- Email: [email protected]
Bone tissue response to an oily calcium hydroxide suspension in tibial defects. An experimental pilot study in minipigs
Accepted Manuscript Bone tissue response to an oily calcium hydroxide suspension in tibial defects. An experimental pilot study in minipigs I. Mihatovic, M. Payer, M. Bertrams, D. Vasiliu, F. Schwarz, J. Becker, S.I. Stratul PII:
S1010-5182(14)00064-X
DOI:
10.1016/j.jcms.2014.02.004
Reference:
YJCMS 1737
To appear in:
Journal of Cranio-Maxillofacial Surgery
Received Date: 17 July 2013 Revised Date:
9 December 2013
Accepted Date: 10 February 2014
Please cite this article as: Mihatovic I, Payer M, Bertrams M, Vasiliu D, Schwarz F, Becker J, Stratul S, Bone tissue response to an oily calcium hydroxide suspension in tibial defects. An experimental pilot study in minipigs, Journal of Cranio-Maxillofacial Surgery (2014), doi: 10.1016/j.jcms.2014.02.004. 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.
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Bone tissue response to an oily calcium hydroxide suspension in tibial defects. An experimental pilot study in minipigs.
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Mihatovic I 1, Payer M 2 , Bertrams M 1 , Vasiliu D 2 , Schwarz F 1, Becker J 1 , Stratul SI 3
Ilja Mihatovic, Marco Bertrams, Frank Schwarz and Jürgen Becker: Department of Oral Surgery, Heinrich Heine University,
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Düsseldorf, Germany
Michael Payer: Department of Oral Surgery and Radiology, School of Dentistry, Medical University of Graz, Austria
Stefan-Ioan Stratul, Daniela Vasiliu: Department of Periodontology, Victor Babes University of Medicine, Timisoara, Romania
Corresponding address:
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Dr. med. dent. Ilja Mihatovic Department of Oral Surgery
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Westdeutsche Kieferklinik Heinrich Heine University
Moorenstr. 5, D-40225 Düsseldorf, Germany
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Tel: +49-211-81-04533 Fax: +49-211-81-04474
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Email: [email protected]
Short title: Bone tissue response to an oily calcium hydroxide suspension Key words: Bone graft, bone regeneration, calcium hydroxide suspension, tibial defect, animal study
ACCEPTED MANUSCRIPT Source of Funding The study was funded by a grant of DFS Diamon GmbH, Riedenburg, Germany.
Conflict of Interests
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The authors report no conflicts of interest related to this study.
Abstract
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Objectives: The present study was conducted to evaluate the bone tissue response after the application of an
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oily calcium hydroxide suspension (OCHS) into defects created in the tibial bone of minipigs.
Materials and methods: Standardized defects (2ccm) were created into the tibia of 4 Goettinger minipigs. Defects in the test group (n=4) were filled with OCHS (Osteora, DFS-Diamon, Riedenburg, Germany). Defects in the control group (n=4) were filled with venous blood. Animals were sacrificed after healing periods of 4 and 8 weeks. Tibias were dissected, soft tissues removed and processed for histological analysis. Digital images (x200)
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were evaluated using the software CellD (Soft Imaging System, Münster, Germany). The following histomorphometrical landmarks were identified: defect size, mineralized tissue, non-mineralized tissue and residual OCHS.
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Results: Healing was uneventful in all four animals. In the test group, new bone formation was observed in the vicinity of the defect margins whereas the centre of the defect was dominated by non-mineralized tissue.
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Mean percentages of mineralized tissue after 4 weeks were 23,01% in the test group vs. 43,45% in the control group. The mean value for residual OCHS was 7.11% at four weeks. After 8 weeks mean percentages of mineralized tissue were 28,15% in the test group vs. 44,39% in the control group as well as 7,05% for residual OCHS.
Conclusion: Within the limits of the present pilot study it can be concluded that OCHS did not have a beneficial effect on new bone formation. To prove an osteoinductive potential of OCHS further studies based on a higher number of samples are needed.
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Introduction
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In oral surgery, the regeneration of extensive bone defects resulting from trauma, tumours or advanced periodontitis still remains a challenge. The reconstruction of the alveolar ridge allowing a rehabilitation
involving endosseous implants requires demanding surgical approaches (Notodihardjo et al. 2012). At the present time transplantation of either autografts, allografts, bone substitutes or distraction osteogenesis are
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routinely used regenerative techniques, each of them having important limitations concerning availability as well as biological or biomechanical drawbacks (Dasmah et al. 2012; Rachmiel et al. 2013; Sbordone et al. 2013).
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Because of these limitations the osteoinductive stimulation of bone formation has gained increasing interest. The application of morphogenic and mitogenic growth factors and their effect on bone formation have been subject to various studies (Jung et al. 2003; Schwarz et al. 2009). Although several studies provided evidence that bone morphogenic proteins (BMP’s), growth/differentiation factor (GDF)-5 or platelet-derived growth factor (PDGF) may provide local bone augmentation (Jung et al. 2008; Dupoirieux et al. 2009; Schwarz et al.
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2009; Schwarz et al. 2010), unpredictable results, treatment costs and uncertain safety arguments prevent the routine implementation of these approaches into daily practice. Thus, there is still a demand for alternatives to
therapy.
