Joshua Chou Maki Komuro Jia Hao Shinji Kuroda Yusuke Hattori Besim Ben-Nissan Bruce Milthorpe Makoto Otsuka

Bioresorbable zinc hydroxyapatite guided bone regeneration membrane for bone regeneration

Authors’ affiliations: Joshua Chou, Advanced Tissue Regeneration and Drug Delivery Group, Faculty of Science, P.O.Box 123, Broadway, Sydney, NSW, 2007 Australia Maki Komuro, Yusuke Hattori, Makoto Otsuka, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, Japan Besim Ben-Nissan, Bruce Milthorpe, Faculty of Science, University of Technology Sydney, Sydney, NSW, Australia Jia Hao, Shinji Kuroda, Oral Implantology and Regenerative Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Key words: bone augmentation, bone regeneration, GBR membrane, hydroxyapatite, implant

Corresponding author: Joshua Chou Advanced Tissue Regeneration and Drug Delivery Group, Faculty of Science, P.O.Box 123, Broadway, Sydney, NSW, 2007 Australia Tel.: +61 2 9514 1729 Fax: +61 2 9514 1460 e-mail: [email protected]

and histological evaluation.

therapy Abstract Objectives: The aim of this study was to investigate the bone regenerative properties of a heat treated cross-linked GBR membrane with zinc hydroxyapatite powders in the rat calvarial defect model over a 6-week period. Material and Methods: In vitro physio-chemical characterization involved X-ray diffraction analysis, surface topology by scanning electron microscopy, and zinc release studies in physiological buffers. Bilateral rat calvarial defects were used to compare the Zn-HAp membranes against the commercially available collagen membranes and the unfilled defect group through radiological Results: The synthesized Zn-MEM (100 lm thick) showed no zinc ions released in the phosphate buffer solution (PBS) buffer, but zinc was observed under acidic conditions. At 6 weeks, both the micro-CT and histological analyses revealed that the Zn-MEM group yielded significantly greater bone formation with 80  2% of bone filled, as compared with 60  5% in the collagen membrane and 40  2% in the unfilled control group. Conclusion: This study demonstrated the use of heat treatment as an alternative method to crosslinking the Zn-MEM to be applied as a GBR membrane. Its synthesis and production are relatively simple to fabricate, and the membrane had rough surface features on one side, which might be beneficial for cellular activities. In a rat calvarial defect model, it was shown that new bone formation was accelerated in comparison with the collagen membrane and the unfilled defect groups. These results would suggest that Zn-MEM has the potential for further development in dental applications.

Date: Accepted 10 October 2014 To cite this article: Chou J, Komuro M, Hao J, Kuroda S, Hattori Y, Ben-Nissan B, Milthorpe B, Otsuka M. Bioresorbable zinc hydroxyapatite gbr membrane for bone regeneration. Clin. Oral Impl. Res. 27, 2016, 354–360 doi: 10.1111/clr.12520

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Innovative techniques and materials in the development of guided bone regeneration (GBR) membranes have substantially opened the possibilities for increasing localized bone volume, thereby making bone augmentation procedures an integral part of contemporary implant therapy. The concept of GBR using bioresorbable membrane(s) to act as a mechanical barrier to create an artificial space, while simultaneously preventing the migration of surrounding soft tissue(s) into the defect, allows for predictable bone formation in various types of osseous defects (Dahlin et al. 1991,1988; Kostopoulos & Karring 1994; Nyman & Lang 1994), while also eliminating the need for revision surgery to remove the membrane. As such, these

membranes have been extensively studied in both animals and in humans in terms of maxillofacial defects and for the treatment of peri-implant bone defects (Kinoshita 2004; Schmidmaier et al. 2006; Moses et al. 2008; Sculean et al. 2008; Thomaidis et al. 2008). Currently, a number of barrier membranes are already being applied in clinical practice, but are presenting specific indications and limitations (Bornstein et al. 2007; Tal et al. 2008a,b). This has led to the ongoing research and development by both commercial and academic institutions to evaluate novel membranes in an effort to overcome the limitations of what is currently being used. The materials used most commonly for the fabrication of bioresorbable membranes are based

