Abstract
Octacalcium phosphate (OCP) and porcine atelocollagen sponge composites (OCP/Col) markedly enhanced bone regeneration in a rat cranial defect model. To assess clinical application, the authors examined whether OCP/Col would enhance bone regeneration in an alveolar cleft model in an adult dog, which was assumed to reflect patients with alveolar cleft. Disks of OCP/Col or collagen were implanted into the defect and bone regeneration by OCP/Col or collagen was investigated 4 months after implantation. Macroscopically, the OCP/Col-treated alveolus was obviously augmented and occupied by radio-opacity, and the border between the original bone and the defect was indistinguishable. Histological analysis revealed it was filled and bridged with newly formed bone; a small quantity of the remaining implanted OCP was observed. X-ray diffraction patterns of the area of implanted OCP/Col indicated no difference from those of dog bone. In the collagen-treated alveolus, the hollowed alveolus was mainly filled with fibrous connective tissue, and a small amount of new bone was observed at the defect margin. These results suggest that bone was obviously repaired when OCP/Col was implanted into the alveolar cleft model in a dog, and OCP/Col would be a significant bone regenerative material to substitute for autogeneous bone.
Reconstructing bone lesions resulting from trauma, neoplasms, or infection is a major problem in reconstructive surgery . Autogeneous bone grafting for patients with cleft lip and palate has become a well-accepted treatment modality to restore the function and structure of the maxillary arch at the cleft site ; but it has disadvantages, such as restricted availability and morbidity associated with collecting bone from a second operative site . Although synthetic and natural biomaterials have been developed and clinically applied to overcome the disadvantages of autogeneous bone grafting, no bone regenerative material comparable to autogeneous bone has been developed.
The authors have investigated octacalcium phosphate (Ca 8 H 2 (PO 4 ) 6 ·5H 2 O; OCP), which is a calcium phosphate and has been suggested to be a precursor of biological apatite in bone . The osteoconductive characteristics of OCP were first established in the subperiosteal region of mouse calvaria . The authors’ previous studies using a rat calvarial defect model demonstrate that implanted OCPs enhance bone regeneration and can be used as an effective scaffold for bone regenerative cytokines . Synthetic OCP enhances bone regeneration more than hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), and implanted OCP is more resorbable than implanted β-TCP or HA , which have been used clinically as a scaffold for bone regeneration .
It was reported that OCP combined with other materials would advance bone regeneration, and excellent osteogenicity is shown by OCP coating on metal implants . The authors have developed a synthetic bone substitute constructed of synthetic OCP and porcine atelocollagen sponge (OCP/Col) to improve handling performance . OCP/Col significantly enhances bone regeneration more than the implantation of OCP, β-TCP collagen composite (β-TCP/Col), or HA collagen composite (HA/Col) in a rat calvarial defect model .
To assess clinical use, the authors have recently reported that implantation of OCP/Col enhances bone healing in a tooth socket model of an adult dog . In this study, they hypothesize that OCP/Col would enhance bone regeneration and restore the structure of the maxillary arch at the cleft site in the artificial cleft of a dog; it was assumed that this would reflect patients with an alveolar cleft. To examine this hypothesis, OCP/Col or collagen is implanted into the artificial alveolar cleft model and bone regeneration by OCP/Col or collagen is investigated macroscopically, radiographically, radiomorphometrically, histologically, and crystallographically 4 months after the implantation of OCP/Col or collagen disks.
Materials and methods
Twelve-month-old male beagle dogs ( n = 9; NARC Co., Chiba, Japan) were used. The principles of laboratory animal care and national laws were followed. All procedures were approved by the Animal Research Committee of Tohoku University.
