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The influence of rifamycin decontamination on incorporation of autologous onlay bone grafts in rats: A histometric and immunohistochemical evaluation
Accepted Manuscript Title: The Influence of Rifamycin Decontamination on Incorporation of Autologous Onlay Bone Grafts in Rats: A Histometric and Immunohistochemical Evaluation ¨ ¸ DDS, PHD Authors: Ufuk Tas¸demir DDS, PHD ˙Ilker Ozec Hasan Esen MD, PHD M.Cihat Avunduk MD, PHD PII: DOI: Reference:
S0003-9969(14)00322-7 http://dx.doi.org/doi:10.1016/j.archoralbio.2014.12.010 AOB 3310
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
16-6-2014 3-12-2014 12-12-2014
¨ ¸ ˙I, Esen H, Avunduk MC, The Influence Please cite this article as: Tas¸demir U, Ozec of Rifamycin Decontamination on Incorporation of Autologous Onlay Bone Grafts in Rats: A Histometric and Immunohistochemical Evaluation, Archives of Oral Biology (2014), http://dx.doi.org/10.1016/j.archoralbio.2014.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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Decontamination solution should considered effect on bone
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healing
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Rifamicyn suitable solution for bone decontamination
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BMP-2 expression was induced by rifamycin solution.
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The Influence of Rifamycin Decontamination on Incorporation of Autologous Onlay Bone Grafts in Rats: A Histometric and Immunohistochemical Evaluation
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Ufuk TAŞDEMİR DDS, PHD1, İlker ÖZEÇ DDS,PHD2, Hasan ESEN MD, PHD3, M.Cihat AVUNDUK MD, PHD4.
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1: Assistant Professor, Pamukkale University, Dentistry Faculty, Oral and Maxillofacial Department.
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2: Associate Professor, Cumhuriyet University, Dentistry Faculty, Oral and Maxillofacial Department. 3: Assistant Professor, Selçuk University, Medicine Faculty, Pathology Department.
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4: Professor, Selçuk University, Medicine Faculty, Pathology Department.
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Assistant Professor
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Correspondance: Ufuk TAŞDEMİR DDS, PHD
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Pamukkale University, Dentistry Faculty, Oral and Maxillofacial Department DENİZLİ /TURKEY
Email: [email protected]
Telephone Number: 05058731895/ 02582961133 Fax: 02582410040
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Abstract Objective: Although It has been shown that rifamycin was an effective agent for bone
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graft decontamination, there is no information about the effects of rifamycin decontamination on bone graft incorporation. The aim of this study was to evaluate the
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influence of rifamycin decontamination on incorporation of autologous onlay bone
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grafts quantitatively.
Design: In 30 rats a standardised 5.0mm diameter bone graft was harvested from the
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right mandibular angle, contaminated in saliva, decontaminated with rifampicin solution and augmented to the left as an onlay graft. Ten animals were sacrificed, respectively at
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7, 14, and 21 days after surgery. In the control group (10 rats) the onlay grafts were either contaminated nor decontaminated and the rats were sacrificed at 21 days after
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surgery. Histological slides were prepared from each grafted site for both
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immunohistochemistry analysis [bone morphogenetic protein-2 (bmp-2) and vascular
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endothelial growth factor (VEGF) anti-bodies] and histometric analysis. Images obtained from the graft incorporation area with the light microscope were transferred to a PC and evaluated using Clemex PE 3.5 image analysis software. Results: Grafts were fully incorporated in all specimens. The results showed that rifamycin decontamination has no detrimental effect of on graft incorporation and the findings revealed a tendency for earlier revascularization and osteogenesis in the decontamination group. Data were analysed using Variance analysis and the Tukey’s test. Conclusions: Rifamycin decontamination has no detrimental effect on autogenous graft incorporation and can be used for graft decontamination confidentially.
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Keywords: Autogenous bone block graft, decontaminaton, rifamycin Introduction
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In oral and maxillofacial surgery, autogenous bone grafts are commonly used for osteoconductive, osteoinductive and osteogenic properties.1 Autogenous bone grafts
graft
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have shown an higher remodelling rate than other grafts.2-4 Cortical bone
remodelling is slow compared to cancelous graft remodelling.5 On the other hand onlay resorption.2 Graft
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bone grafting was associated with a variable degree of graft
resorption rate depends on graft type, vascularization, stabilization and infection.6 An
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average resorption rate of 87% for block grafts ın the mandible was observed previously
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study.2
Bone grafts are inadvertently dropped to the floor of the operation room or into
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the oral cavity and may be contaminated with saliva.6 Despite the rarity of this event, at
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least one in four orthopedic surgeons are faced with this situation during their careers.7
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Previously, 58% to-60% of dropped grafts have been shown to grow positive cultures.8,9 Bone grafts harvested from the oral cavity or dropped into the oral cavity, may cause contamination and augmentation failure.10 Contamination is associated with infection and may impair osteogenesis.11 Chemical and antibiotic solutions are commonly used for decontamination. The
microbiological and histological effects of these solutions were evaluated in the literature. Yaman et all.,12 reported that these decontamination solutions were compared, and rifamycin solution seems to be the appropriate agent for decontamination. Rifamycin SV is the semi-synthetic antibiotic group procured from Streptomyces mediterranei and derived from natural rifamycin B. Rifamycin SV has a
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large
spectrum
of
bacterial
action
on
gram-positive
and
gram-negative
microorganisms.13 In dentistry, rifamycin has been used for locally washing fistulas, maxillary sinuses, osteomyelitis, abscess wounds,
pulp cavities and root canals.14
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Rifamycin ’s topical application on bone tissue is well tolerated and decreases the
incidence of infective complications.13,14 However ın the literature, studies showed
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only antimicrobial effects of local antibiotics or influence of these solution on bone
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structure or bone cells. There was no enough information about local antibiotic decontamination effect on bone graft incorparion, healing process of autogenous bone
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block grafts or release of growth factors such as BMPs.
Decontamination solution may damage the structure of bone grafts, endangering
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the success of reconstruction.12 The literature contains insufficient data on the osteoinductive and structural qualities of decontaminated bone block grafts. The
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purpose of this study was to evaluate the effects of rifamycin solution on the
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incorporation of onlay autogenous bone block grafts. Materials and Methods
All animal procedures were approved by the Institutional Animal Care ε Use Committee of Cumhuriyet University, and their care was in accordance with institution guidelines. Forty Wistar albino rats were used for this study. The rats were at the adult stage and weighed approximately 250 g. The animals were kept in cages and fed a solid diet and water ad libitum. The 40 rats were divided into one control group (n = 10) and three experimental groups (each group: n = 10). In the experimental group, bone grafts were contaminated with saliva, decontaminated with rifamycin solution, and fixed to the mandible as onlay
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grafts. The Rats were sacrificed on the 7th, 14th, and 21st days after surgery. Ten rats were used in the control group: onlay grafts were neither contaminated nor decontaminate, and the rats were sacrificed 21 days after surgery.
