Bone formation and osseointegration with titanium implant using granular- and block-type porous hydroxyapatite ceramics (IP-CHA)

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1 Dental Materials Journal 2013; 32(5): Bone formation and osseointegration with titanium implant using granular- and block-type porous hydroxyapatite ceramics (IP-CHA) Masahiko MINAMI, Masaaki TAKECHI, Kouji OHTA, Akira OHTA, Yoshiaki NINOMIYA, Megumi TAKAMOTO, Akiko FUKUI, Misato TADA and Nobuyuki KAMATA Departments of Oral and Maxillofacial Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi, Minami-Ku, Hiroshima , Japan Corresponding author, Masaaki TAKECHI; The aim of the present study was to examine whether interconnected porous hydroxyapatite ceramics (IP-CHA) could be used as bone substitute for implant treatment in reconstructive surgery. We firstly assessed if surround of the titanium surface placed into granular or block-type IP-CHA can observe new bone formation in a rabbit bone defect model. Subsequently, osseointegration and stability of titanium implant inserted into block-type IP-CHA was investigated in a rabbit onlay graft model. Direct contact between new bone and the surface of the titanium in granular- or block-type IP-CHA was found in a rabbit bone defect. Further, new bone formation was found in direct contact with the implant surface in the block-type IP-CHA in an onlay graft model, and the implant stability quotient (ISQ) values were significantly increased after surgery. Therefore, IP-CHA may be a useful material for implant treatment in reconstructive surgery strategies. Keywords: Interconnected porous hydroxyapatite ceramics (IP-CHA), Titanium implant, New bone formation INTRODUCTION Reconstructive surgery is required for bone defects after tumor resection in patients who undergo oral maxillofacial surgery. Graft is used in reconstructive surgery to restore the jaw form, while occulusal function is restored with the use of implants. Presently, autogenous bone grafting is the gold standard method among available graft materials, because of its osteogenic potential, mechanical properties, and lack of adverse immunological response. However, that has some limitations, such as additional surgery needed for harvesting, the limited availability of grafts of sufficient size and shape, and the risk of donor site morbidity, such as long-term pain, fracture, nerve damage, and infection 1,2). Bone augmentation is necessary in cases with inadequate jaw bone volume for implant treatment. Onlay grafting has been used extensively for reconstructive surgery, as it can restore bone volume by laying autogenous bone directly onto the surface of the host bone 3,4). Although, small bone grafts incorporate well with recipient bone, there are problems regarding resorption of the grafted bone during healing 5). Hydroxyapatite ceramics (HA) materials have been used extensively as a substitute for bone grafting because the crystalline phase of natural bone is similar to that of HA 6,7). Since the 1980s, first-generation conventional porous calcium hydroxyapatite ceramics (C-CHA) have been used in orthopedic, dental, and craniofacial surgery 8,9). However, few reports have indicated that the pores of C-CHA are totally filled with newly formed host bone, probably owing to the closed structure and few inter-pore connections 10). Bone substitute of block- or granular-type may be utilized for implant treatment in bone defect after tumor rejection. On the other hand, block-type bone substitute is hardly resorbed has been to be expected in the implant treatment with horizontal or vertical insufficient bone volume. Recently, interconnected porous hydroxyapatite ceramics (IP-CHA) materials with high porosity have been developed and used successfully in the field of orthopedics 11,12). IP-CHA consists of a porous sintered body made of HAs with a unique pore structure 11), thus it may undergo extensive incorporation into host bone more rapidly than C-CHA 13,14). Therefore, we hypothesized that IP-CHA would be a useful bone substitute for reconstructive surgery in dental implant treatment. We firstly assessed if surround of the titanium surface placed into granular or block-type IP-CHA can observe new bone formation in a rabbit bone defect model. Subsequently, osseointegration and stability of titanium implant inserted into block-type IP-CHA was investigated in a rabbit onlay graft model. MATERIALS AND METHODS Specimens IP-CHA (NEOBONE ; MMT Co. Ltd., Osaka, Japan) has three-dimensional structure with spherical pores of uniform size (average 150 µm, porosity 75%) which are interconnected by window-like holes (average diameter 40 µm) 11). We used granular-type or block-type IP-CHA in following three experiments. Color figures can be viewed in the online issue, which is available at J-STAGE. Received Jun 28, 2012: Accepted Jun 25, 2013 doi: /dmj JOI JST.JSTAGE/dmj/

