D. S. Thoma A. Kruse C. Ghayor R. E. Jung F. E. Weber

Bone augmentation using a synthetic hydroxyapatite/silica oxide-based and a xenogenic hydroxyapatite-based bone substitute materials with and without recombinant human bone morphogenetic protein-2

Authors’ affiliations: D. S. Thoma, R. E. Jung, Department of Fixed and Removable Prothodontics and Dental Material Science, Dental School, University of Zurich, Zurich, Switzerland A. Kruse, C. Ghayor, F. E. Weber, Department of Cranio-Maxillofacial and Oral Surgery, Section Oral Biotechnology & Bioengineering, University Hospital Zurich, Z€ urich, Switzerland A. Kruse, C. Ghayor, F. E. Weber, Dental School, University of Zurich, Zurich, Switzerland F. E. Weber, CABMM, Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland

Key words: bone augmentation, bone substitute material, nanocrystalline hydroxyapatite

Corresponding author: F. E. Weber Department of Cranio-Maxillofacial and Oral Surgery, Oral Biotechnology & Bioengineering, University Hospital, Frauenklinikstrasse 24, 8091 Z€ urich, Switzerland Tel.: +41 44 255 5055 Fax: +41 44 255 4179 e-mail: [email protected]

Results: The mean number of points of the test grid coinciding with bone within the cylinder

Abstract Aim: To test whether or not bone regeneration using deproteinized bovine bone mineral (DBBM) is comparable to hydroxyapatite/silica oxide (HA/SiO) and to test the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) as an adjunct to DBBM for localized bone regeneration. Materials and methods: In each of the 10 rabbits, 4 titanium cylinders were placed on the external cortical plates of their calvaria. Four treatment modalities were randomly allocated: (i) empty, (ii) HA/SiO, (iii) DBBM, and (iv) DBBM plus rhBMP-2 (DBBM/BMP). The animals were sacrificed at week 8. Descriptive histology and histomorphometric assessment using a superimposed test grid of points and cycloids were performed. reached 124  35 bone points for empty controls, 92  40 bone points for DBBM, 98  44 bone points for synthetic HA/SiO, and 146  34 bone points DBBM/BMP. The P-value for DBBM with and without BMP reached a borderline statistical significance of 0.051. However, the area of bone regeneration within the cylinders peaked for DBBM/BMP and was statistically significantly higher compared with empty cylinders (P < 0.05). The bone-to-bone substitute contact ranged between 32.9%  21.7 for DBBM, 39.6  18.4% for HA/SiO, and 57.8%  10.2 for DBBM/BMP. The differences between DBBM/BMP and controls (DBBM, HA/SiO) were statistically significant (P < 0.05). Conclusions: DBBM and HA/SiO rendered comparable amounts of bone regeneration. The addition of rhBMP-2 to DBBM resulted in more favorable outcomes with respect to the area of bone regeneration and to bone-to-implant contact, thereby indicating the potential of this growth factor to enhance bone regeneration within this animal model.

Introduction

Date: Accepted 3 July 2014 To cite this article: Thoma DS, Kruse A, Ghayor C, Jung RE, Weber FE. Bone augmentation using a synthetic hydroxyapatite/silica oxidebased and a xenogenic hydroxyapatite-based bone substitute materials with and without recombinant human bone morphogenetic protein-2. Clin. Oral Impl. Res. 00, 2014, 1–7. doi: 10.1111/clr.12469

The success of dental implants has expanded the treatment options in partially and fully edentulous patients over the course of the years. Initially, a sufficient amount of boneto-place implants completely surrounded by native bone was considered a prerequisite. The introduction of guided bone regeneration (GBR) procedures has further expanded the applicability of dental implants also to sites where previously implants could not be placed (Dahlin et al. 1988; Linde et al. 1993). In the beginning, autogenous bone blocks were used to obtain the sufficient amount of bone