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Surgical Procedure
The rats were anesthetized with an intraperitoneal injection of 3 mg/kg Xylazine
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(Rompun 2%; Bayer, İstanbul, Turkey) and 90 mg/kg Ketamine HCl (Ketalar;
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Eczacıbaşı- Warner Lambert, İstanbul, Turkey). The right mandible was used as a donor site, and the contralateral angle of the mandible was the recipient area.
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The skin in the mandible was shaved and disinfected with iodine. An incision was made inferior to the angle of the mandible extending to the mandibular bone and
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the periosteum of the mandible was ablated. Autogenous bone block grafts with a standardized thickness of 5 mm were harvested from right mandible ramus region using
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a surgical trephine with an internal diameter of 5 mm. The bone block was
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contaminated with the rat’ s saliva for 15 seconds and then decontaminated with
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rifamycin solution (Rif 250 mg; Abdiİbrahim, İstanbul, Turkey) for 10 minutes. The bone block was then drilled in the center with a 0.5 mm carbide bur, allowing a silk (Doğsan, Istanbul, Turkey) 4-0 suture thread to go through the mandible and the bone block. The receptor area was also drilled with a 0.5 mm carbide bur to allow for placement and stabilization of the graft in close contact with the mandibular bone surface. (Figure 1) The donor and recipient areas were sutured with 4-0 Vicryl (Pegelak, poly-glycolide-co-lactide) [PGLA], Doğsan, Trabzon, Turkey) and allowed to heal by primary intent. Histologic and Immunohistochemical Analysis
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The rats were sacrificed on the 7th, 14th, and 21st days after surgery with an overdose of sodium pentobarbital. The mandible bone was excised and separated into hemimandibles to gather with the surrounding tissue and fixed in 10% buffered
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paraformaldehyde for 48 hours; they were then decalcified in ethylenediamine tetraacetic acid (EDTA) solution. The tissue specimens were prepared in an autotechnicon,
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embedded in paraffin, and sectioned with a microtome. The sections were stained with
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haematoxylin-eosin. The stained specimens were investigated using a Nikon microscope. After each specimen was stained, the same area was photographed using a
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Nikon camera. A Coolpix 5000 photograph attachment was made. A photograph of the Nikon micrometer microscope slide was also taken during the procedure. All photo-
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graphs were then transferred into a PC environment and analyzed (Clemex Vision Lite 3.5 Image Analysis, Clemex Technologies, Longueuil, Quebec, Canada). The length
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was calibrated by comparing the photograph of the specimen with the photograph of the
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Nikon micro-meter microscope slide, which was taken under the same magnification. A
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0.4 mm2 area was designated using the Clemex Vision Lite 3.5 Image Analy-sis program, and osteoblasts, osteoclasts, and new bone areas were marked with the same Image Analysis program in a 0.4 mm2 area (Figure 2). Damaged cells were not evaluated. The marked cells were counted automatically with the same image analysis program. Histological procedure was done different deparment and pathologists blinded to animal group’ s information and
measurements evaluated with Image Analysis
program for reproducibility of procedure. For immunohistochemical staining, the sections (5 µm) were stained with hematoxylin and eosin and monoclonal antibodies for the immunohistochemical analysis of Bone Morphogenetic Protein-2 (BMP-2) expression (Monoclonal Anti-
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human Pro-BMP-2 Antibody [ MAB2260, R&D Systems, Inc.] and Anti-human Vascular Endotelial Growth Factor (VEGF) Antibody [AB-293-NA, R&D Systems, Inc.] Immunostained cells were evaluated in the subepithelial connective tissue beneath
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the sulcular epithelium using a light microscope (Nikon Eclipse E400 light microscope,
Nikon, Tokyo, Japan). For each specimen, the same area was photographed after
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staining by using a photograph attachment (Nikon Coolpix 5000 photograph
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attachment, Nikon). Photographs of the microscope slides were also taken during the procedure. All photographs were then transferred into a PC environment and analyzed
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with an image analysis program (Clemex Vision Lite 3.5 Image Analysis, Clemex, Quebec, Canada). The length was calibrated by comparing the photograph of the
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specimen with the photograph of the microscope slide, which was taken under the same magnification. First, a 0.4 mm2 area was designated using the image analysis program,
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and then positive-stained cells were marked with the same image analysis program in a
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0.4-mm2 area. (Figure 3-4). Damaged cells were not evaluated. The marked cells were
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counted automatically with the same image analysis program. The measurements were repeated 5 times, and then the average data were obtained. The mean (SD) was calculated for each group. The data were analyzed using
variance analysis and Tukey’ s test. Probabilities of less than .05 were accepted as significant.
Results
There were no surgical complications during the operation. After the intervention, the animals recovered without post-operative signs of infection. One rat was lost after general anesthesia was administered, and it was replaced with another rat.
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Histometric Results
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The histometric values for osteoblastic count were higher for group 3 than for the
control group (Table 1). No statistically significant differences were found in terms of
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new bone area and osteoclastic count between the control group and group 3.
Immunohistochemical Results
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No statistically significant differences were seen in terms of the VEGF count between group 3 and the control group. The BMP-2 count was more statistically significantly in
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group 3 and group 2 than in the control group (Table 2). Discussion
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One of the main goals of this study was to analyze the healing of autogenous bone block
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grafts. This bone block graft harvesting and fixation methodology was described by
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Jardini et al.,5 to specify the early healing model of onlay autogenous bone block grafts. We are of the opinion that no studies have examined the incorporation of decontaminated onlay autogenous bone block grafts in rats. Bonfante et al.,15 Jardini et al.,5 and our study showed that this fixation
procedure is satisfactory with rats due to the feature’ s of the recipient area and the block graft, such as graft thickness, mastication forces, and micromovements. In our study the recipient and donor areas which originated from membranous bone, were selected in the mandible, and our results showed that rat models tolerated this bilateral procedure without significant donor site morbidity.
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Membranous bone orgin grafts showed less resorption than grafts of endochonral origin. In our study bone block grafts, orginated from membranous bone, were harvested from the mandible to maintain a larger volume. In our study, we did
decontamination agents between graft resorption ın the longterm result.
