Histological and histomorphometrical comparative study of β-tricalcium phosphate block grafts and periosteal expansion osteogenesis for alveolar bone augmentation


This study evaluated local tissue reaction around the β-tricalcium phosphate (β-TCP) block and compared results with β-TCP block grafting and periosteal expansion osteogenesis (PEO). The mandibular premolars were extracted from five dogs and buccal corticotomy was performed. Narrow alveolar ridge models were created at 4 weeks. The β-TCP block graft, such as veneer graft, was used on the right side and PEO using β-TCP block on the left side. Changes of alveolar width, histological findings and histomorphometrical analysis were evaluated. There were no problems with materials at any of the sites at any time. In both groups, the width increased after surgery and results were stable 8 weeks after surgery. Newly formed bone tissue was observed inside the β-TCP block in both sides. Histological findings differed especially at the division between mandibular bone and β-TCP block. Histomorphometric analyses revealed that β-TCP had been absorbed (mean decrease 28%) and new bone had formed (mean increase 43%) at 8 weeks postoperatively on both sides. The β-TCP block worked as a space-maker under the soft tissue, including the periosteum, and acted as a substitute for original bone. This bone substitute was effective material for bone augmentation in both methods.

Alveolar bone augmentation is one of the standard treatments for dental implantation when height and/or width of alveolar bone are insufficient. Such insufficient volume can be overcome by augmentation procedures, with or without guided bone regeneration techniques . Autografts are considered the gold standard for the reconstruction of maxillofacial bone , but autogenous bone-grafting can involve donor-site morbidity and resorption of the grafted bone , and this technique cannot be used for simultaneous soft tissue augmentation so the amount of bone augmentation is usually limited.

Recently, bone substitute materials have been advocated as alternatives to autografts and allografts . Highly purified β-tricalcium phosphate (β-TCP) has attracted attention because of its biocompatibility and biodegradability . During the bone remodeling process, β-TCP is gradually degraded and finally replaced by mature new bone in animal experiments . This material acts as an osteoconductive material; it allows osteoprogenitor cells to grow on its surface or within its pores and differentiate into osteoblasts, eventually leading to bone deposition . This material has been shown to have good biocompatibility and osteoconductivity in the clinical setting .

Distraction osteogenesis (DO) is an alternative method for increasing bone volume before implant placement . This method utilizes a biological process in which new bone formation occurs between segments that are gradually separated; bone formation continues as long as the strained tissue is incrementally activated . This gradual traction of pedicled bone fragment is followed by simultaneous osteogenesis (bone) and histiogenesis (functional soft tissue matrix) . Adequate bone volume and maturity in the distracted region may provide a better implant success rate than following bone-grafting due to greater bone resorption expected in the long term . There is still significant disagreement about various treatment parameters such as surgical technique, type of distraction device and minimal bone height and width necessary to perform the distraction.

Mechanical strain is also known to induce subperiosteal bone formation. Recent in vivo studies have shown that tensile strain on the periosteum, which causes tenting of the subperiosteal capsule, is sufficient to produce bone formation, without corticotomy or local harvesting of the bone . These studies indicate a new technical aspect of DO or tissue expansion, with the controlled guided formation of new bone.

In a previous report, the authors investigated the utility of periosteal expansion osteogenesis (PEO), based on the concept of DO, using a highly purified β-TCP block, instead of an original bone segment, in a dog model . The highly purified β-TCP block worked as an activator of soft tissue, including the periosteum, and as a space-maker to induce an osteoblastic response in the periosteum.

The purpose of this study was to evaluate the local tissue reaction around and inside the β-TCP block, and to compare different techniques of β-TCP block grafting and PEO in a dog model. The authors also investigated the stability of bone volume and determined whether PEO and the β-TCP block may serve as alternative techniques to generate sufficient volume of hard tissue to allow stable osseointegration of dental implants.

Materials and methods

Preparation of the β-TCP block

β-TCP (OSferion) was obtained from Olympus Terumo Biomaterials (Tokyo, Japan). Fine β-TCP powder was synthesized by wet milling (a mechanochemical method). Calcium-deficient hydroxyapatite (HA) was obtained by milling dibasic calcium phosphate dihydrate and calcium carbonate at a molar ratio of 2:1 with pure water and zirconium beads, followed by drying at 80 °C. This crystalline solid was converted to β-TCP by calcination at 750 °C for 1 h. Upon sintering the β-TCP powder at 1050 °C for 1 h, a porous β-TCP block was obtained, which was then characterized by assessment of the surface area and pore size distribution of the porous structure. The porous blocks (15 mm × 10 mm × 3 mm) were manufactured at extraordinarily high purity.

Surgical protocol

The protocol and guidelines for this study were approved by the Institutional Animal Care and Use Review Committee of Kyushu Dental College, Kitakyushu, Japan. Five female beagle dogs (weighing 10–15 kg) were used. The animals were anaesthetized by intramuscular administration of ketamine hydrochloride (50 mg/kg), followed by diazepam (5 mg) and atropine sulfate (0.5 mg), without endotracheal intubation. Before the operation, 10 mg/kg pentobarbital sodium was injected intravenously. Immediately after the operation, the dogs received cefazolin sodium (20 mg/kg) subcutaneously, which was continued until postoperative day 3. The operation was performed under standard sterile conditions, and local anaesthesia using 2% lidocaine with epinephrine was used during all surgical procedures. The mandibular premolars were extracted and a buccal corticotomy was performed in each dog. Narrow alveolar ridge models were created at 4 weeks, when wound healing was complete. The experiments were designed in a split-mouth manner. That is, a veneer graft using a β-TCP block was performed on one side (graft side) and PEO using a β-TCP block was performed on the other side (PEO side).