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achieve good bone regeneration as well as the patients desire for a minimally invasive and comfortable surgical
A promising input had been raised several years ago with the idea of using oily calcium hydroxide suspensions
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for tissue regenerative purposes (Freeman et al. 1994). Several experimental studies have shown that calcium hydroxide (CH) may possess antimicrobial (Haenni et al. 2003; Evanov et al. 2004) and anti-inflammatory properties (Charles et al. 2004). When applied on amputated dental pulp or into the root canal close to the apex, CH has been reported to result in a destruction of the vital tissue, leading to the formation of a necrotic layer and, subsequently, formation of a hard tissue barrier below the exposure site (Holland et al. 1977; Tepel et al. 1994). Additionally, CH also seems to have a positive influence on the healing of periapical lesions (De Moor et al. 2002; De Rossi et al. 2005). These effects may be mainly due to the alkaline properties of CH leading to a neutralization of the acidic metabolites of macrophages and osteoclasts (Wikesjo et al. 1999). However, the CH mechanism used to promote the repair of bone tissues may not only do so by providing rich Ca2+ and
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ACCEPTED MANUSCRIPT alkaline environment mineral deposition, but also by stimulating the calcification enzyme activity of osteoblasts (Liang et al. 2000). So far, several groups have reported positive effects in the treatment of periodontal defects (Kasai et al. 2006; Schwarz et al. 2006; Stratul et al. 2006), but little, controversial, information is available about CH on bone regeneration (Stavropoulos et al. 2007; Busenlechner et al. 2008). Until now, effects of CH on
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osseous healing have been evaluated in open models (Kohal et al. 1997) or external applications with GBRtechniques, without positive outcome. (Stavropoulos et al. 2007; Busenlechner et al. 2008). Yet, in vitro data indicate that oily CH-suspensions may have stimulatory effects on cell growth, differentiation and metabolism of progenitor cells. Kasaj 2007 et al. further observed that the addition of an oily CH-suspension enhances
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mitogenic responses of human PDL cells by activating extracellular signal-related kinase (ERK1/2) and increasing cell proliferation (Kasaj et al. 2007).
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To review the potential of CH to promote bone formation, the aim of the present pilot study was to investigate the bone tissue response to an oily CH-suspension focusing on the process of resorption and bone formation using an animal model.
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Materials and Methods:
Animals
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Four Goettinger minipigs (weight between 15,5 kg and 19,5 kg) were used in the present study. The study protocol was approved by the Committee on Animal Ethics of the Victor Babes University of Medicine and
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Pharmacy Timisoara, Romania
Surgical Procedure:
All animals received premedication using 1% Atropin (0,04 mg/kg) by means of an intramuscular injection. Following intramuscular sedation with 2% Acepromazine (1,1 mg/kg) and 10% Ketamine (20mg/kg), anaesthesia was initiated using Thiopental-sodium (4mg/kg) intravenously. During surgery, inhalation anaesthesia was performed by the use of oxygen and Halothane (1-1,5%). To maintain hydration, all animals received 300-500 ml physiological saline solution at the end of surgery while anaesthetized. Postoperative analgesia was ensured by intramuscular injection of Ketonal (0,05mg/kg). Control radiographs of the
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ACCEPTED MANUSCRIPT experimental sites were taken immediately after surgery. None of the animals showed any disorder of movement postoperatively. Prophylactic administration of clindamycin (Clerobe,Pharmacia Tiergesundheit, Erlangen, Germany) (11.0 mg/kg body weight), was performed intra- and postoperatively for 3 days. Until suture removal after 7 days, all animals were kept under observation in individual boxes before being
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held in one group. The temperature of the operated animals was maintained at an optimal value by setting the environment temperature at 22 degrees Celsius.
Following disinfection of the surgical area (Cutasept®, Bode, Hamburg, Germany), an incision penetrating skin, muscular layers and periosteum was performed in order to reflect a flap exposing the medial face of the tibia.
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Under continuous irrigation with sterile 0.9% physiologic saline a hole was drilled and a central screw was inserted marking the centre of the future defect. By means of a diamond disk a rectangular cortical lid (2 cm
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length/2 cm width) on the medial plate of the diaphysis was created and carefully removed. The bone marrow was entirely excavated until a defect of 2ccm was established (Fig. 1a). Defect volume was finally validated by filling the defect with exactly 2 ml of sterile saline. The defects in the test group were entirely filled with OCHS (Osteora, DFS-Diamon GmbH, Riedenburg, Germany) (Fig. 1c) . Defects in the control group were filled with venous blood collected from the retroauricula artery (Fig. 1b). Subsequently, the cortical lids were repositioned
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using the central titanium screw (Fig. 1d). Wound closure was accomplished layer by layer using resorbable sutures (Resorba®, Nürnberg, Germany).
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Animal preparation and retrieval of specimens
The animals were sacrificed after a healing period of 4 and 8 weeks, respectively, 2 animals (test group/control)
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were randomly assigned to each healing period. The euthanasia of the animals was performed by intracardiac injection of 20-40 ml of 20% Kaliumchlorid solution. The treated tibias were dissected from the limbs, the soft tissues were removed and the blocks containing the experimental specimens were obtained. All specimens were fixed in 4% neutral-buffered formalin solution for 4 weeks.
Histological preparation All specimens were dehydrated using ascending grades of alcohol and xylene, infiltrated and embedded in methyl- methacrylate (MMA; Technovit 9100 NEU, Heraeus Kulzer, Wehrheim, Germany) for non-decalcified sectioning. During this procedure, any negative influence of polymerisation heat was avoided due to controlled
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ACCEPTED MANUSCRIPT polymerization in a cold atmosphere (−4°C). ALer 20 hours, the specimens were completely polymerized. The diaphysis of each tibia was sectioned transversally using a diamond wire saw (Exakt®, Apparatebau, Norderstedt, Germany). The sectioning was performed in the direction perpendicular to the long axis of the bone starting immediately at the end of the epiphysis and descending towards the diaphysis, up to the middle
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of the diaphysis below the bone plate covering the operated site. As a result, sections of approximately 500 μm in thickness were created. Subsequently, all specimens were glued with acrylic cement (Technovit 7210 VLC, Heraeus Kulzer) to silanized glass slides (Super Frost, Menzel GmbH, Braunschweig, Germany) and ground to a final thickness of approximately 40 μm. The specimens were stained with Toluidin Blue (TB; quality and
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quantity of mineralized tissue/newly formed bone). The decision to use Toluidine Blue (TB) for
histomorphometry was based on previous publications on bone regeneration of our study group (Schwarz et al.