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not focus on bone graft resorption and futher studies are necessary to explain the role of
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Rifamycin SV is commonly used in oral and maxillofacial surgery for irrigating
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abscessed cavities, for infected alveoli following extractions, for traumatic lesions, and for treating pericoronaritis.16 It is extremely rare for side effects to occur after the local
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administration of rifamycin. Very rarely, anaphylactic reactions have been reported after the application of rifamycin SV.13,17 In our study, there were no side effects resulting
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from the rifamycin solution. Rifamycin SV’ s local action is advanced by a slow resorption rate, which depends on vascularition, contact surface, administered volume,
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and frequency of administration.13
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Local and systemic antibiotics used for local proflaxis or cure infection in bone
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augmentation procedures. Vascularation was poor at the initial stage of bone graft healing area and resultant to decreased systemic antibiotic penetration and therapeutic effect.18 Local antibiotics were preferred for reduce risk of initial surgical infection, adverse systemic effects, systemic toxicity and necessary high dose of antibiotic intake.19
Cancellous and cortical bone grafts were used for local antibiotic delivery. Witso
et al.,20 reported that cortical bone allograft is an efficient vehicle for local rifamycin delivery. Our decontamination procedure adds another benefit in that autogenous bone block graft may be an effective vehicle for local rifamycin delivery at the operation area, and it can avoid systemic effects, cross infection and foreign body reaction.
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The ideal disinfectant would eliminate the organisms on the graft while maintaining the viability of bone and osteoprogenitor cells.21 Previous studies have reported on the decontamination agents effects on the bone’ s histological and
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morphological features.7,11,12,21 Yaman et al.,12 reported that no histological damage to Haversian canals was found when there was contact with 10% povidone-iodine,
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rifamycin, neomycin and cephazolin sodium solutions. Bruce et al.,7 did not
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recommended chlorhexidine because of decreasing chondrocyte viability. Hooe et al.,21 reported that the autoclave and ethyl alcohol/ethanol caused changes in the bony
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histologic examination. Verdugo et al.,11 reported that osteoblastic growth occurred with povidone-iodine and %2 chlorhexidine. Cabral et al.,22 reported that chlorhexidine was
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more cytotoxic to bone cells than povidone-iodine.
Hooe et al.,21 and Mollina et al.,9 reported that antibiotics are inefficient for
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decontamination because of short exposure time, but Yaman et al.,12 and Sivolella et
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al.,16 have established positive decontamination benefits with microorganisms tested
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using rifamycin solution for 10 minutes and 30 minutes. Rifamycin SV is locally well tolerated which is used topically to prevent local sepsis.13 de Carvalho et al.,14 reported that rifamycin irrigation demonstrated better bone formation in the alveolar socket. Previous study reported that the direct application of rifamycin on bone tissue does not appear to have a negative effect on osteogenesis.23 Our results show that rifamycin has no detrimental effect on bone volume, bone cells, or vascularization in the incorporation area. Our results showed that rifamycin enhanced osteoblastic count in group 3. Previous studies reported that doxycyclines and tetracyclines stimulate cell proliferation24 and enhance osteoblastic activity and osteoid formation in alveolar
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bone.25 Almazin et al.,26 reported that minocycline had a cytotoxic effect on osteoblastic proliferation, and doxycycline improved maturation and differentiation of osteoblasts. Interestingly in the present study, the BMP-2 count was higher for group 3 and
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group 2 than for the control group. Vertugo et al.,11 reported that BMP could counteract
exposure to 5.25% NaOCL. Schmidlin et al.,27 reported that PVP-1 treatment decreases
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the osteogenic cues already deposited into the extracellular matrix by inactivating
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autologous BMP. Park et al.,28 reported that doxycycline diminished BMP-2 expression. The mechanisms underlying the influence of rifamycin over BMP-2 remain unclear.
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This effect of rifamycin may have a molecular basis, such as doxycyclines and SMAD. SMADs are groups of functionally and structurally associated intracellular effectors
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that, intercede signaling, are initiated by BMPs, and regulate cell-definite commitment.25 Liu et al.,29 showed that the ectopic expression of SMAD1C can be
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stimulated, with doxycycline bypassing BMP signaling in the induction of osteoblastic
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differentiation and bone formation.
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Conclusion
Rifamycin decontamination has no detrimental effects on autogenous graft incorporation and can be used for graft decontamination with confidence. Unexpectedly, rifamycin decontamination enhanced BMP-2 and osteoblastic numbers on the graft incorporation side. Future studies will focus on the relation of antibiotics and bone growth factors.
Acknowledgements This work was supported by the Scientific Research Project Fund of Cumhuriyet University under Project number DİŞ-074. Ethical approval
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İlker ÖZEÇ, Asistant Professor, DDS, Phd, Medical Ethics Committee of Medical Faculty, University of Cumhuriyet. Reference No: B. 30.2.CUM.0.01.00.00-50/ 338
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02-07-2009.
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Conflict of interest
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None of the authors have any financial interest either directly or indirectly in any company or any of the products mentioned in this manuscript.
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References
North Am 2010; 22: 347-352.
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1) Deatherage J. Bone materials avaible for alveolar grafting.Oral Maxillofac Surg Clin
2) Sbordone C, Toti P, Guidetti F, Califano L, Santoro A, Sbordone L. Volume
d
changes of iliac crest autogenous bone grafts after vertical and horizontal alveolar
te
ridge augmentation of atrophic maxillae and mandibles: a 6-year computerized
Ac ce p
tomographic follow up. Journal oral and maxillofacial surgery 2012; 70: 2559-2565. 3) Sbordone C, Toti P, Guidetti F, Califano L, Bufo P, Sbordone L. Volume changes of autogenous bone after sinüs lifting and grafting procedures:A 6- year computerized tomographic follow up. Journal of Cranio-Maxillofacial Surgery 2013;41:235-241.
4) Sbordone L, Levin L, Guidetti L, Sbordone C, Glikman A, Schwartz-Arad D. Apical and marginal bone alterations aroun implants in maxillary sinus augmentation grafted with autogenous bone or bovine bone material and simultaneous or delayed dental implant positioning. Clinical oral implants research 2011; 22: 485-491.
13
Page 13 of 20
5) Jardini MA, De Marco AC, Lima LA. Early healing pattern of autogenous bone grafts with and without e-PTFE membranes: a histomorphometric study in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 100: 666-73.
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6) Misch CE. Contemporary Implant Dentistry. Third edition, Mosby Elsevier, Canada, USA, 2008: p840.