OCP was prepared by mixing a calcium and phosphate solution as described previously . The sieved granules (particle size 300–500 μm) of OCP, obtained from dried OCP, were sterilized by heating at 120 °C for 2 h. A previous study showed that such heating does not affect physical properties such as the crystalline structure or the specific surface area of OCP granules , although increasing the temperature above 100 °C induced collapse of the OCP structure through dehydration . The authors have previously described the preparation of OCP/Col and collagen disks . Collagen was prepared from NMP collagen PS (Nippon Meat Packers, Tsukuba, Ibaraki, Japan), a lyophilized powder of pepsin-digested atelocollagen isolated from porcine dermis. NMP collagen PS was dissolved in distilled water and adjusted to 3% of the final concentration at pH 7.4. The concentrated collagen was lyophilized, and a disk was molded (9 mm diameter, 1 mm thick). OCP/Col was prepared from NMP collagen PS and OCP granules. OCP was added to the concentrated collagen and mixed. The OCP/collagen mixture was lyophilized, and the disk was molded (9 mm diameter, 1 mm thick). The molded collagen and OCP/Col underwent dehydrothermal treatment (150 °C, 24 h) in a vacuum drying oven DP32 (Yamato Scientific, Tokyo, Japan) and were sterilized using electron beam irradiation (5 kGy) ( Fig. 1 ).
Implantation procedure
The artificial maxillary alveolar cleft was prepared first ( Fig. 2 ). General anesthesia was administered with intravenous sodium pentobarbital (0.5 ml/kg), followed by intramuscular atropine sulfate (0.5 mg) and ketamine hydrochloride (20 mg/kg). After disinfection of the oral cavity and injection of local anesthesia (2% lidocaine with 1/80,000 epinephrine), an artificial maxillary alveolar cleft was prepared, as previously reported . After extraction of the left second and third maxillary incisors, buccal mucoperiosteum was ablated toward the floor of the nasal cavity, and palatal mucoperiosteum was ablated toward the left palatal fissure. The exposed maxillary bone was then resected to have an open connection between the oral and nasal cavity. The resected area was the buccal bone of the left second and third maxillary incisors to the left palatal fissure ( Fig. 2 ). The mean width of the artificial cleft on the alveolar and nasal sides was 15 mm and 7 mm, respectively. To confirm the condition of the prepared alveolar cleft, maxillary occlusal radiographs were taken by dental radiography (SANKO X-ray MFG) with instant film for occlusal radiography (Hanshin Technical Laboratory, Ltd., Nishinomiya, Hyogo, Japan) under standardized conditions (50 kV, 10 mA, 0.8–1.0 s) immediately after operation. To close the operative wound, the ablated mucoperiosteal flap was folded back to cover the bone margin and sutured. Finally, a gauze tamponade, containing gentamicin sulfate ointment, was applied to the defect. During the operation, flomoxef sodium (1000 mg; Flumarin ® ; Shionogi Co., Ltd., Osaka, Japan) as an antibiotic, methylprednisolone sodium succinate (125 mg) as an anti-edematous drug, and ketoprofen (50 mg) as an analgesic were administered. A soft diet was fed to rest the operative wounds for 2 weeks after implantation. Oral cefcapene pivoxil hydrochloride hydrate was used postsurgically for 3 days. Three days after the operation, the gauze tamponade was removed.
Four months after preparation of the artificial maxillary alveolar cleft, randomly selected experimental dogs were divided into the OCP/Col-treated group ( n = 6) and the collagen-treated group ( n = 3), and implant surgery for OCP/Col or collagen disks was performed under general anesthesia ( Fig. 3 ). An incision on the vestibular side was made along the gingival border and the crest of the prepared alveolar ridge. Posteriorly, the incision was extended to the canine teeth where it was angled up into the sulcus of the second premolar. Anteriorly, the incision was extended along the gingival border to the center of the first incisor. On the palatal side, mucoperiosteal flaps were raised along the edges of the cleft. Wide exposure of the cleft area was achieved with these incisions. Mucoperiosteal flaps were raised to identify the edges of the floor of the nasal cavity and left palatal fissure. To confirm the condition of the prepared alveolar cleft, maxillary occlusal radiographs were taken with instant film under the same conditions (50 kV, 10 mA, 0.8–1.0 s) as before preparation of the recipient bed. The artificial maxillary alveolar cleft was re-prepared by removing surplus regenerated bone, and the recipient bed was prepared, after which, 15–20 disks of OCP/Col were implanted into the artificial maxillary alveolar cleft (OCP/Col-treated group) just to the volume of the recipient bed ( Fig. 3 ). The lateral mucoperiosteal flap was advanced to cover the cleft and was sutured to the palatal flap. As a control, 25 collagen disks were implanted into the artificial maxillary alveolar cleft (collagen-treated group). Maxillary occlusal radiographs were taken, under the same conditions, after preparing the recipient bed, and immediately after implantation of OCP/Col or collagen.