2 754 Dent Mater J 2013; 32(5): Animals Adult male Japanese white rabbits weighting from 3.0 kg to 3.5 kg at weeks of age were used in this study. All animals were housed individually and fed a commercial diet. The study protocol was reviewed and approved by the Ethics Committee for Experimental Animals of Hiroshima University, and all animals were treated according to the guidelines of the Institutional Animal Care and Use Committee. Experiment 1: Titanium bar fixation with a granulartype IP-CHA in rabbit bone defect model A granular-type IP-CHA ranged between mm was used in this study. Anesthesia was induced in 3 rabbits by an intravenous injection of sodium pentobarbital (Nembutal, Abbot Co., Chicago, IL), at a dose of 0.2 mg/kg body weight. A skin incision of 10 cm was made from the right upper medial thigh to the distal third of the leg, then the medial femoral condyle was exposed, and a cylindrical bone defect with a diameter of 7 mm and depth of 10 mm was made (Fig. 1a). A granular-type IP-CHA was closely packed into the bone defect, then the periosteum and skin were closed in separate layers (Fig. 1b). After 4 weeks, the condyle was exposed again under the anesthesia. A hole in 2 mm diameter and 8 mm high was made using a steel bar in the center of granular specimens, and a titanium bar with the same dimensions (JIS type II) was placed into the hole (n=3). After 8 weeks, these rabbits were euthanized with an overdose of pentobarbital given by intravenous injection. Fig. 1 Placement of titanium bar into granular-type IP- CHAs in bone defect. a: Cylindrical bone defect was made in medial femoral condyle. granular-type IP-CHAs were then closely placed into the defect. b: A titanium bar was placed into the hole in the center of granulartype IP-CHAs. Experiment 2: Titanium implant fixation with a blocktype IP-CHA in rabbit bone defect model A cylindrical block-type of IP-CHA in diameter 7 mm and height 10 mm were used in next study. In 10 rabbits, a skin incision 10 cm long was made from the right upper medial thigh to distal third of the leg, then the medial femoral condyle was exposed, and a cylindrical bone defect with a diameter of 7 mm and depth of 10 mm was made. In the experimental group, we inserted an IP-CHA block fixed with a titanium implant with a diameter of 3.8 mm and length of 8 mm (GENESiO, GC, Tokyo), into the bone defect (IP- CHA groups, n=5) (Fig. 2a, b, c). In the control group (n=5), titanium implant with a diameter of 3.8 mm and length of 8 mm (GENESiO, GC, Tokyo) was directly inserted into a drill holes in the femoral condyle using standard protocol. After 8 weeks, all animals were euthanized with an overdose of pentobarbital given by intravenous injection Experiment 3: Titanium implant fixation with a blocktype IP-CHA in rabbit onlay grafting model A cylindrical block-type of IP-CHA in diameter 7 mm and height 5 mm were used in onlay grafting models. The bilateral mandibular premolar teeth of rabbits (n=5) were extracted under anesthesia. After 1 week, buccal skin was incised, and the fascial periosteal flap folded back. Next, a block-type of IP-CHA was placed on the cortical bone surface in the lateral mandibular, and titanium implant with a diameter of 3.8 mm and length of 8 mm (GENESiO) was inserted through the IP-CHA and into the surface of the contralateral cortex (Fig. 3a, b). After 6 or 12 weeks, rabbits were euthanized with an overdose of pentobarbital given by intravenous injection. Histological examination Samples were processed for histological examinations after extraction of the IP-CHA and implants. At 3 days before euthanasia, the rabbits were intravenously injected with calcein (Sigma, St. Louis, MO) in a 10 mg/ ml solution for new bone labelling. Resected specimens were fixed in 10% buffered formalin for 7 days, then dehydrated in graded ethanol and embedded in polyester resin (Rigolac; Oken Co., Tokyo, Japan). Sections about 200 µm thick were cut with a low-speed diamond saw, and ground by hand parallel to the long axis of the implants to approximately 70 µm. The sections were observed under a light microscope after staining with 5 % toluidine blue. Calcein labeling was observed using a fluorescent microscope.

3 Dent Mater J 2013; 32(5): Fig Insertion of block-type IP-CHA fixed with titanium implant into femoral condyle. a: Block-type IP-CHA fixed with titanium implant. b and c: Block-type IP-CHA fixed with titanium implant was inserted into the defect in the medial condyle. Histomorphometric evaluation The bone-implant contact (BIC) ratio was calculated for the quantitative evaluation of new bone formation (Toluidine blue staining, original 100 magnification). The ratio was defined as the length of implant surface (A) in length of new bone-titanium contact (B) (B/A 100(%)), and was calculated using Image-J (National Institutes of Health, Bethesda, MD, USA). Fig. 3 Placement of implant into block-type IP-CHA as onlay bone graft substitute. a and b: A titanium implant was inserted through the block-type IP-CHA into the surface of the contralateral cortex. Resonance frequency analysis (RFA) Resonance frequency analysis (RFA) has been established as a non-invasive and non-destructive quantitative measurement of implant stability. RFA performed to determine implant stability using an OsstellTM Mentor (Integration Diagnostics AB, Göteborg, Sweden) and which convert RFA values into implant stability quotient (ISQ) values. ISQ measurement was described as follows. The cover screw of implant were exposed by incision of skin, and removed from implant fixture. A magnetic peg was inserted using plastic screw driver. The probe was held 3 mm from the peg, then. ISQ values ranging from 1 to 100 were displayed on the screen of the analyzer, with higher values indicating greater dental implant stability. Each determination was repeated 3 times to assess precision, and the average ISQ value from those was calculated. Statistical analysis Data were analyzed using Student s t-test or one-way