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7) Bruce B, Sheibani-Rad S, Appleyard D, et al. Are Dropped Osteoarticular Bone
us
Fragments Safely Reimplantable in Vivo. J Bone Joint Surg Am 2011; 93: 430-8. 8) Cooper DE, Arnoczky SP, Warren RF. Contaminated patellar tendon grafts:
vitro study. Arthroscopy 1991; 7: 272-4.
an
Incidence of positive cultures and efficacy of an antibiotic solution soak—An in
M
9) Molina ME, Nonweiller DE, Evans JA, et al. Contaminated anterior cruciate
378.
d
ligament grafts: The efficacy of 3 sterilization agents. Arthroscopy 2000; 16: 373-
te
10) Tezulas E, Dilek OC. Decontamination of autogenous bone grafts collected from
Ac ce p
dental implant sites via osteotomy: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106: 679-84.
11) Verdugo F, Sáez-Rosón A, Uribarri A, et al. Bone microbial decontamination agents in osseous grafting: an in vitro study with fresh human explants. J Periodontol 2011; 82: 863-71.
12) Yaman F, Unlü G, Atilgan S, et al. Microbiologic and histologic assessment of intentional bacterial contamination of bone grafts. J Oral Maxillofac Surg 2007; 65: 1490-4. 13) Cardot E, Tillie-Leblond I, Jeannin P, et al. Anaphylactic reaction to local administration of rifamycin SV. J Allergy Clin Immunol 1995; 95: 1–7.
14
Page 14 of 20
14) de Carvalho PS, Mariano RC, Okamoto T. Treatment of fibrinolytic alveolitis with rifamycin B diethylamide associated with gelfoam: a histological study. Braz Dent J 1997; 8: 3-8. S, Bosco
AF, Luize
DS, de
Almeida
JM, Cestari
TM.
Influence of nicotine on healing process of autogenous bone block grafts in
the
ip t
15) Bonfante
cr
mandible: a histomorphometric study in rats. Int J Oral Maxillofac Implants 2008;
us
23: 437-44.
16) Sivolella S, Berengo M, Scarin M, et al. Autogenous particulate bone collected with
an
a piezo-electric surgical device and bone trap: a microbiological and histomorphometric study. Archives of Oral Biology 2006; 51: 883—891.
M
17) Laxenaire MC, Mouton C, Frederic A, et al. Anaphylactic shock after tourniquet removal in ortophedic surgery. Ann Fr Anesth Reanim1996; 15: 179–84.
demineralized
te
lincomycin-ımpregnated
d
18) Tabrizi R, Khorshidi H, Shahidi S, Gholami M, Kalbasi S, Khayati A. Use of freeze-dried
bone
allograft
in
the
Ac ce p
periodontal defect after third molar surgery. J Oral Maxillofac Surg 2014; 72: 850857.
19) Rathbone CR1, Cross JD, Brown KV, Murray CK, Wenke JC. Effect of various concentrations of antibiotics on osteogenic cell viability and activity. J Orthop Res 2011: 7: 1070-4.
20) Witso E, Persen L, Benum P, et al. Cortical allograft as a vehicle for antibiotic delivery. Acta Orthop 2005; 76: 481-6. 21) Hooe W, Steinberg B. Management of contaminated bone grafts: an experimental in vitro study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996; 82: 34-7.
15
Page 15 of 20
22) Cabral CT, Fernandes MH. In vitro comparison of chlorhexidine and povidoneiodine onthe longterm proliferation andfunctional activity of human alveolar bone ce lls. Clin Oral Investig 2007; 11: 155-64.
ip t
23) Varlet A, Dauchy P, Hingrez M. Osteogenesis induced by the addition of
Rev Chir Orthop Reparatrice Appar Mot 1985; 71: 73-8.
cr
demineralised bone matrix to plaster pellets with antibiotics. Animal experiment.
us
24) Gomes PS, Santos JD, Fernandes MH. Cell-induced response by tetracyclines on human bone marrow colonized hydroxyapatite and Bonelike. Acta Biomater
an
2008; 4: 630-7.
25) Polson A, Bouwsma O, McNamara T, et al. Enhancement of alveolar bone
M
formation after tetracycline administration in squirrel monkeys. J Appl Res Clin Dent 2005; 2: 32–42.
d
26) Almazin SM, Dziak R, Andreana S, et al. The effect of doxycycline hyclate,
te
chlorhexidine gluconate, and minocycline hyrochlride on osteoblastic proliferation
Ac ce p
and differentiation in vitro. J Periodontol 2009; 80: 999-1005. 27) Schmidlin PR, Imfeld T, Sahrmann P, et al. Effect of short-time povidone-iodine application on osteoblast proliferation and differentation. Open Dent J 2009; 3: 20812.
28) Park JB: Effects of doxycycline, minocycline, and tetracycline on cell proliferation, differentiation, and protein expression in osteoprecursor cells. J Craniofac Surg 2001; 22: 1839-42. 29) Liu Z, Shi W, Ji X, et al. Molecules mimicking Smad1 interacting with Hox stimulate bone formation. J Biol Chem 2004; 279: 11313-19.
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0.4mm2 area. ( n=10 in each group) Group 2
(21 Day)
(7 day)
(14 Day)
Mean (SD)
Mean (SD)
Mean (SD)
Mean (SD)
51,88 (10,91)
42,33 (13,72)
51,77 (12,89)
63,60 (13,63)
Count 4,88 (1,90)
3,66 (1,50)
Count
P Value
(21 Day)
F: 4,46 P: 0,012*
6,50 (1,26)
F: 5,86 P: 0,003*
Bone 233950,3(46824,08) 179681,5(21358,65) 227600,2(49481,20) 266732,2(63270,38) F: 5,15
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New
4,55 (1,33)
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Osteoclast
Group 3
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Group 1
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Osteoblast
Control Group
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Variable
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Table 1. Mean (SD) Histometric results of incorporation regions selected
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Area (mm2)
P: 0,005*
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Data were analysed using Variance analysis and the Tukey’s test. The level of significance was set at P<0.05.( *: P<0.05)
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n=10 in each group) Group 1
Group 2
Group 3
Group
(7 day)
(14 Day)
(21 Day)
(21 Day)
Mean (SD)
Mean (SD)
Mean (SD)
56,55
73,00
AntiBMP-2
36,88 (5,15)
Labelling
(19,02)
AntiVEGF
7,77 (4,29)
8,66 (4,60)
9,60 (5,33)
F: 29,16 P: 0,01*
F: 1,48 P: 0,235
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(Cell Count)
5,44 (3,16)
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Labelling
(16,75)
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(Cell Count)
91,00 (6,12)
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Mean (SD)
P Value
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Control
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Variable
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Table 2. Mean (SD) Immunhistochemical measurements of incorporation area (
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Data were analysed using Variance analysis and the Tukey’s test. The level of significance was set at P<0.05.( *: P<0.05)
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S0003-9969(14)00322-7 http://dx.doi.org/doi:10.1016/j.archoralbio.2014.12.010 AOB 3310
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
16-6-2014 3-12-2014 12-12-2014
¨ ¸ ˙I, Esen H, Avunduk MC, The Influence Please cite this article as: Tas¸demir U, Ozec of Rifamycin Decontamination on Incorporation of Autologous Onlay Bone Grafts in Rats: A Histometric and Immunohistochemical Evaluation, Archives of Oral Biology (2014), http://dx.doi.org/10.1016/j.archoralbio.2014.12.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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Decontamination solution should considered effect on bone
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healing
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Rifamicyn suitable solution for bone decontamination
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BMP-2 expression was induced by rifamycin solution.