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2010; Mihatovic et al. 2011; Iglhaut et al. 2012). With this technique, old bone can be identified by a light blue staining, whereas new bone stains dark blue due to its higher protein content (Schenk et al. 1984).
Histological analysis
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Histomorphometrical analysis as well as microscopic observations was performed by one experienced investigator masked to the specific experimental conditions. For image acquisition, a colour CCD camera (Color View III, Olympus, Hamburg, Germany) was mounted on a binocular light microscope (Olympus BX50, Olympus). Digital images (original magnification ×200) were evaluated using a software program (CellD®, Soft
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Imaging System, Münster, Germany).
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For the analysis, 6 sections of each experimental specimen, i.e. 2x OCHS and 2x control, were selected to be able to represent the entire dimension of the defect. As a result, a total of 24 sections were examined. The following histomorphometrical landmarks were analysed within the sections: defect size (DS), mineralized(MT), non-mineralized (NMT) tissue and residual OCHS (OCHS) within the defect. Moreover, the reintegration of the cortical bone lid, as well as new bone formation in the vicinity of the defect, was examined.
Statistical Analysis The statistical analysis was performed using a commercially available software program (PASW Statistics 20.0, SPSS Inc., Chicago, IL, USA). Mean values and standard deviations among the analysed sections were calculated for each variable and group.
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Results
Clinical Healing The postoperative healing was uneventful in all 4 animals. No allergies or foreign body reactions were observed
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throughout the whole study period. All wounds healed eventless and no signs of inflammation or infection occurred. After one week, all experimental sites revealed complete wound closure without any signs of local
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irritation.
Histological Evaluation
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The mean values, standard deviation (SD) and medians for MT, NMT and OCHS in both groups after 4 and 8 weeks are presented in the table.
4 weeks
After 4 weeks, sections of both groups revealed a well reintegrated cortical bone lid indicated by newly built
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trabecular bone adjacent to the former gap of separation (Fig. 5a). In various sections of both groups an excessive bone formation around the cortical bone lid on the averted side of the defect close to the former attached titanium osteosynthesis screw was observed (Fig.5b). However, both groups contained sections
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showing non-mineralized defect gaps (Fig.5c). In test as well as control groups, tissue response was characterized by new bone formation close to the defect margins showing trabecular bone in the vicinity of the
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former defect margins, supporting fine branches of newly formed bone being orientated towards the centre of the defect (Fig. 2a,b). However, in the test group the defect centre was dominated by non-mineralized tissue. In sections of the test group only small amounts of residual OCHS were detected being surrounded by a layer of mineralized tissue (Fig. 4a). Mean percentages of mineralized tissue were 25,51% in the test group vs. 42,44% in the control group. The mean value for residual OCHS was 7.11% at 4 weeks.
8 weeks At 8 weeks, the maturation of bone adjacent to the defect margins into woven bone could be recognized in both groups as well as proceeding bone formation towards the centre of the defect (Fig. 3a,b). Concerning the
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both groups only a couple of sections exhibited dense bone formation inside the centre of the defect. Comparing both groups, the amount of mineralized tissue seemed to be higher in the control group, especially in the centre of the defect. In both groups the value for mineralized tissue only slightly increased between the healing periods 4 and 8 weeks. In the test group, the quantity of residual OCHS only slightly decreased
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indicating only minimal resorption between 4 and 8 weeks of healing (Fig. 4b). Mean percentages of
mineralized tissue were 26,23% in the test group vs. 41,84% in the control group as well as 7,04% for residual
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OCHS.
Discussion
The present study was conducted to evaluate the bone tissue response to the application of an oily calcium
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hydroxide suspension (OCHS) in defects in the tibiae of minipigs. To ensure best possible biological analogy and transfer of experimental findings, minipigs were chosen since bone regeneration in pigs and humans (1.0– 1.5 mm/day vs. 1.2–1.5 mm/day) is considered to be comparable (Albrektsson 1988). The defect model was
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adapted to a study design performed by Merten et al. (2001) who created tibial marrow defects in minipigs and filled them with two different tricalcium phosphate ceramics. Simultaneously dental screw implants were
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inserted in each ceramic and fixed within the orthotopically replanted corticalis lids (Merten et al. 2001). The results of the present study show that the application of OCHS was associated with good biocompatibility and no signs of inflammation. In both test and control groups normal bone regeneration after a healing period of four and eight weeks was observed. However, histomorphometric analysis indicated that in the test group quality and quantity of new bone formation tended to be slightly inferior to the control group. These observations might be due to the chosen defect model. The configuration of this closed defect model served as an ideal setting for spontaneous healing with a cancellous environment surrounded by tibial bone marrow containing multipotent progenitor cells favouring bone regeneration in the control group (Suzuki et al. 2001; Takemoto et al. 2010). In the test group, the application of OCHS was associated with slightly lower amounts of
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ACCEPTED MANUSCRIPT new bone which might point to an obstructive effect in the early phase of wound healing. This observation might be due to the fact that OCHS initially competed with blood clot formation within the defect. The assumption that OCHS inhibits angiogenesis in early stages of wound healing, especially when the resorption process of the grafting material has not advanced yet, is in accordance with the results of two other
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experimental studies evaluating OCHS effects applied in “closed models” in rats and dogs. The data illustrated obstructive effects of OCHS on bone formation in combination with GBR on cortical bones (Stavropoulos et al. 2007; Busenlechner et al. 2008). Stavropoulos et al. reported on OCHS interfering with bone regeneration by showing only minimal new bone formation as well as nearly no signs of resorption (Stavropoulos et al. 2007).