Tissue preparations and radiographic analysis
Four months after implant surgery, the dogs were killed by intravenous injection of an overdose of sodium pentobarbital, followed by intramuscular ketamine hydrochloride. The maxilla and surrounding tissues were resected and fixed with 10% formalin neutral buffer solution, pH 7.4, by infiltration for 4 weeks at 4 °C. The excised maxillae were radiographed by microradiography (Softex CMBW-2; Softex, Tokyo, Japan) with X-ray (FR; Fuji Photo Film, Tokyo, Japan) under standardized conditions (40 kV, 5 mA, 60 s), and neither OCP/Col nor collagen showed radio-opacity.
Radiomorphometric analysis
The radiographs taken after fixations were scanned. An area of interest was defined and the percentages of radio-opacity within the area of interest (radio-opacity percentage) were quantified as follows ( Fig. 4 ). First, a perpendicular line was drawn from the apex of the left maxillary first incisor ( A ) to the midsagittal line, and the intersection was defined as A 0 . Second, a perpendicular line was drawn from the apex of the right maxillary third incisor to the midsagittal line, and the intersection of the midsagittal line and the mesial side of the left maxillary canine were defined as M and C 0 , respectively. Third, a tangential line was drawn from the crown of the left maxillary first incisor to that of the left maxillary canine, and their intersections were defined as A 1 and C 1 , respectively. Finally, an enclosed area between A 1 , A , A 0 , M , C 0 , C 1 , and A 1 was defined as the area of interest on condition that from A 1 to A and from C 0 to C 1 and was traced outward form of tooth. Radio-opacity percentage was calculated as the radio-opaque area in the area of interest/the area of interest × 100 in the OCP/Col-treated and collagen-treated groups.
Statistical analysis
Radiomorphometric data were analyzed using a computer software package (Excel v.X; Microsoft Co., Redmond, WA, USA). All values were reported as the means ± standard deviation (SD). The χ 2 test was applied to examine whether each group had a normal distribution, then the F -test examined whether the standard deviations of two normally distributed groups were equal or not. The mean values of the OCP/Col-treated and collagen-treated groups were compared using unpaired t -tests. Statistical significance was accepted at p < 0.05.
Histology
Each sample was decalcified in formic acid and sodium citrate solutions for 12 weeks at 4 °C. The samples were dehydrated in a graded series of ethanol, embedded in paraffin and sectioned horizontally at a thickness of 10 μm. The sections were stained with hematoxylin and eosin, and photographs were taken with a photomicroscope (Leica DFC300 FX; Leica Microsystems Japan, Tokyo, Japan).
X-ray diffraction
OCP/Col implants in the dog maxilla were examined with X-ray diffraction (XRD). A specimen was collected after 4 months from an area that corresponded to OCP/Col implantation. XRD samples were obtained before implantation of OCP/Col, OCP, HA, collagen, and the maxillary bone of a dog. Powder XRD patterns were recorded using step-scanning at 0.02° intervals from 3.5 to 80°, with Cu Kα X-rays on a diffractometer (Mini Flex; Rigaku Electrical Co., Ltd., Tokyo, Japan) at 30 kV, 15 mA. The XRD patterns of the implanted OCP/Col were compared with the maxillary bone of a dog, OCP/Col disks, OCP granules, HA granules, and collagen disks.
Results
Macroscopic view
The prepared alveolar cleft before implantation of OCP/Col or collagen disks (4 months after initial preparation) became markedly hollow toward the palatal and apical sides and easily compressible. The oral vestibule of the alveolar cleft became narrow. Four months after OCP/Col implantation, the OCP/Col-treated alveolus was obviously augmented and the depth of the oral vestibule became increased; the implanted OCP/Col was united with the surrounding bone and had no mobility. In the collagen-treated alveolus, the hollowed alveolar ridge and narrowed oral vestibule was against the prepared alveolus before implantation ( Fig. 5 ).