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The Influence of Rifamycin Decontamination on Incorporation of Autologous Onlay Bone Grafts in Rats: A Histometric and Immunohistochemical Evaluation
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Ufuk TAŞDEMİR DDS, PHD1, İlker ÖZEÇ DDS,PHD2, Hasan ESEN MD, PHD3, M.Cihat AVUNDUK MD, PHD4.
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1: Assistant Professor, Pamukkale University, Dentistry Faculty, Oral and Maxillofacial Department.
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2: Associate Professor, Cumhuriyet University, Dentistry Faculty, Oral and Maxillofacial Department. 3: Assistant Professor, Selçuk University, Medicine Faculty, Pathology Department.
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4: Professor, Selçuk University, Medicine Faculty, Pathology Department.
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Assistant Professor
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Correspondance: Ufuk TAŞDEMİR DDS, PHD
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Pamukkale University, Dentistry Faculty, Oral and Maxillofacial Department DENİZLİ /TURKEY
Email: [email protected]
Telephone Number: 05058731895/ 02582961133 Fax: 02582410040
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Abstract Objective: Although It has been shown that rifamycin was an effective agent for bone
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graft decontamination, there is no information about the effects of rifamycin decontamination on bone graft incorporation. The aim of this study was to evaluate the
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influence of rifamycin decontamination on incorporation of autologous onlay bone
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grafts quantitatively.
Design: In 30 rats a standardised 5.0mm diameter bone graft was harvested from the
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right mandibular angle, contaminated in saliva, decontaminated with rifampicin solution and augmented to the left as an onlay graft. Ten animals were sacrificed, respectively at
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7, 14, and 21 days after surgery. In the control group (10 rats) the onlay grafts were either contaminated nor decontaminated and the rats were sacrificed at 21 days after
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surgery. Histological slides were prepared from each grafted site for both
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immunohistochemistry analysis [bone morphogenetic protein-2 (bmp-2) and vascular
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endothelial growth factor (VEGF) anti-bodies] and histometric analysis. Images obtained from the graft incorporation area with the light microscope were transferred to a PC and evaluated using Clemex PE 3.5 image analysis software. Results: Grafts were fully incorporated in all specimens. The results showed that rifamycin decontamination has no detrimental effect of on graft incorporation and the findings revealed a tendency for earlier revascularization and osteogenesis in the decontamination group. Data were analysed using Variance analysis and the Tukey’s test. Conclusions: Rifamycin decontamination has no detrimental effect on autogenous graft incorporation and can be used for graft decontamination confidentially.
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Keywords: Autogenous bone block graft, decontaminaton, rifamycin Introduction
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In oral and maxillofacial surgery, autogenous bone grafts are commonly used for osteoconductive, osteoinductive and osteogenic properties.1 Autogenous bone grafts
graft
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have shown an higher remodelling rate than other grafts.2-4 Cortical bone
remodelling is slow compared to cancelous graft remodelling.5 On the other hand onlay resorption.2 Graft
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bone grafting was associated with a variable degree of graft
resorption rate depends on graft type, vascularization, stabilization and infection.6 An
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average resorption rate of 87% for block grafts ın the mandible was observed previously
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study.2
Bone grafts are inadvertently dropped to the floor of the operation room or into
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the oral cavity and may be contaminated with saliva.6 Despite the rarity of this event, at
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least one in four orthopedic surgeons are faced with this situation during their careers.7
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Previously, 58% to-60% of dropped grafts have been shown to grow positive cultures.8,9 Bone grafts harvested from the oral cavity or dropped into the oral cavity, may cause contamination and augmentation failure.10 Contamination is associated with infection and may impair osteogenesis.11 Chemical and antibiotic solutions are commonly used for decontamination. The
microbiological and histological effects of these solutions were evaluated in the literature. Yaman et all.,12 reported that these decontamination solutions were compared, and rifamycin solution seems to be the appropriate agent for decontamination. Rifamycin SV is the semi-synthetic antibiotic group procured from Streptomyces mediterranei and derived from natural rifamycin B. Rifamycin SV has a
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large
spectrum
of
bacterial
action
on
gram-positive
and
gram-negative
microorganisms.13 In dentistry, rifamycin has been used for locally washing fistulas, maxillary sinuses, osteomyelitis, abscess wounds,
pulp cavities and root canals.14
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Rifamycin ’s topical application on bone tissue is well tolerated and decreases the
incidence of infective complications.13,14 However ın the literature, studies showed
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only antimicrobial effects of local antibiotics or influence of these solution on bone
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structure or bone cells. There was no enough information about local antibiotic decontamination effect on bone graft incorparion, healing process of autogenous bone
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block grafts or release of growth factors such as BMPs.
Decontamination solution may damage the structure of bone grafts, endangering
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the success of reconstruction.12 The literature contains insufficient data on the osteoinductive and structural qualities of decontaminated bone block grafts. The
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purpose of this study was to evaluate the effects of rifamycin solution on the
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incorporation of onlay autogenous bone block grafts. Materials and Methods
All animal procedures were approved by the Institutional Animal Care ε Use Committee of Cumhuriyet University, and their care was in accordance with institution guidelines. Forty Wistar albino rats were used for this study. The rats were at the adult stage and weighed approximately 250 g. The animals were kept in cages and fed a solid diet and water ad libitum. The 40 rats were divided into one control group (n = 10) and three experimental groups (each group: n = 10). In the experimental group, bone grafts were contaminated with saliva, decontaminated with rifamycin solution, and fixed to the mandible as onlay
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grafts. The Rats were sacrificed on the 7th, 14th, and 21st days after surgery. Ten rats were used in the control group: onlay grafts were neither contaminated nor decontaminate, and the rats were sacrificed 21 days after surgery.