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Stavropoulos suggested that a reason for minor OCHS degradation was due to the compromised blood supply of the environment inside the capsules applied (Stavropoulos et al. 2007). In the present study the histological
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analysis exhibited an obvious degradation process of the grafting material at 4 and 8 weeks after surgical application. The results of the test group showed lower amounts of mineralized tissue after 4 and 8 weeks in comparison to control sites, but an inhibition of bone formation, as observed in the previous studies with closed models, could not be detected. Hence, any obstructive effect of OCHS might have only affected the first phase of wound healing in this specific defect model resulting in delayed bone formation. Ito et al. conducted a
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study in rats comparing wound healing of extraction sockets filled with OCHS and untreated sockets. They reported that after 1 month of healing, sockets previously filled with OCHS showed statistically significantly larger amounts of bone fill than control groups (Ito et al. 2001). In another descriptive study (Schwarz et al.
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2006), three-wall intrabony (4x4x 4mm) periodontal defects were produced bilaterally in a dog model and were randomly treated with either access flap surgery and the application of OCHS, or access flap surgery alone.
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After 2 months of healing, larger amounts of regenerated bone and newly formed cementum were observed in the sites treated with OCHS than that found in the control sites, in which healing was predominantly characterized by the formation of a long junctional epithelium along the previously denuded root surface and only minimal bone regeneration (Schwarz et al. 2006). However, studies which indicated that OCHS facilitates new bone formation were all open defect models. In these cases it meant that the defect and its filling were exposed to an oral environment. Therefore, the positive effect of OCHS could also be related to antibacterial effects of CaOH (Bystrom et al. 1985) - a result of the stable pH gradient of 7.5–9 - before being washed out of defects and pockets. Moreover, the results from in vitro studies showed beneficial effects of continuous OCHS presence on cell growth and differentiation of osseous and undifferentiated progenitor cells (Kasaj et al. 2006).
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ACCEPTED MANUSCRIPT In order to gain additional data on the impact of OCHS on tissue response without any external factors, the present pilot study was designed as a closed inductive trial in an ideal cancellous environment surrounded by tibial bone marrow containing multipotent progenitor cells (Suzuki et al. 2001; Takemoto et al. 2010). The evaluation of the histological sections of the present experimental investigation revealed only a slight gain of
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osseous cell maturation and metabolism related to the application of OCHS. Thus, the results observed in cell cultures differ from results after the application of OCHS in animal models. We can only speculate upon the reasons - one might be the absence of inflammatory cells in in vitro studies (Kohal et al. 1997). Despite the data, CaOH is clinically successful in the field of endodontics and has been shown to promote calcified tissue
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formation in various indications (e.g., dentin barrier, apical seal, apexification, radicular growth, healing of periapical lesions) (Tepel et al. 1994). Periodontal regeneration after OCHS application into periodontal pocket
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defects has been shown in experimental and clinical studies (Kasaj et al. 2006; Schwarz et al. 2006; Stratul et al. 2006).
Summarizing the results of the present investigation, the application of OCHS into tibial defects was inferior to venous blood alone, but showed biocompatibility, decent resorption and new bone formation.
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Conclusion:
Within the limits of the present pilot study it can be concluded that OCHS did not have a beneficial effect on new bone formation. To prove an osteoinductive potential of OCHS further studies based on a higher number
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Acknowledgements
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of samples are needed.
We kindly appreciate the skills and commitment of Mr. Vladimir Golubovic in the preparation of the histological specimens.
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4 weeks
NMT MT OCHS
mean 67,35 25,51 7,11
SD 10,91 15,32 5,5
median 70,16 26,44 6,24
8 weeks
NMT MT OCHS
67,05 26,23 7,04
16,19 12,45 6,66
71,19 22,25 4,61
4 weeks
NMT MT
57,55 42,44
13,12 13,53
60,88 39,12
8 weeks
NMT MT
58,15 41,84
23,19 22,27
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61,26 38,75
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control group
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test group
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Figure legends Empty defect configuration in the diaphysis of the tibia
Figure 1b.
Control group filled with venous blood
Figure 1c.
Defect filled with OCHS
Figure 1d.
Repositioned cortical bone plate
Figure 2.
Region of Interest (ROI)
Figure 3a.
Test group after a healing period of 4 weeks
Figure 3b.
Control group after 4 weeks
Figure 4a.
Test group after 8 weeks
Figure 4b.
Control group after 8 weeks
Figure 5a.
Residual OCHS surrounded by mineralized matrix (test group)
Figure 5b.
Residual OCHS surrounded by mineralized matrix (higher magnification
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Figure 1a.
view x 200, test group) Figure 6a.
Both groups revealed a well reintegrated cortical bone lid indicated by
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newly built trabecular bone adjacent to the former gap of separation (test group)
Excessive bone formation around the cortical bone lid on the averted
side
of the defect (test group)
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Figure 6b.
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S1010-5182(14)00064-X
DOI:
10.1016/j.jcms.2014.02.004
Reference:
YJCMS 1737
To appear in:
Journal of Cranio-Maxillofacial Surgery
Received Date: 17 July 2013 Revised Date:
9 December 2013
Accepted Date: 10 February 2014
Please cite this article as: Mihatovic I, Payer M, Bertrams M, Vasiliu D, Schwarz F, Becker J, Stratul S, Bone tissue response to an oily calcium hydroxide suspension in tibial defects. An experimental pilot study in minipigs, Journal of Cranio-Maxillofacial Surgery (2014), doi: 10.1016/j.jcms.2014.02.004. 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.
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Bone tissue response to an oily calcium hydroxide suspension in tibial defects. An experimental pilot study in minipigs.