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

Chou et al  Zinc hydroxyapatite GBR membrane

on natural or synthetic polymers; collagen and aliphatic polyesters are both well documented for medical purposes (Bunyaratavej & Wang 2001). To accelerate bone formation, researchers have investigated ways to combine the membrane with bioactive compounds (Hao et al. 2013). Although the concept of adding biological enhancements using growth factors to enhance the proliferation and differentiation of osteogenic cells seems promising, the results are often mixed. In a study where collagen membrane was compared with or without the addition of recombinant-human bone morphogenetic protein-2 (rhBMP-2), the results showed no statistically significant differences in both clinical and radiological outcomes (Jung et al. 2009). However, in vitro and in vivo studies have demonstrated improved bone formation where platelet-derived growth factor (PDGFBB; Lee et al. 2001), basic fibroblast growth factor (FGF2; Hong et al. 2010), and rhBMP-2 (Linde & Hedner 1995; Zellin & Linde 1997; Jung et al. 2009) were added to the membrane. Furthermore, maintaining the therapeutic concentration of growth factors within the bone defect is challenging, due to rapid clearance of the stimulant (Lee et al. 2001). Additionally, the current use of growth factors is limited because of high costs and a short shelf life (Alt et al. 2006; Dahabreh et al. 2009). This has necessitated the need for biologically active GBR membranes that are biocompatible, cell occlusive, and present adequate space-making and clinical handling properties (Gottlow 1993; Scantlebury 1993). Gelatin, which is a denatured form of collagen, is used extensively in pharmaceutical developments owing to its biocompatibility (Kuijpers et al. 2000) and its ability to be synthesized into a membrane. However, gelatin alone does not possess the required mechanical strength to function as a GBR membrane. Noritake et al. (2011) demonstrated the strengthening capacity of gelatin membrane by incorporating tricalcium phosphate particles into the membrane, cross-linked by glutaraldehyde, which showed promising results as compared with the collagen membrane (Noritake et al. 2011). To provide the required mechanical strength and the ability to stimulate bone formation, in this study, zinc hydroxyapatite (Zn-HAp) was incorporated into the gelatin membrane and evaluated for its therapeutic efficacy. In past studies, Zn-HAp has been shown to stimulate bone formation through the actions of zinc ions accelerating the proliferation and differentiation of osteogenic cells, while also depressing the actions of bone resorption

cells (Yamaguchi & Yamaguchi 1986; Hall et al. 1999; Ikeuchi et al. 2003; Ganss & Jheon 2004; Hyun-Ju et al. 2010). Additionally, another study suggests that the use of a zinc phosphate mineralized membrane can inhibit oral bacterial colonization and prevent inflammation as a result of membrane exposure (Chou et al. 2007). As such, the aim of this study was to evaluate the synthesis of a bioresorbable Zn-HAp GBR membrane and investigate its bone-healing ability compared to a collagen membrane in a rat calvarial defect model.

observe the difference in release under different simulated physiological conditions. Zinc hydroxyapatite membrane (Zn-MEM) (1 9 1 cm) was immersed in 10 ml of each respective buffer solution at 37°C at 120 rpm in a shaking water bath. The buffer solutions were collected every 24 h for 7 days and replaced with fresh buffer; zinc ion concentration was quantified using a commercially available zinc assay kit (Wako Chemical Co. Ltd, Tokyo, Japan) through the use of a UV-Vis spectrophotometer. Animal care and surgery

Material and methods Fabrication of Zn-HAp GBR membrane

The zinc hydroxyapatite powder was produced as a mixture containing CaO: CaHPO4: ZnO = 0.7: 2: 0.3 (molar ratio) (Wako Chemicals Co., Tokyo, Japan). The mixed powder samples (10 g in total) were ground in an agate centrifugal ball mill that contained a number of agate balls (diameter and number of balls: 10 mm95, 15 mm910, 20 mm95). Gelatin membranes were produced by completely dissolving gelatin blocks (MediGelatin, Nippi Inc., Tokyo, Japan) in a 60°C water bath for 3 h at a 5% (wt) concentration. The dissolved gelatin solution was cooled to approximately 40°C in ice, and then mixed with Zn-HAp powders at a concentration of 70 mg/ml before being poured into a petri dish. The mixture was air-dried in a clean bench under constant airflow for 12 h. Afterward, the membranes were collected and were allowed to swell in 4°C distilled water for one hour before being subjected to freezedrying for 12 h. Finally, the membranes were cross-linked by heat at 150°C for 5 h. The collagen membrane control group was purchased commercially (Koken Tissue Guide; Koken Co. Ltd, Tokyo, Japan). Physicochemical evaluation of Zn-HAp membrane