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Surgical Procedure
The rats were anesthetized with an intraperitoneal injection of 3 mg/kg Xylazine
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(Rompun 2%; Bayer, İstanbul, Turkey) and 90 mg/kg Ketamine HCl (Ketalar;
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Eczacıbaşı- Warner Lambert, İstanbul, Turkey). The right mandible was used as a donor site, and the contralateral angle of the mandible was the recipient area.
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The skin in the mandible was shaved and disinfected with iodine. An incision was made inferior to the angle of the mandible extending to the mandibular bone and
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the periosteum of the mandible was ablated. Autogenous bone block grafts with a standardized thickness of 5 mm were harvested from right mandible ramus region using
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a surgical trephine with an internal diameter of 5 mm. The bone block was
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contaminated with the rat’ s saliva for 15 seconds and then decontaminated with
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rifamycin solution (Rif 250 mg; Abdiİbrahim, İstanbul, Turkey) for 10 minutes. The bone block was then drilled in the center with a 0.5 mm carbide bur, allowing a silk (Doğsan, Istanbul, Turkey) 4-0 suture thread to go through the mandible and the bone block. The receptor area was also drilled with a 0.5 mm carbide bur to allow for placement and stabilization of the graft in close contact with the mandibular bone surface. (Figure 1) The donor and recipient areas were sutured with 4-0 Vicryl (Pegelak, poly-glycolide-co-lactide) [PGLA], Doğsan, Trabzon, Turkey) and allowed to heal by primary intent. Histologic and Immunohistochemical Analysis
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The rats were sacrificed on the 7th, 14th, and 21st days after surgery with an overdose of sodium pentobarbital. The mandible bone was excised and separated into hemimandibles to gather with the surrounding tissue and fixed in 10% buffered
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paraformaldehyde for 48 hours; they were then decalcified in ethylenediamine tetraacetic acid (EDTA) solution. The tissue specimens were prepared in an autotechnicon,
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embedded in paraffin, and sectioned with a microtome. The sections were stained with
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haematoxylin-eosin. The stained specimens were investigated using a Nikon microscope. After each specimen was stained, the same area was photographed using a
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Nikon camera. A Coolpix 5000 photograph attachment was made. A photograph of the Nikon micrometer microscope slide was also taken during the procedure. All photo-
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graphs were then transferred into a PC environment and analyzed (Clemex Vision Lite 3.5 Image Analysis, Clemex Technologies, Longueuil, Quebec, Canada). The length
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was calibrated by comparing the photograph of the specimen with the photograph of the
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Nikon micro-meter microscope slide, which was taken under the same magnification. A
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0.4 mm2 area was designated using the Clemex Vision Lite 3.5 Image Analy-sis program, and osteoblasts, osteoclasts, and new bone areas were marked with the same Image Analysis program in a 0.4 mm2 area (Figure 2). Damaged cells were not evaluated. The marked cells were counted automatically with the same image analysis program. Histological procedure was done different deparment and pathologists blinded to animal group’ s information and
measurements evaluated with Image Analysis
program for reproducibility of procedure. For immunohistochemical staining, the sections (5 µm) were stained with hematoxylin and eosin and monoclonal antibodies for the immunohistochemical analysis of Bone Morphogenetic Protein-2 (BMP-2) expression (Monoclonal Anti-
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human Pro-BMP-2 Antibody [ MAB2260, R&D Systems, Inc.] and Anti-human Vascular Endotelial Growth Factor (VEGF) Antibody [AB-293-NA, R&D Systems, Inc.] Immunostained cells were evaluated in the subepithelial connective tissue beneath
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the sulcular epithelium using a light microscope (Nikon Eclipse E400 light microscope,
Nikon, Tokyo, Japan). For each specimen, the same area was photographed after
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staining by using a photograph attachment (Nikon Coolpix 5000 photograph
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attachment, Nikon). Photographs of the microscope slides were also taken during the procedure. All photographs were then transferred into a PC environment and analyzed
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with an image analysis program (Clemex Vision Lite 3.5 Image Analysis, Clemex, Quebec, Canada). The length was calibrated by comparing the photograph of the
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specimen with the photograph of the microscope slide, which was taken under the same magnification. First, a 0.4 mm2 area was designated using the image analysis program,
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and then positive-stained cells were marked with the same image analysis program in a
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0.4-mm2 area. (Figure 3-4). Damaged cells were not evaluated. The marked cells were
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counted automatically with the same image analysis program. The measurements were repeated 5 times, and then the average data were obtained. The mean (SD) was calculated for each group. The data were analyzed using
variance analysis and Tukey’ s test. Probabilities of less than .05 were accepted as significant.
Results
There were no surgical complications during the operation. After the intervention, the animals recovered without post-operative signs of infection. One rat was lost after general anesthesia was administered, and it was replaced with another rat.
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Histometric Results
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The histometric values for osteoblastic count were higher for group 3 than for the
control group (Table 1). No statistically significant differences were found in terms of
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new bone area and osteoclastic count between the control group and group 3.
Immunohistochemical Results
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No statistically significant differences were seen in terms of the VEGF count between group 3 and the control group. The BMP-2 count was more statistically significantly in
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group 3 and group 2 than in the control group (Table 2). Discussion
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One of the main goals of this study was to analyze the healing of autogenous bone block
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grafts. This bone block graft harvesting and fixation methodology was described by
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Jardini et al.,5 to specify the early healing model of onlay autogenous bone block grafts. We are of the opinion that no studies have examined the incorporation of decontaminated onlay autogenous bone block grafts in rats. Bonfante et al.,15 Jardini et al.,5 and our study showed that this fixation
procedure is satisfactory with rats due to the feature’ s of the recipient area and the block graft, such as graft thickness, mastication forces, and micromovements. In our study the recipient and donor areas which originated from membranous bone, were selected in the mandible, and our results showed that rat models tolerated this bilateral procedure without significant donor site morbidity.
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Membranous bone orgin grafts showed less resorption than grafts of endochonral origin. In our study bone block grafts, orginated from membranous bone, were harvested from the mandible to maintain a larger volume. In our study, we did
decontamination agents between graft resorption ın the longterm result.