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Mihatovic I 1, Payer M 2 , Bertrams M 1 , Vasiliu D 2 , Schwarz F 1, Becker J 1 , Stratul SI 3
Ilja Mihatovic, Marco Bertrams, Frank Schwarz and Jürgen Becker: Department of Oral Surgery, Heinrich Heine University,
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Düsseldorf, Germany
Michael Payer: Department of Oral Surgery and Radiology, School of Dentistry, Medical University of Graz, Austria
Stefan-Ioan Stratul, Daniela Vasiliu: Department of Periodontology, Victor Babes University of Medicine, Timisoara, Romania
Corresponding address:
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Dr. med. dent. Ilja Mihatovic Department of Oral Surgery
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Westdeutsche Kieferklinik Heinrich Heine University
Moorenstr. 5, D-40225 Düsseldorf, Germany
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Tel: +49-211-81-04533 Fax: +49-211-81-04474
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Email: [email protected]
Short title: Bone tissue response to an oily calcium hydroxide suspension Key words: Bone graft, bone regeneration, calcium hydroxide suspension, tibial defect, animal study
ACCEPTED MANUSCRIPT Source of Funding The study was funded by a grant of DFS Diamon GmbH, Riedenburg, Germany.
Conflict of Interests
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The authors report no conflicts of interest related to this study.
Abstract
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Objectives: The present study was conducted to evaluate the bone tissue response after the application of an
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oily calcium hydroxide suspension (OCHS) into defects created in the tibial bone of minipigs.
Materials and methods: Standardized defects (2ccm) were created into the tibia of 4 Goettinger minipigs. Defects in the test group (n=4) were filled with OCHS (Osteora, DFS-Diamon, Riedenburg, Germany). Defects in the control group (n=4) were filled with venous blood. Animals were sacrificed after healing periods of 4 and 8 weeks. Tibias were dissected, soft tissues removed and processed for histological analysis. Digital images (x200)
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were evaluated using the software CellD (Soft Imaging System, Münster, Germany). The following histomorphometrical landmarks were identified: defect size, mineralized tissue, non-mineralized tissue and residual OCHS.
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Results: Healing was uneventful in all four animals. In the test group, new bone formation was observed in the vicinity of the defect margins whereas the centre of the defect was dominated by non-mineralized tissue.
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Mean percentages of mineralized tissue after 4 weeks were 23,01% in the test group vs. 43,45% in the control group. The mean value for residual OCHS was 7.11% at four weeks. After 8 weeks mean percentages of mineralized tissue were 28,15% in the test group vs. 44,39% in the control group as well as 7,05% for residual OCHS.
Conclusion: Within the limits of the present pilot study it can be concluded that OCHS did not have a beneficial effect on new bone formation. To prove an osteoinductive potential of OCHS further studies based on a higher number of samples are needed.
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Introduction
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In oral surgery, the regeneration of extensive bone defects resulting from trauma, tumours or advanced periodontitis still remains a challenge. The reconstruction of the alveolar ridge allowing a rehabilitation
involving endosseous implants requires demanding surgical approaches (Notodihardjo et al. 2012). At the present time transplantation of either autografts, allografts, bone substitutes or distraction osteogenesis are
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routinely used regenerative techniques, each of them having important limitations concerning availability as well as biological or biomechanical drawbacks (Dasmah et al. 2012; Rachmiel et al. 2013; Sbordone et al. 2013).
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Because of these limitations the osteoinductive stimulation of bone formation has gained increasing interest. The application of morphogenic and mitogenic growth factors and their effect on bone formation have been subject to various studies (Jung et al. 2003; Schwarz et al. 2009). Although several studies provided evidence that bone morphogenic proteins (BMP’s), growth/differentiation factor (GDF)-5 or platelet-derived growth factor (PDGF) may provide local bone augmentation (Jung et al. 2008; Dupoirieux et al. 2009; Schwarz et al.
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2009; Schwarz et al. 2010), unpredictable results, treatment costs and uncertain safety arguments prevent the routine implementation of these approaches into daily practice. Thus, there is still a demand for alternatives to
therapy.
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achieve good bone regeneration as well as the patients desire for a minimally invasive and comfortable surgical
A promising input had been raised several years ago with the idea of using oily calcium hydroxide suspensions
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for tissue regenerative purposes (Freeman et al. 1994). Several experimental studies have shown that calcium hydroxide (CH) may possess antimicrobial (Haenni et al. 2003; Evanov et al. 2004) and anti-inflammatory properties (Charles et al. 2004). When applied on amputated dental pulp or into the root canal close to the apex, CH has been reported to result in a destruction of the vital tissue, leading to the formation of a necrotic layer and, subsequently, formation of a hard tissue barrier below the exposure site (Holland et al. 1977; Tepel et al. 1994). Additionally, CH also seems to have a positive influence on the healing of periapical lesions (De Moor et al. 2002; De Rossi et al. 2005). These effects may be mainly due to the alkaline properties of CH leading to a neutralization of the acidic metabolites of macrophages and osteoclasts (Wikesjo et al. 1999). However, the CH mechanism used to promote the repair of bone tissues may not only do so by providing rich Ca2+ and
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ACCEPTED MANUSCRIPT alkaline environment mineral deposition, but also by stimulating the calcification enzyme activity of osteoblasts (Liang et al. 2000). So far, several groups have reported positive effects in the treatment of periodontal defects (Kasai et al. 2006; Schwarz et al. 2006; Stratul et al. 2006), but little, controversial, information is available about CH on bone regeneration (Stavropoulos et al. 2007; Busenlechner et al. 2008). Until now, effects of CH on
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osseous healing have been evaluated in open models (Kohal et al. 1997) or external applications with GBRtechniques, without positive outcome. (Stavropoulos et al. 2007; Busenlechner et al. 2008). Yet, in vitro data indicate that oily CH-suspensions may have stimulatory effects on cell growth, differentiation and metabolism of progenitor cells. Kasaj 2007 et al. further observed that the addition of an oily CH-suspension enhances
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mitogenic responses of human PDL cells by activating extracellular signal-related kinase (ERK1/2) and increasing cell proliferation (Kasaj et al. 2007).