4 756 Dent Mater J 2013; 32(5): analysis of variance (ANOVA), with the results presented as the mean±standard deviation. RESULTS No discharge from the surgical lesions was observed and all of the animals were in good general condition at all times points. Experiment 1: Bone formation around titanium bar fixation with a granular-type Firstly, we examined bone formation around the titanium bars inserted into granular-type IP-CHA packed into bone defects. Figure 4a) shows that bone formation was observed in nearly all pores of the IP-CHA, while direct contact between new bone and the surface of the titanium bar was found at 8 weeks after the second-operation in Fig. 4b). Mean BIC at 8 weeks was 71.6±7.5%. Experiment 2: Osseointegration and stability of titanium implant with a block-type IP-CHA in bone defect To examine whether block-type IP-CHA as well as granular-type IP-CHA could be used as bone substitute for implant treatment in bone defect, we examined bone formation and stability around titanium implants inserted into block-type IP-CHA in the rabbit bone defect model. At 8 weeks, Fig. 5a) shows that bone ingrowth was observed in the majority of pores in the block-type IP-CHA. Furthermore, as shown in Fig. 5b) and c), new bone formation that showed an immature pattern and woven bone structure was found in direct contact with the implant surface in the block-type IP- CHA. Figure 6 demonstrated that mean BIC for the IP-CHA and control groups after 8 weeks was 66.3±3.4% and 51.6±8.2%, respectively, with that in IP-CHA groups significantly higher. Figure 7 shows that the ISQ values for the implants in the IP-CHA groups showed an increasing tendency after 8 weeks, though there were no significant differences between the IP- CHA and control groups. Fig. 4 Section of granular-type IP-CHAs fixed with titanium bar. a: Bone ingrowth in the pores of the granulartype IP-CHA by toluidine blue staining ( 4 magnification). b: Titanium bar surface placed in granular-type IP-CHAs by toluidine blue staining ( 40 magnification). Experiment 3: Osseointegration and stability of titanium implant in a block-type IP-CHA as onlay grafts Finally, to examine whether block-type IP-CHA could be used as onlay grafting substitute in implant treatment, titanium implants were inserted into block-type IP-CHA placed on the cortical bone surface, and implant stability was examined after surgery. As shown in Fig. 8 a) and b), a higher levels of new bone formation from host bone was observed in the pores of the IP-CHA group after 12 weeks as compared to 6 weeks, and bone ingrowth was also observed in the pores of the block-type IP-CHA in Fig. 8 c). In addition, direct contact between the implant surface and new bone was found in the block-type IP-CHA in Fig. 8d). Figure 9 demonstrated that the ISQ values were significantly increased at 12 weeks after surgery. DISCUSSION Calcium hydroxyapatite ceramics is composed of nontoxic materials, and generally provokes no reactions from human tissues, thus it is considered to be represent a good starting point for creating bone substitutes 13,14). C-CHA have been used in orthopedic, craniofacial, and dental applications 8,9). However, few studies have reported that C-CHA was fully filled by newly formed bone, which may be due to its structure and the limited connectivity between pores 10). Inter-pore connections that are less than 2 or 3 µm in diameter do not allow for cell migration or vascularization into the pores, events that are essential for new bone formation 13). However, IP-CHA, a second generation porous calcium hydroxyapatite was developed by adopting the form-

5 Dent Mater J 2013; 32(5): Fig. 5 Fig Section of block-type IP-CHA fixed with implant. a: Bone ingrowth in the pores of the block-type IP-CHA fixed with titanium implant by toluidine blue staining and merged images with calcein labeling (green) ( 4 magnification). b: Bone formation in the pores of block-type IP-CHA, as shown by toluidine blue staining and merged images with calcein labeling (green) ( 40 magnification). c: The surface of the implant in the block-type IP-CHA by toluidine blue staining and merged images with calcein labeling (green) ( 20 magnification ). BIC ratio at 8 weeks after implantation of titanium implant (n=5). All data are expressed as the mean±sd (error bars). *Significantly different from control group (Student s t-test: p<0.05). Fig. 7 ISQ values for titanium implant at 4 and 8 weeks after surgery (n=5). All data are expressed as the mean±sd (error bars). *Significantly different from value obtained on day of placement of IP-CHA fixed with implant (Student s t-test: p<0.05).