Zn-HAp powders were evaluated by powder Xray diffraction analysis (XRD) (RINT- UltimaIII; Rigaku Co., Tokyo, Japan; CuKa radiation, 40 kV, 40 mA) with a step-scanning integration time of one minute at intervals of 2° (2h). The peak pattern was then matched with the JCPDS database. The surface morphological features and the thickness of the membrane were observed under a scanning electron microscope (JEOL JSM-7600F, Field Emission SEM, 10 kV, Tokyo, Japan). The in vitro release of zinc ions from the membrane was evaluated in a phosphate buffer solution (PBS; pH 7.1) and an acetate buffer (pH 4.5) to

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

All studies were approved and in accordance with the guidelines prepared by the Animal Ethics Committee at Musashino University, Japan. Thirty, non-adult, 15-week-old Wistar rats (400 g) were randomly selected and divided into the following experimental groups: (i) empty defect (Control); (ii) collagen membrane (derived from bovine type I collagen and cross-linked with glutaraldehyde); and (iii) zinc hydroxyapatite membrane (Zn-MEM). These animals were selected because they provide adequate size and tissue volume for evaluating the membranes, and a sufficient area that is widely accepted for determining tissue response to implanted biomaterials. Before implantation, the membranes were aseptically cut into 1.6 9 0.8 cm samples. The condition of the animals was monitored on a daily basis, and the weight of the rats was measured weekly. The surgical procedure involved the rats being anesthetized intramuscularly using a combination of ketamine (40 mg/kg) and xylazine (4 mg/kg). After shaving and alcohol disinfection, a cutaneous flap was created by making a midcoronal incision through the skin, where the periosteum was incised and elevated to expose the calvarial bone on both sides of the midline. Two symmetrical, full-thickness bone defects with outer diameters of 5 mm were created with a trephine bur under continuous saline irrigation. The defects were either left empty or covered with collagen membranes or Zn-MEM. Extreme care was taken to suture the periosteum back to its original place to prevent any infiltration of soft tissue cells; the surgeries were generally uneventful. X-ray scans were taken every 2 weeks, and the animals were sacrificed at 8 weeks post-surgery and evaluated radiologically and histologically. Radiological and histological assessment of bone mineral formation

The defect site was evaluated radiologically by X-ray CT (Latheta 200; Aloka, Tokyo,

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Japan), and bone mineral formations were measured by a software program based on the CT scans with a voxel size of 40 lm/pixel (Latheta 2.0; Aloka, Tokyo, Japan) (Chou et al. 2013; Kawao et al. 2013). VG Studio Max 2.0 was used to make a 3D reconstruction from the resulting set of scans every 2 weeks from initiation until week 6. The region of interest (ROI) was defined as a cylinder 5 mm in diameter and of a height that could cover the entire thickness of the calvarial defect. Mask work was performed to binarize the newly formed bone in the ROI by a threshold of intensity, and then the bone volume was measured. The defect closure was calculated based on the percentage of the defect filled with new bone formation by calculating BV/TV based on the bone and implant volume (BV) and the total defect volume (TV). After 8 weeks, the animals were sacrificed and the calvariae were excised; the residual soft tissues were carefully removed and fixed in 10% formalin (Wako Chemical Co. Ltd., Tokyo, Japan). The samples were fixed for 4 weeks and subsequently decalcified in 5% formic acid (Wako Chemical Co. Ltd.) for 5 weeks. After decalcification, the samples were dehydrated in ascending grades of ethanol and embedded in paraffin. Sections of the central defect area were prepared by cutting 5-lm thick coronal slices and staining them with hematoxylin and eosin (H&E). Statistical analysis

Statistical analysis was performed by using SPSS ver. 11.5 for Windows. After confirming that the data within each group were normally distributed with equal variances across groups, ANOVA with a Scheffe test was used for comparing the significance among the groups. P values

Bioresorbable zinc hydroxyapatite guided bone regeneration membrane for bone regeneration.

The aim of this study was to investigate the bone regenerative properties of a heat treated cross-linked GBR membrane with zinc hydroxyapatite powders...
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