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To review the potential of CH to promote bone formation, the aim of the present pilot study was to investigate the bone tissue response to an oily CH-suspension focusing on the process of resorption and bone formation using an animal model.
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Materials and Methods:
Animals
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Four Goettinger minipigs (weight between 15,5 kg and 19,5 kg) were used in the present study. The study protocol was approved by the Committee on Animal Ethics of the Victor Babes University of Medicine and
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Pharmacy Timisoara, Romania
Surgical Procedure:
All animals received premedication using 1% Atropin (0,04 mg/kg) by means of an intramuscular injection. Following intramuscular sedation with 2% Acepromazine (1,1 mg/kg) and 10% Ketamine (20mg/kg), anaesthesia was initiated using Thiopental-sodium (4mg/kg) intravenously. During surgery, inhalation anaesthesia was performed by the use of oxygen and Halothane (1-1,5%). To maintain hydration, all animals received 300-500 ml physiological saline solution at the end of surgery while anaesthetized. Postoperative analgesia was ensured by intramuscular injection of Ketonal (0,05mg/kg). Control radiographs of the
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ACCEPTED MANUSCRIPT experimental sites were taken immediately after surgery. None of the animals showed any disorder of movement postoperatively. Prophylactic administration of clindamycin (Clerobe,Pharmacia Tiergesundheit, Erlangen, Germany) (11.0 mg/kg body weight), was performed intra- and postoperatively for 3 days. Until suture removal after 7 days, all animals were kept under observation in individual boxes before being
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held in one group. The temperature of the operated animals was maintained at an optimal value by setting the environment temperature at 22 degrees Celsius.
Following disinfection of the surgical area (Cutasept®, Bode, Hamburg, Germany), an incision penetrating skin, muscular layers and periosteum was performed in order to reflect a flap exposing the medial face of the tibia.
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Under continuous irrigation with sterile 0.9% physiologic saline a hole was drilled and a central screw was inserted marking the centre of the future defect. By means of a diamond disk a rectangular cortical lid (2 cm
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length/2 cm width) on the medial plate of the diaphysis was created and carefully removed. The bone marrow was entirely excavated until a defect of 2ccm was established (Fig. 1a). Defect volume was finally validated by filling the defect with exactly 2 ml of sterile saline. The defects in the test group were entirely filled with OCHS (Osteora, DFS-Diamon GmbH, Riedenburg, Germany) (Fig. 1c) . Defects in the control group were filled with venous blood collected from the retroauricula artery (Fig. 1b). Subsequently, the cortical lids were repositioned
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using the central titanium screw (Fig. 1d). Wound closure was accomplished layer by layer using resorbable sutures (Resorba®, Nürnberg, Germany).
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Animal preparation and retrieval of specimens
The animals were sacrificed after a healing period of 4 and 8 weeks, respectively, 2 animals (test group/control)
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were randomly assigned to each healing period. The euthanasia of the animals was performed by intracardiac injection of 20-40 ml of 20% Kaliumchlorid solution. The treated tibias were dissected from the limbs, the soft tissues were removed and the blocks containing the experimental specimens were obtained. All specimens were fixed in 4% neutral-buffered formalin solution for 4 weeks.
Histological preparation All specimens were dehydrated using ascending grades of alcohol and xylene, infiltrated and embedded in methyl- methacrylate (MMA; Technovit 9100 NEU, Heraeus Kulzer, Wehrheim, Germany) for non-decalcified sectioning. During this procedure, any negative influence of polymerisation heat was avoided due to controlled
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ACCEPTED MANUSCRIPT polymerization in a cold atmosphere (−4°C). ALer 20 hours, the specimens were completely polymerized. The diaphysis of each tibia was sectioned transversally using a diamond wire saw (Exakt®, Apparatebau, Norderstedt, Germany). The sectioning was performed in the direction perpendicular to the long axis of the bone starting immediately at the end of the epiphysis and descending towards the diaphysis, up to the middle
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of the diaphysis below the bone plate covering the operated site. As a result, sections of approximately 500 μm in thickness were created. Subsequently, all specimens were glued with acrylic cement (Technovit 7210 VLC, Heraeus Kulzer) to silanized glass slides (Super Frost, Menzel GmbH, Braunschweig, Germany) and ground to a final thickness of approximately 40 μm. The specimens were stained with Toluidin Blue (TB; quality and
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quantity of mineralized tissue/newly formed bone). The decision to use Toluidine Blue (TB) for
histomorphometry was based on previous publications on bone regeneration of our study group (Schwarz et al.
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2010; Mihatovic et al. 2011; Iglhaut et al. 2012). With this technique, old bone can be identified by a light blue staining, whereas new bone stains dark blue due to its higher protein content (Schenk et al. 1984).
Histological analysis
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Histomorphometrical analysis as well as microscopic observations was performed by one experienced investigator masked to the specific experimental conditions. For image acquisition, a colour CCD camera (Color View III, Olympus, Hamburg, Germany) was mounted on a binocular light microscope (Olympus BX50, Olympus). Digital images (original magnification ×200) were evaluated using a software program (CellD®, Soft
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Imaging System, Münster, Germany).
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For the analysis, 6 sections of each experimental specimen, i.e. 2x OCHS and 2x control, were selected to be able to represent the entire dimension of the defect. As a result, a total of 24 sections were examined. The following histomorphometrical landmarks were analysed within the sections: defect size (DS), mineralized(MT), non-mineralized (NMT) tissue and residual OCHS (OCHS) within the defect. Moreover, the reintegration of the cortical bone lid, as well as new bone formation in the vicinity of the defect, was examined.