On the graft side, a horizontal incision around the mucogingival junction was made and the mucoperiosteal flap was reflected, exposing the lateral surface of the mandible. The β-TCP block was placed at the bone surface, and two titanium screws (diameter, 2.0 mm; length, 4–6 mm; Medartis, Basel, Switzerland) were inserted at the inferior border of the block to avoid inferior displacement ( Fig. 1 ). Corticotomy was not performed for the lateral surface of the mandibular bone. On the PEO side, the β-TCP block was placed at the bone surface and two titanium screws were inserted at the inferior border of the block to avoid inferior displacement. Two more titanium screws (diameter, 2.0 mm; length, 12 mm; Medartis) were inserted from the lingual aspect, to push the block to the buccal side ( Fig. 2 ). After checking the movement of the block, the screws were turned back to the initial position. After a latency period of 8 days, during which primary wound healing occurred, the lingual screws were activated to push the β-TCP block laterally and expand the lateral soft tissue by 0.5 mm/day for 6 days.

Fig. 1
Intraoperative photograph. The block was placed under the mucoperiosteum. Two titanium positioning screws were placed at the inferior border of the block.

Fig. 2
The β-TCP block was displaced laterally by pressure from the titanium screws at PEO side.

8 weeks after lingual screw adjustments were halted, the dogs were killed using an intramuscular administration of midazolam and ketamine and an intravenous injection of pentobarbital sodium. Both mandibular experimental areas, including peripheral soft tissues and the β-TCP blocks, were removed carefully.

Evaluation of changes in alveolar bone width

Alveolar width was measured using bone calipers (YDM, Tokyo, Japan). The measurement point was 3 mm inferior from the top of the alveolar ridge and the middle point of the β-TCP block. These measurements were made before the operation (T1), immediately after the operation (T2), after expansion (T3), and before death (T4).

Tissue preparation and histological evaluation

All resected materials, including the β-TCP block were fixed in 10% neutral buffered formalin for at least 7 days. The titanium screws were removed from the mandibular bone. Each specimen was divided into two by a vertical cut through the middle. One specimen was decalcified, embedded in paraffin, sectioned at a thickness of 3 μm, and stained with hematoxylin and eosin (HE). The second specimen was fixed in 70% ethanol without decalcification, and immersed in Villanueva bone stain solution (Maruto, Tokyo, Japan). The specimen was dehydrated through a graded ethanol series, embedded in methylmethacrylate (Wako, Pure Chemical Industries, Osaka, Japan), and cut into 5-μm sections.

Histomorphometric analyses

Histological and histomorphometric analyses were performed using a light microscope with a digital camera (Olympus DP12; Olympus, Tokyo, Japan) and digital image editing software (Adobe Photoshop CS2; Adobe, San Jose, CA). Morphometric measurements were made on three vertical sections per specimen, closest to the centre of the β-TCP area. The area of an unused β-TCP block was measured as a control.

Statistical analyses

Nonparametric analyses were used because blocks were used on the same individual and because the experimental groups were not large enough to use parametric analyses. The Wilcoxon t -test was used for intergroup analyses (graft vs. PEO). P values < 0.05 were deemed to be statistically significant.


No complication involving the materials was observed at the sites of intervention, before, during, or at the end of the experimental phase. Infection within or around the β-TCP block was not observed. The alveolar form at the experimental region changed dramatically following lateral expansion with the β-TCP block, and the amount of augmentation was significantly different.

Changes in alveolar width are shown in Table 1 . Alveolar width increased after surgery in both groups. In the PEO group, the alveolar width increased further following lateral expansion. Both groups showed stable results at week 8 after surgery.

Table 1
Changes of alveolar width.
T1 T2 T3 T4
Graft side 2.9 ± 0.3 6.4 ± 1.0 6.5 ± 0.8 6.2 ± 1.1
PEO side 2.7 ± 0.4 6.3 ± 0.9 10.7 ± 1.1 10.0 ± 1.3 (mm)

On the graft side, newly formed bone tissue was clearly observable inside the β-TCP block ( Fig. 3 a ). Osteoblasts lining the new bone and osteoids adjacent to the β-TCP were observed in HE-stained, decalcified specimens. Prominent new bone formation, including osteoblastic cells and a considerable number of osteocytes, was also observed in the β-TCP ( Fig. 4 ). No fibrous or granulation tissue was observed between the β-TCP block and the new bone. A few multinucleated giant cells were observed adjacent to osteoblastic cells in this area. No fibrous tissue layer was observed between the pre-existing bone and the β-TCP block; direct contact with the lateral surface of the bone was apparent. At the border of the block and bone, newly formed bone had progressed into the pores of the block ( Fig. 5 a ).

Fig. 3
Histological findings with HE stain. New bone formation was observed around the β-TCP block, as well as the replacement of large parts of the β-TCP with newly formed bone. (a) Graft side. (b) PEO side. Original magnification 1×.

Fig. 4
Prominent new bone formation was observed on the β-TCP. There was no fibrous tissue between the β-TCP (TCP) and the newly formed bone (NB) (HE staining). Original magnification 200×.

Feb 8, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Histological and histomorphometrical comparative study of β-tricalcium phosphate block grafts and periosteal expansion osteogenesis for alveolar bone augmentation
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