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

needed prior to implant placement (Misch & Misch 1995; Cordaro et al. 2002). This was followed by the introduction of bone substitute materials intended to serve as replacements for autogenous bone. A number of prerequisites are necessary for the successful use of bone substitute materials intended for primary bone augmentation including: (i) biocompatibility, (ii) easy clinical and technical handling, and (iii) osteoconduction. Deproteinized bovine bone minerals (DBBMs) are characterized by a high number of preclinical and clinical data, mainly for guided bone regeneration (GBR) with simultaneous implant placement and for sinus elevation procedures, thus

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Thoma et al  Bone augmentation with HA/SiO and DBBM

demonstrating to fulfill the required criteria. While bone substitute materials are regularly used in unison with implant placement (Hammerle et al. 2002), only limited clinical evidence is available for primary augmentative procedures using bone substitute materials alone (Hammerle et al. 2008; Dahlin & Johansson 2011). More recently, bone substitute materials were combined with biologic mediators to further enhance the outcomes (Jung et al. 2003; Thoma et al. 2010; Urban et al. 2013). Among the growth factors considered, rhBMP-2 and rhPDGF-BB were widely used (Jung et al. 2008a,b), combining them with various bone substitute materials (Schmoekel et al. 2004, 2005; Jung et al. 2005, 2007, 2008a,b; Simion et al. 2006, 2008, 2009; Valderrama et al. 2010; Thoma et al. 2012). Still, some patients may be reluctant to the use of xenogenic bone substitute materials, hence offering the advantage of neglecting the risk of disease transmission and the dependency of animal-derived materials. The latter one could cause ethical conflicts as such or based on religious platforms regarding implantation of materials derived from another living organism. To overcome these potential concerns, many synthetic calcium phosphatebased materials are under investigation and brought on the market as unobjectionable bone substitute materials. Hydroxyapatite/silica oxide (HA/SiO)-based materials were documented in preclinical and clinical studies (Behnia et al. 2013; Reichert et al. 2013; Shakibaie 2013). Non-sintered HA/SiO is characterized by osteoinductivity and biodegradability with a high turnover rate, osteoconductivity, and biomimetic properties (Werner et al. 2002; Gotz et al. 2008; Canullo & Dellavia 2009; Heinemann et al. 2009; Kruse et al. 2011). However, the number of scientific evidence is rather low, and more data are needed to confirm material characteristics, bone formation potential, and to evaluate the additional effect of biologic mediators to bone substitute materials alone. The aim of this study was therefore to test whether or not bone regeneration using DBBM is comparable to HA/SiO and to assess the effect of rhBMP-2 as an adjunct to DBBM for localized bone regeneration.

Materials and methods Ethical approval

Prior to the start of the study, the experimental protocol was approved by the local ethical committee (Veterin€ aramt Kanton Z€ urich; file 107/2012).

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Bone substitute materials

Bone substitute materials of comparable size were used: synthetic hydroxyapatite/silica oxide-based granules (HA/SiO; Nanoboneâ granules of 0.25–1 mm, Artoss, Rostock, Germany) and xenogenic hydroxyapatite particles (DBBM; BioOssâ granules of 0.25–1 mm, Geistlich Biomaterials, Wolhusen, Switzerland). Bone morphogenetic protein

Non-glycosylated rhBMP-2 was expressed in E. coli and refolded as previously described (Weber et al. 2002). RhBMP-2 was desalted through solvent exchange to 1 mM HCl using centrifugal filters (MWCO 10 kDa, Millipore). For loading with growth factor, 100 lg rhBMP-2 dissolved in 3 ml 1 mM HCl was mixed with 2 g of DBBM granules. Following absorption for 1 h at room temperature, the material and rhBMP-2 were frozen in liquid nitrogen and freeze-dried overnight. 100 mg of the resulting DBBM/BMP was needed to fill one cylinder and was loaded with 5 lg of rhBMP-2. Animal model