Statistical Analysis The statistical analysis was performed using a commercially available software program (PASW Statistics 20.0, SPSS Inc., Chicago, IL, USA). Mean values and standard deviations among the analysed sections were calculated for each variable and group.
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Results
Clinical Healing The postoperative healing was uneventful in all 4 animals. No allergies or foreign body reactions were observed
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throughout the whole study period. All wounds healed eventless and no signs of inflammation or infection occurred. After one week, all experimental sites revealed complete wound closure without any signs of local
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irritation.
Histological Evaluation
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The mean values, standard deviation (SD) and medians for MT, NMT and OCHS in both groups after 4 and 8 weeks are presented in the table.
4 weeks
After 4 weeks, sections of both groups revealed a well reintegrated cortical bone lid indicated by newly built
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trabecular bone adjacent to the former gap of separation (Fig. 5a). In various sections of both groups an excessive bone formation around the cortical bone lid on the averted side of the defect close to the former attached titanium osteosynthesis screw was observed (Fig.5b). However, both groups contained sections
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showing non-mineralized defect gaps (Fig.5c). In test as well as control groups, tissue response was characterized by new bone formation close to the defect margins showing trabecular bone in the vicinity of the
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former defect margins, supporting fine branches of newly formed bone being orientated towards the centre of the defect (Fig. 2a,b). However, in the test group the defect centre was dominated by non-mineralized tissue. In sections of the test group only small amounts of residual OCHS were detected being surrounded by a layer of mineralized tissue (Fig. 4a). Mean percentages of mineralized tissue were 25,51% in the test group vs. 42,44% in the control group. The mean value for residual OCHS was 7.11% at 4 weeks.
8 weeks At 8 weeks, the maturation of bone adjacent to the defect margins into woven bone could be recognized in both groups as well as proceeding bone formation towards the centre of the defect (Fig. 3a,b). Concerning the
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both groups only a couple of sections exhibited dense bone formation inside the centre of the defect. Comparing both groups, the amount of mineralized tissue seemed to be higher in the control group, especially in the centre of the defect. In both groups the value for mineralized tissue only slightly increased between the healing periods 4 and 8 weeks. In the test group, the quantity of residual OCHS only slightly decreased
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indicating only minimal resorption between 4 and 8 weeks of healing (Fig. 4b). Mean percentages of
mineralized tissue were 26,23% in the test group vs. 41,84% in the control group as well as 7,04% for residual
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OCHS.
Discussion
The present study was conducted to evaluate the bone tissue response to the application of an oily calcium
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hydroxide suspension (OCHS) in defects in the tibiae of minipigs. To ensure best possible biological analogy and transfer of experimental findings, minipigs were chosen since bone regeneration in pigs and humans (1.0– 1.5 mm/day vs. 1.2–1.5 mm/day) is considered to be comparable (Albrektsson 1988). The defect model was
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adapted to a study design performed by Merten et al. (2001) who created tibial marrow defects in minipigs and filled them with two different tricalcium phosphate ceramics. Simultaneously dental screw implants were
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inserted in each ceramic and fixed within the orthotopically replanted corticalis lids (Merten et al. 2001). The results of the present study show that the application of OCHS was associated with good biocompatibility and no signs of inflammation. In both test and control groups normal bone regeneration after a healing period of four and eight weeks was observed. However, histomorphometric analysis indicated that in the test group quality and quantity of new bone formation tended to be slightly inferior to the control group. These observations might be due to the chosen defect model. The configuration of this closed defect model served as an ideal setting for spontaneous healing with a cancellous environment surrounded by tibial bone marrow containing multipotent progenitor cells favouring bone regeneration in the control group (Suzuki et al. 2001; Takemoto et al. 2010). In the test group, the application of OCHS was associated with slightly lower amounts of
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ACCEPTED MANUSCRIPT new bone which might point to an obstructive effect in the early phase of wound healing. This observation might be due to the fact that OCHS initially competed with blood clot formation within the defect. The assumption that OCHS inhibits angiogenesis in early stages of wound healing, especially when the resorption process of the grafting material has not advanced yet, is in accordance with the results of two other
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experimental studies evaluating OCHS effects applied in “closed models” in rats and dogs. The data illustrated obstructive effects of OCHS on bone formation in combination with GBR on cortical bones (Stavropoulos et al. 2007; Busenlechner et al. 2008). Stavropoulos et al. reported on OCHS interfering with bone regeneration by showing only minimal new bone formation as well as nearly no signs of resorption (Stavropoulos et al. 2007).
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Stavropoulos suggested that a reason for minor OCHS degradation was due to the compromised blood supply of the environment inside the capsules applied (Stavropoulos et al. 2007). In the present study the histological
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analysis exhibited an obvious degradation process of the grafting material at 4 and 8 weeks after surgical application. The results of the test group showed lower amounts of mineralized tissue after 4 and 8 weeks in comparison to control sites, but an inhibition of bone formation, as observed in the previous studies with closed models, could not be detected. Hence, any obstructive effect of OCHS might have only affected the first phase of wound healing in this specific defect model resulting in delayed bone formation. Ito et al. conducted a
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study in rats comparing wound healing of extraction sockets filled with OCHS and untreated sockets. They reported that after 1 month of healing, sockets previously filled with OCHS showed statistically significantly larger amounts of bone fill than control groups (Ito et al. 2001). In another descriptive study (Schwarz et al.
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2006), three-wall intrabony (4x4x 4mm) periodontal defects were produced bilaterally in a dog model and were randomly treated with either access flap surgery and the application of OCHS, or access flap surgery alone.
