2.02 ± 5.01 3.86 ± 4.2 16.7 0.001

Table 3. Results obtained by Dunn’s multiple comparison test.

Dunn’s multiple comparison test Difference % Changes

Cerasorb®/Fortoss® 0.378 0.245

Cerasorb®/Perioglass® 0.018 0.018

Fortoss®/erioglass® 0.496 0.669

B. G. Köseoğlu et al. / Open Journal of Stomatology 3 (2013) 447-451

450

frequency changes might be expected [24].

Non-steroidal anti-inflammatory drugs (NSAIDs) in-

clude variety of chemicals with analgesic, antipyretic and

anti-inflammatory effects [16]. In one of our previous

studies, we examined genotoxic effects of the etodolac,

nimesulid and naproxen sodium, and concluded that

short term use of selective and non-selective NSAIDs

was not associated with a significant genotoxic effect

that could be detected using the SCE method in pe-

ripheric lymphocytes [16]. However, findings from

Özkul et al. [25], althought statistically insignificant

were opposite to ours in case of naproxen sodium.

The patients included in our study were under antibi-

otic and analgesic therapy after the surgeries. Following

the results of previously mentioned studies, Amoxicillin

2 gr/day (for 7 days) and Naproxen sodium 1100 mg/day

(as needed) were prescribed. Due to the fact that all of

the patients were under the same therapy, with changes

of SCE frequency in working subgroups, the results of

the study suggests that synthetic bone graft materials

might be genotoxic.

The results before and after the treatment have shown

the statistical significance (p = 0.003) in the Perioglass®

subgroup. Also, multiple subgroup comparison tests

were statistically significant p = 0.001, while compari-

son between subgroups have shown that a meaningful

low value was in the Cerasorb® versus Perioglass® sub-

group (p = 0.018). This finding suggests, that the bone

augmentation with synthetic bone grafts might have an

influence on the sisters chromatid exchange frequency.

Although studies of bone grafts are limited to exami-

nations of their biocompatibility, osteoinductive and os-

teoconductive qualities by radiolographic, histologic or

histomorphometric analysis, a review of the English lit-

erature suggests that this could be the first study investi-

gating a genotoxic influence of the synthetic bone grafts

by sisters chromatide exchange method. Due to the fact

that the examined materials in the previous studies have

shown good results as the bone substitutes [26,27], we

wonder whether the resorption rate of the materials play

a role in their genotoxicity. A TCPs show bioresorbabil-

ity during bone regeneration and completely substitute

for a bone tissue after bone stimulation, while hydroxyl

apatite (HA) remains in the body for a long time after

replantation, due to a low grade of bioresorbability [26].

Low grade of resorption is also characteristic of bioac-

tive glasses, where radiographic density and volume are

more expressed in bone sites treated by these materials

[27]. These senses might explain a meaningful low val-

ues (p = 0.018) in the Cerasorb® (TCP) versus Pe-

rioglass® (bioactive glass) and Cerasorb® versus For-

toss® (TCP + HA) (p = 0.245), as compared with the

results with the Fortoss® versus Perioglass® subgroups (p

= 0.669).

Although this is just hypothesis, because we did not

look at the resorption rate of the materials, we are of the

opinion that the bioresorption of the synthetic bone sub-

stitutes did not have any influence on sisters chromatide

exchange frequency, due to the fact that the blood sam-

ples were taken two months after the bone augmentation,

which is not long enough a period for the graft replace-

ment with the authentic bone tissue.

We hope that with these findings, we will open a gate

to some other researchers and studies that will examinate

the genotoxic influence of other bone synthetic substi-

tutes.

5. CONCLUSION

Although further investigations may be necessary, our

results suggests that some alloplasts such as bioactive

glass ceramic might have an influence on SCE in periph-

eral lymphocytes.

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