Ten New Zealand white rabbits were sedated by ketamine and further anaesthetized by an isoflurane-N2O inhalation method. The surgical area was clipped and prepared with iodine for aseptic surgery. A linear incision was made from the nasal bone to the midsagittal crest. After the deflection of the soft tissues, a subperiosteal dissection of the operation site was performed (occipital, frontal, and parietal bones). To place the specially designed cylinder made of c.p. titanium with a machined surface, four evenly distributed 6-mm diameter circular slits with a 1 mm sink depth were created and perforations of the external cortical plate inside the slits were prepared. The cylinders measured 7 mm in height and 7 mm in outer diameter and exhibited a screw design toward the bone site and a small shoulder for a titanium lid toward the covering skin flap. The surgical area was rinsed with saline to remove bone debris, and 4 cylinders were screwed in the prepared slits, providing primary stability. Next, the cylinders were either left empty or filled with DBBM, synthetic HA/SiO-based bone substitute or DBBM/BMP. The cylinder fillings were assigned in a random systematic manner. For the first animal, the treatment modalities were randomly chosen. For the consecutive animals, the randomly chosen order was stepwise rotated by one cylinder. The cylinders were left open toward the bone but were closed with a titanium lid toward

the covering skin-periosteal flap, which was designed to press fit into the opening. After placement of the materials, the soft tissues were closed with sutures. Analgesia was provided by injection (50 mg/kg of Novalginâ, Aventis Pharma AG, Zurich, Switzerland). At week 8, the rabbits were sacrificed after sedation with an overdose of barbiturates (Ketaminâ, Pfizer AG, Zurich, Switzerland) and the calvarial bone was excised. Histology

The samples were first prepared with a sequential water substitution process, which involved 48 h in 40% ethanol, 72 h in 70% ethanol (changed 24 h), 72 h in 96% ethanol, and finally 72 h in 100% ethanol. Samples were placed in xylene for 72 h for defatting of the recovered bone (changed every 24 h). Next, infiltration was performed by placing the samples in methyl methacrylate (MMA) for 72 h (Fluka 64200) followed by 3 days in 100 ml MMA + 2 g di-benzoylperoxid (Fluka 38581), at 4°C. Samples were embedded by placing them in 100 ml MMA + 3 g di-benzoylperoxid + 10 ml plastoid N or dibuthylphtalat (Merck 800 19.25) and allowing polymerization to occur at 37°C in a airtight water bath according to standard procedures (Schenk et al. 1984). The specimens were sectioned in the frontal plane through the middle of the cylinders. Sections of 200 mm thickness were obtained, ground, and polished to a uniform thickness of 60–80 mm. The specimens were surface stained with toluidine blue (Schenk et al. 1984). Histomorphometry

Quantitative evaluation of bone regeneration was assessed by standard histomorphometric techniques. Measurements were obtained on a picture mosaic taken from the entire area of interest via a light microscope at a magnification of 1609, using a superimposed test grid of points and cycloid lines. The numbers of test points overlying the profiles of the different components (i.e., mineralized bone tissue, non-mineralized tissue, and graft particles) were counted. Test points are defined and symbolized according to the standard nomenclature of the International Society for Stereology (Exner 1987). Points overlying bone tissue were counted as bone points. The graft-to-bone contact was calculated by the number of intersections between graft particles and the outlines of either mineralized bone or non-mineralized tissue. In addition, a quantitative evaluation of the area of bone augmentation within the cylinders was carried out to extract a

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Thoma et al  Bone augmentation with HA/SiO and DBBM

clinically relevant parameter. First digital images were obtained and processed with an image analysis program. The pixel counts were directly carried out on the digital images and comprised the area of newly formed bone, including bone marrow and osteointegrated bone substitute particles.

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Statistical analysis

The primary unit of analysis was the animal. Mean values and standard deviations were calculated for the percentage of bone tissue formation, for the absolute bone points, and for bone-to-bone substitute contact in the middle section within the original defect. The significance of differences was evaluated by the Friedman test followed by Wilcoxon signed rank test. A Bonferroni–Holm procedure was applied to account for the number of the groups, and the P-values were corrected accordingly. The limit for significance after correction was set to

silica oxide-based and a xenogenic hydroxyapatite-based bone substitute materials with and without recombinant human bone morphogenetic protein-2.

To test whether or not bone regeneration using deproteinized bovine bone mineral (DBBM) is comparable to hydroxyapatite/silica oxide (HA/SiO) and to t...
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