6 758 Dent Mater J 2013; 32(5): Fig. 8 Section of block-type IP-CHA fixed with implant in onlay graft model. a and b: Bone formation in the IP-CHA pores at 6 and 12 weeks by toluidine blue staining and merged images with calcein labeling (green) ( 4 magnification ). c: Bone formation in the pores of IP-CHA, as shown by toluidine blue staining and merged images with calcein labeling (green) ( 40 magnification ). d: The implant surface in IP-CHA blocks by toluidine blue staining and merged images with calcein labeling (green) ( 40 magnification ). Fig. 9 ISQ values of titanium implant at 6 and 12 weeks after surgery (n=5). Data are shown as the mean±sd (error bars). *Significantly different from ISQ value on the day of placement of IP-CHA fixed with implant (Student s t-test: p<0.05). gel technique. It has a three-dimensional structure with spherical pores of uniform size interconnected by window-like holes that have diameters greater than 10 μm 11). A previous in vivo study that used a rabbit model reported that mature bone ingrowth in IP-CHA inserted into bone defect was seen in all of the pores 13). In agreement with those findings, bone ingrowth was observed in the majority of interconnected pores of the granular and block-type IP-CHA and placed into bone defects in the present study. Therefore, IP-CHA inserted into a bone defect has potential to induce new bone ingrowth from host bone. To investigate whether hydroxyapatite can be used for implant treatment after tumor resection as a graft substitute, Schliephake et al. examined new bone formation around implant inserted into blocktype C-CHA using a pig model. They found very little contact between the titanium implants and ingrown bone in the blocks, which had a small pore size ( µm in diameter). On the other hand, hydroxyapatite

7 Dent Mater J 2013; 32(5): blocks with a larger pore size (260 µm in diameter) were penetrated by bone that extended into the central pores, while the titanium implants inserted into these blocks were in close contact with the newly formed bone 15). In the present study, new bone formation was observed to be in direct contact with the surface of titanium implants inserted into the block-type IP-CHA. Furthermore, the mean BIC of the IP-CHA groups was significantly higher than that in the control group after 8 weeks. IP-CHA has an average pore diameter of µm pore diameter and the pore are entirely interconnected which allow for osteoblastic cells to deeply invade into those near the implant surface. Previously, our group also showed that IP-CHA promoted differentiation of osteoblasts derived from human jaw bones 16,17). Also, acceleration of osseointegration and increases in bone-implant contact can be achieved by implants and hydroxyapatite because of the osteoconductive capacity of hydroxyapatite 18,19). Thus, bone ingrowth into IP-CHA should be sufficient to accelerate osseointegration of titanium implants. Although, autogenous bone augmentation, such as the onlay graft method, has been used to overcome problems with insufficient bone volume to make placement of implants possible, a small portion of autogenous grafted bone becomes resorbed during healing. In a previous clinical study, histologic evaluation of hydroxyapatite as an onlay bone graft substitute in clinical cases over a period of 9 years revealed that HA graft particles had no signs of active resorption 20). However, few in vivo studies have been conducted to determine whether titanium implant inserted into hydroxyapatite block as an onlay bone graft substitute undergoes osseointegration. In the present study, new bone formation from host bone was observed in the pores of IP-CHA placed on cortical bone, while the ISQ values of implants inserted into blocktype IP-CHA within the bone cortex were significantly increased at 12 weeks after surgery. On the other hand, Park et al. reported that ISQ values measured with an Osstell device had a significant correlation with BIC values in vivo study 21). When used as an onlay graft material, a block-type IP-CHA promotes osseoconduction from the host bone surface and periosteum, thereby facilitating osseointegration of an implant inserted into the block and cortical bone. Clinically, IP-CHA has been used in the fields of orthopedic surgery, including cases with osteonecrosis of the femoral condyle, juxta-articular intraosseous lesions related to rheumatoid arthritis, and benign bone tumor 13). Our groups performed implant placement and maxillary sinus floor augmentation with mixed grafts composed of cortical bone and IP-CHA granules in a female patient, and evaluated the clinical behavior and the histological aspects. Implant stability was increased at 9 months after fixture installation compared with the first operation, while histological analysis revealed there was new bone formation in the majority of pores of IP-CHA 22). Those results along with those obtained in the present study provide evidence for the ability of IP-CHA to serve as a substitute for implant treatment in reconstructive surgery. CONCLUSION Our findings are the first to show a titanium implant inserted into IP-CHA undergoes osseointegration and is related to new bone formation in rabbits. IP-CHA may be a useful substitute for implant treatment in reconstructive surgery. 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