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not focus on bone graft resorption and futher studies are necessary to explain the role of
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Rifamycin SV is commonly used in oral and maxillofacial surgery for irrigating
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abscessed cavities, for infected alveoli following extractions, for traumatic lesions, and for treating pericoronaritis.16 It is extremely rare for side effects to occur after the local
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administration of rifamycin. Very rarely, anaphylactic reactions have been reported after the application of rifamycin SV.13,17 In our study, there were no side effects resulting
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from the rifamycin solution. Rifamycin SV’ s local action is advanced by a slow resorption rate, which depends on vascularition, contact surface, administered volume,
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and frequency of administration.13
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Local and systemic antibiotics used for local proflaxis or cure infection in bone
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augmentation procedures. Vascularation was poor at the initial stage of bone graft healing area and resultant to decreased systemic antibiotic penetration and therapeutic effect.18 Local antibiotics were preferred for reduce risk of initial surgical infection, adverse systemic effects, systemic toxicity and necessary high dose of antibiotic intake.19
Cancellous and cortical bone grafts were used for local antibiotic delivery. Witso
et al.,20 reported that cortical bone allograft is an efficient vehicle for local rifamycin delivery. Our decontamination procedure adds another benefit in that autogenous bone block graft may be an effective vehicle for local rifamycin delivery at the operation area, and it can avoid systemic effects, cross infection and foreign body reaction.
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The ideal disinfectant would eliminate the organisms on the graft while maintaining the viability of bone and osteoprogenitor cells.21 Previous studies have reported on the decontamination agents effects on the bone’ s histological and
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morphological features.7,11,12,21 Yaman et al.,12 reported that no histological damage to Haversian canals was found when there was contact with 10% povidone-iodine,
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rifamycin, neomycin and cephazolin sodium solutions. Bruce et al.,7 did not
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recommended chlorhexidine because of decreasing chondrocyte viability. Hooe et al.,21 reported that the autoclave and ethyl alcohol/ethanol caused changes in the bony
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histologic examination. Verdugo et al.,11 reported that osteoblastic growth occurred with povidone-iodine and %2 chlorhexidine. Cabral et al.,22 reported that chlorhexidine was
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more cytotoxic to bone cells than povidone-iodine.
Hooe et al.,21 and Mollina et al.,9 reported that antibiotics are inefficient for
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decontamination because of short exposure time, but Yaman et al.,12 and Sivolella et
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al.,16 have established positive decontamination benefits with microorganisms tested
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using rifamycin solution for 10 minutes and 30 minutes. Rifamycin SV is locally well tolerated which is used topically to prevent local sepsis.13 de Carvalho et al.,14 reported that rifamycin irrigation demonstrated better bone formation in the alveolar socket. Previous study reported that the direct application of rifamycin on bone tissue does not appear to have a negative effect on osteogenesis.23 Our results show that rifamycin has no detrimental effect on bone volume, bone cells, or vascularization in the incorporation area. Our results showed that rifamycin enhanced osteoblastic count in group 3. Previous studies reported that doxycyclines and tetracyclines stimulate cell proliferation24 and enhance osteoblastic activity and osteoid formation in alveolar
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bone.25 Almazin et al.,26 reported that minocycline had a cytotoxic effect on osteoblastic proliferation, and doxycycline improved maturation and differentiation of osteoblasts. Interestingly in the present study, the BMP-2 count was higher for group 3 and
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group 2 than for the control group. Vertugo et al.,11 reported that BMP could counteract
exposure to 5.25% NaOCL. Schmidlin et al.,27 reported that PVP-1 treatment decreases
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the osteogenic cues already deposited into the extracellular matrix by inactivating
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autologous BMP. Park et al.,28 reported that doxycycline diminished BMP-2 expression. The mechanisms underlying the influence of rifamycin over BMP-2 remain unclear.
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This effect of rifamycin may have a molecular basis, such as doxycyclines and SMAD. SMADs are groups of functionally and structurally associated intracellular effectors
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that, intercede signaling, are initiated by BMPs, and regulate cell-definite commitment.25 Liu et al.,29 showed that the ectopic expression of SMAD1C can be
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stimulated, with doxycycline bypassing BMP signaling in the induction of osteoblastic
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differentiation and bone formation.
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Conclusion
Rifamycin decontamination has no detrimental effects on autogenous graft incorporation and can be used for graft decontamination with confidence. Unexpectedly, rifamycin decontamination enhanced BMP-2 and osteoblastic numbers on the graft incorporation side. Future studies will focus on the relation of antibiotics and bone growth factors.
Acknowledgements This work was supported by the Scientific Research Project Fund of Cumhuriyet University under Project number DİŞ-074. Ethical approval
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İlker ÖZEÇ, Asistant Professor, DDS, Phd, Medical Ethics Committee of Medical Faculty, University of Cumhuriyet. Reference No: B. 30.2.CUM.0.01.00.00-50/ 338
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02-07-2009.
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Conflict of interest
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None of the authors have any financial interest either directly or indirectly in any company or any of the products mentioned in this manuscript.
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References
North Am 2010; 22: 347-352.
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1) Deatherage J. Bone materials avaible for alveolar grafting.Oral Maxillofac Surg Clin
2) Sbordone C, Toti P, Guidetti F, Califano L, Santoro A, Sbordone L. Volume
d
changes of iliac crest autogenous bone grafts after vertical and horizontal alveolar
te
ridge augmentation of atrophic maxillae and mandibles: a 6-year computerized
Ac ce p
tomographic follow up. Journal oral and maxillofacial surgery 2012; 70: 2559-2565. 3) Sbordone C, Toti P, Guidetti F, Califano L, Bufo P, Sbordone L. Volume changes of autogenous bone after sinüs lifting and grafting procedures:A 6- year computerized tomographic follow up. Journal of Cranio-Maxillofacial Surgery 2013;41:235-241.
4) Sbordone L, Levin L, Guidetti L, Sbordone C, Glikman A, Schwartz-Arad D. Apical and marginal bone alterations aroun implants in maxillary sinus augmentation grafted with autogenous bone or bovine bone material and simultaneous or delayed dental implant positioning. Clinical oral implants research 2011; 22: 485-491.
13
Page 13 of 20
5) Jardini MA, De Marco AC, Lima LA. Early healing pattern of autogenous bone grafts with and without e-PTFE membranes: a histomorphometric study in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 100: 666-73.
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6) Misch CE. Contemporary Implant Dentistry. Third edition, Mosby Elsevier, Canada, USA, 2008: p840.