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After 2 months of healing, larger amounts of regenerated bone and newly formed cementum were observed in the sites treated with OCHS than that found in the control sites, in which healing was predominantly characterized by the formation of a long junctional epithelium along the previously denuded root surface and only minimal bone regeneration (Schwarz et al. 2006). However, studies which indicated that OCHS facilitates new bone formation were all open defect models. In these cases it meant that the defect and its filling were exposed to an oral environment. Therefore, the positive effect of OCHS could also be related to antibacterial effects of CaOH (Bystrom et al. 1985) - a result of the stable pH gradient of 7.5–9 - before being washed out of defects and pockets. Moreover, the results from in vitro studies showed beneficial effects of continuous OCHS presence on cell growth and differentiation of osseous and undifferentiated progenitor cells (Kasaj et al. 2006).
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ACCEPTED MANUSCRIPT In order to gain additional data on the impact of OCHS on tissue response without any external factors, the present pilot study was designed as a closed inductive trial in an ideal cancellous environment surrounded by tibial bone marrow containing multipotent progenitor cells (Suzuki et al. 2001; Takemoto et al. 2010). The evaluation of the histological sections of the present experimental investigation revealed only a slight gain of
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osseous cell maturation and metabolism related to the application of OCHS. Thus, the results observed in cell cultures differ from results after the application of OCHS in animal models. We can only speculate upon the reasons - one might be the absence of inflammatory cells in in vitro studies (Kohal et al. 1997). Despite the data, CaOH is clinically successful in the field of endodontics and has been shown to promote calcified tissue
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formation in various indications (e.g., dentin barrier, apical seal, apexification, radicular growth, healing of periapical lesions) (Tepel et al. 1994). Periodontal regeneration after OCHS application into periodontal pocket
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defects has been shown in experimental and clinical studies (Kasaj et al. 2006; Schwarz et al. 2006; Stratul et al. 2006).
Summarizing the results of the present investigation, the application of OCHS into tibial defects was inferior to venous blood alone, but showed biocompatibility, decent resorption and new bone formation.
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Conclusion:
Within the limits of the present pilot study it can be concluded that OCHS did not have a beneficial effect on new bone formation. To prove an osteoinductive potential of OCHS further studies based on a higher number
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Acknowledgements
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of samples are needed.
We kindly appreciate the skills and commitment of Mr. Vladimir Golubovic in the preparation of the histological specimens.
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study in dogs. Clin Oral Implants Res 21:90-99, 2010 Schwarz, F, Ferrari, D, Sager, M, Herten, M, Hartig, B and Becker, J: Guided bone regeneration using rhGDF-5and rhBMP-2-coated natural bone mineral in rat calvarial defects. Clin Oral Implants Res 20:1219-1230, 2009 Schwarz, F, Stratul, SI, Herten, M, Beck, B, Becker, J and Sculean, A: Effect of an oily calcium hydroxide suspension (Osteoinductal) on healing of intrabony periodontal defects. A pilot study in dogs. Clin Oral Investig 10:29-34, 2006 Stavropoulos, A, Geenen, C, Nyengaard, JR, Karring, T and Sculean, A: Oily calcium hydroxide suspension (Osteoinductal) used as an adjunct to guided bone regeneration: an experimental study in rats. Clin Oral Implants Res 18:761-767, 2007
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ACCEPTED MANUSCRIPT Stratul, SI, Schwarz, F, Becker, J, Willershausen, B and Sculean, A: Healing of intrabony defects following treatment with an oily calcium hydroxide suspension (Osteoinductal). A controlled clinical study. Clin Oral Investig 10:55-60, 2006 Suzuki, A, Zheng, YW, Fukao, K, Nakauchi, H and Taniguchi, H: Clonal expansion of hepatic stem/progenitor
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cells following flow cytometric cell sorting. Cell Transplant 10:393-396, 2001 Suzuki, A, Zheng, YW, Fukao, K, Nakauchi, H and Taniguchi, H: Hepatic stem/progenitor cells with high proliferative potential in liver organ formation. Transplant Proc 33:585-586, 2001
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Tepel, J, Darwisch el Sawaf, M and Hoppe, W: Reaction of inflamed periapical tissue to intracanal medicaments
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and root canal sealers. Endod Dent Traumatol 10:233-238, 1994
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Wikesjo, UM and Selvig, KA: Periodontal wound healing and regeneration. Periodontol 2000 19:21-39, 1999
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4 weeks
NMT MT OCHS
mean 67,35 25,51 7,11
SD 10,91 15,32 5,5
median 70,16 26,44 6,24
8 weeks
NMT MT OCHS
67,05 26,23 7,04
16,19 12,45 6,66
71,19 22,25 4,61
4 weeks
NMT MT
57,55 42,44
13,12 13,53
60,88 39,12
8 weeks
NMT MT
58,15 41,84
23,19 22,27
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61,26 38,75
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Figure legends Empty defect configuration in the diaphysis of the tibia
Figure 1b.
Control group filled with venous blood
Figure 1c.
Defect filled with OCHS
Figure 1d.
Repositioned cortical bone plate
Figure 2.
Region of Interest (ROI)
Figure 3a.
Test group after a healing period of 4 weeks
Figure 3b.
Control group after 4 weeks
Figure 4a.
Test group after 8 weeks
Figure 4b.
Control group after 8 weeks
Figure 5a.
Residual OCHS surrounded by mineralized matrix (test group)
Figure 5b.
Residual OCHS surrounded by mineralized matrix (higher magnification
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Figure 1a.
view x 200, test group) Figure 6a.
Both groups revealed a well reintegrated cortical bone lid indicated by
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newly built trabecular bone adjacent to the former gap of separation (test group)
Excessive bone formation around the cortical bone lid on the averted
side
of the defect (test group)
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Figure 6b.
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