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7) Bruce B, Sheibani-Rad S, Appleyard D, et al. Are Dropped Osteoarticular Bone
us
Fragments Safely Reimplantable in Vivo. J Bone Joint Surg Am 2011; 93: 430-8. 8) Cooper DE, Arnoczky SP, Warren RF. Contaminated patellar tendon grafts:
vitro study. Arthroscopy 1991; 7: 272-4.
an
Incidence of positive cultures and efficacy of an antibiotic solution soak—An in
M
9) Molina ME, Nonweiller DE, Evans JA, et al. Contaminated anterior cruciate
378.
d
ligament grafts: The efficacy of 3 sterilization agents. Arthroscopy 2000; 16: 373-
te
10) Tezulas E, Dilek OC. Decontamination of autogenous bone grafts collected from
Ac ce p
dental implant sites via osteotomy: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106: 679-84.
11) Verdugo F, Sáez-Rosón A, Uribarri A, et al. Bone microbial decontamination agents in osseous grafting: an in vitro study with fresh human explants. J Periodontol 2011; 82: 863-71.
12) Yaman F, Unlü G, Atilgan S, et al. Microbiologic and histologic assessment of intentional bacterial contamination of bone grafts. J Oral Maxillofac Surg 2007; 65: 1490-4. 13) Cardot E, Tillie-Leblond I, Jeannin P, et al. Anaphylactic reaction to local administration of rifamycin SV. J Allergy Clin Immunol 1995; 95: 1–7.
14
Page 14 of 20
14) de Carvalho PS, Mariano RC, Okamoto T. Treatment of fibrinolytic alveolitis with rifamycin B diethylamide associated with gelfoam: a histological study. Braz Dent J 1997; 8: 3-8. S, Bosco
AF, Luize
DS, de
Almeida
JM, Cestari
TM.
Influence of nicotine on healing process of autogenous bone block grafts in
the
ip t
15) Bonfante
cr
mandible: a histomorphometric study in rats. Int J Oral Maxillofac Implants 2008;
us
23: 437-44.
16) Sivolella S, Berengo M, Scarin M, et al. Autogenous particulate bone collected with
an
a piezo-electric surgical device and bone trap: a microbiological and histomorphometric study. Archives of Oral Biology 2006; 51: 883—891.
M
17) Laxenaire MC, Mouton C, Frederic A, et al. Anaphylactic shock after tourniquet removal in ortophedic surgery. Ann Fr Anesth Reanim1996; 15: 179–84.
demineralized
te
lincomycin-ımpregnated
d
18) Tabrizi R, Khorshidi H, Shahidi S, Gholami M, Kalbasi S, Khayati A. Use of freeze-dried
bone
allograft
in
the
Ac ce p
periodontal defect after third molar surgery. J Oral Maxillofac Surg 2014; 72: 850857.
19) Rathbone CR1, Cross JD, Brown KV, Murray CK, Wenke JC. Effect of various concentrations of antibiotics on osteogenic cell viability and activity. J Orthop Res 2011: 7: 1070-4.
20) Witso E, Persen L, Benum P, et al. Cortical allograft as a vehicle for antibiotic delivery. Acta Orthop 2005; 76: 481-6. 21) Hooe W, Steinberg B. Management of contaminated bone grafts: an experimental in vitro study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996; 82: 34-7.
15
Page 15 of 20
22) Cabral CT, Fernandes MH. In vitro comparison of chlorhexidine and povidoneiodine onthe longterm proliferation andfunctional activity of human alveolar bone ce lls. Clin Oral Investig 2007; 11: 155-64.
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23) Varlet A, Dauchy P, Hingrez M. Osteogenesis induced by the addition of
Rev Chir Orthop Reparatrice Appar Mot 1985; 71: 73-8.
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demineralised bone matrix to plaster pellets with antibiotics. Animal experiment.
us
24) Gomes PS, Santos JD, Fernandes MH. Cell-induced response by tetracyclines on human bone marrow colonized hydroxyapatite and Bonelike. Acta Biomater
an
2008; 4: 630-7.
25) Polson A, Bouwsma O, McNamara T, et al. Enhancement of alveolar bone
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formation after tetracycline administration in squirrel monkeys. J Appl Res Clin Dent 2005; 2: 32–42.
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26) Almazin SM, Dziak R, Andreana S, et al. The effect of doxycycline hyclate,
te
chlorhexidine gluconate, and minocycline hyrochlride on osteoblastic proliferation
Ac ce p
and differentiation in vitro. J Periodontol 2009; 80: 999-1005. 27) Schmidlin PR, Imfeld T, Sahrmann P, et al. Effect of short-time povidone-iodine application on osteoblast proliferation and differentation. Open Dent J 2009; 3: 20812.
28) Park JB: Effects of doxycycline, minocycline, and tetracycline on cell proliferation, differentiation, and protein expression in osteoprecursor cells. J Craniofac Surg 2001; 22: 1839-42. 29) Liu Z, Shi W, Ji X, et al. Molecules mimicking Smad1 interacting with Hox stimulate bone formation. J Biol Chem 2004; 279: 11313-19.
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0.4mm2 area. ( n=10 in each group) Group 2
(21 Day)
(7 day)
(14 Day)
Mean (SD)
Mean (SD)
Mean (SD)
Mean (SD)
51,88 (10,91)
42,33 (13,72)
51,77 (12,89)
63,60 (13,63)
Count 4,88 (1,90)
3,66 (1,50)
Count
P Value
(21 Day)
F: 4,46 P: 0,012*
6,50 (1,26)
F: 5,86 P: 0,003*
Bone 233950,3(46824,08) 179681,5(21358,65) 227600,2(49481,20) 266732,2(63270,38) F: 5,15
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New
4,55 (1,33)
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Osteoclast
Group 3
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Group 1
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Osteoblast
Control Group
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Variable
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Table 1. Mean (SD) Histometric results of incorporation regions selected
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Area (mm2)
P: 0,005*
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Data were analysed using Variance analysis and the Tukey’s test. The level of significance was set at P<0.05.( *: P<0.05)
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n=10 in each group) Group 1
Group 2
Group 3
Group
(7 day)
(14 Day)
(21 Day)
(21 Day)
Mean (SD)
Mean (SD)
Mean (SD)
56,55
73,00
AntiBMP-2
36,88 (5,15)
Labelling
(19,02)
AntiVEGF
7,77 (4,29)
8,66 (4,60)
9,60 (5,33)
F: 29,16 P: 0,01*
F: 1,48 P: 0,235
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(Cell Count)
5,44 (3,16)
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Labelling
(16,75)
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(Cell Count)
91,00 (6,12)
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Mean (SD)
P Value
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Control
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Variable
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Table 2. Mean (SD) Immunhistochemical measurements of incorporation area (
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Data were analysed using Variance analysis and the Tukey’s test. The level of significance was set at P<0.05.( *: P<0.05)
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