Evaluation of osteoinductive calcium phosphate ceramics repairing alveolar cleft defects in dog model

Abstract

BACKGROUND:

Alveolar cleft repair is an important step in the sequence of treatments for cleft lip and palate. Intrinsically osteoinductive materials have been the subject of research interest.

OBJECTIVE:

The aim of this study was to explore the use of osteoinductive biphasic calcium phosphate (BCP) ceramics to repair alveolar cleft defects in dogs.

METHODS:

We prepared two kinds of BCP ceramic with different physical characteristics: osteoinductive BCP (OBCP) and non-osteoinductive BCP (NBCP). Bilateral alveolar cleft models were surgically established in dogs. On one side, OBCP was implanted in the defect; on the opposite side NBCP was implanted as a control. The materials were also implanted in the femoral muscles to test their properties at non-osseous sites. The osteogenic ability of materials was evaluated with imaging, spiral CT, histology and fluorescent dye tests.

RESULTS:

At the muscular implantation sites, new bone formed in all of the OBCP samples, but none in the NBCP samples. Imaging and spiral CT revealed good appearance and continuity of the alveolar cleft postoeration, with normal eruption of the bilateral permanent teeth in the groups. Histological and fluorescent dye testing revealed new bone formation in both groups in situ. However, earlier osteogenesis initiation and bone remodeling were superior with OBCP. Osteogenic process in the intramuscular samples with OBCP was similar to that seen in situ.

CONCLUSIONS:

Our findings indicated that osteoinductive biphasic calcium phosphate ceramics (OBCP) have superior characteristics in alveolar cleft repair compared with non-osteoinductive calcium phosphate ceramics (NBCP).

Keywords

Osteoinductive calcium phosphate ceramic alveolar cleft

1. Introduction

Alveolar cleft repair is an important step in the sequence of treatments for cleft lip and palate [1]. Bone substitutes and their potential applications have been the subject of research interest in alveolar bone grafting [2–4].

For decades, intrinsically osteoinductive calcium phosphate theory was beginning to gain popularity. Increasing data have shown that the optimization of physical and chemical properties could make the calcium phosphate ceramics have intrinsical osteoinductity which can form ectopic bone in non-osseous tissues without the addition of any growth factors or cells [5–8].

Therefore, intrinsically osteoinductive calcium phosphate ceramics may be a better candidate as a bone substitute than other options. This is the first study to investigate the clinical effects of osteoinductive biphasic calcium phosphate (OBCP) ceramics for alveolar cleft repair in a surgically established dog model, and meanwhile, the two groups materials were implanted in the femoral muscle the difference between in situ and ectopic bone formation. We want to gain more insight into the processes: (i) early and later-term results of in situ bone defect repair, (ii) in situ versus ectopic bone formation, and (iii) the impact of implanting material on the eruption of adjacent teeth.

2. Materials and methods

2.1. Preparation of materials

The starting apatite powders were prepared with a wet precipitation method [5,6]. With stearic acid as macroporous pore former, 30% hydrogen peroxide as microporous pore former and 4 wt% polyvinyl alcohol as binder, experimental group, OBCP, were sintered according to a certain process. At the same time, control group, NBCP were made by the mixing formula without hydrogen peroxide and changing the sintering process.

2.2. Animal experiments

2.2.1. Animal choice

Dogs have periodontal and craniofacial structures and bone healing mechanisms similar to those of humans [9,10] and dogs at 6 months of age are also experiencing dental transition equivalent to that of 9 to 11-year-old children, which is the age when bone grafting surgery is generally performed in children with alveolar defects [11,12]. Obviously, the previous adult dog models of permanent dentition defects could not truly simulate the clinical situation of hard and soft tissues in children [13,14]. Therefore, in our study, we used puppies (3 months of age) to surgically establish the alveolar cleft model, and performed a second surgery during the dental transitional period (6 months of age).

Nine beagle dogs (all male, 95 to 100 days old, weighing 6.9 to 8.0 kg) were provided by the Dongguan Songshan Lake Pearl Laboratory Animal Science and Technology Co., Ltd. The experimental protocol was approved by the Ethics Committee of Second People’s Hospital of Shenzhen, China.

2.2.2. First-stage surgery to establish the cleft model

Nine 3-month-old beagle puppies were anesthetized with 3% sodium pentobarbital at a dose of 1 mg/kg. The bilateral third milk incisors were extracted and a fissure bur was used to remove a bone segment from the distal aspect of the second milk incisor to the mesial aspect of the milk canine tooth, leaving the nasal floor mucosa intact. Mucoperiosteal flaps were sutured to cover the mesial and distal bone wounds to form bilateral alveolar cleft models (Fig. 2(a)).

2.2.3. Second-stage surgery to repair the alvelar cleft

The second-stage surgery was performed 3 months after cleft modeling. The dogs were anesthetized with 3% sodium pentobarbital at a dose of 1 mg/kg. In each dog, OBCP was implanted in one side alveolar cleft and NBCP on the other side. Simultaneously, a muscle pocket was made in the bilateral femoral muscles, with implantation of OBCP in one pocket and NBCP in the other (Fig. 2(b)).

Three dogs were sacrificed at each evaluation time point postoperation (4, 8 and 12 weeks). Bone and teeth adjacent to the grafting region of the maxillary alveolar cleft were sectioned and the materials in the femoral muscles were collected.

2.3. Sample analysis

2.3.1. Fluorescent dye labeling

Fluorescent dye was injected intravenously to dogs in 3 consecutive days before the observation times: xylenol orange at 4 weeks postoperation, tetracycline hydrochloride at 8 weeks postoperation and calcein 12 weeks postoperation [7]. Hard-tissue sections were made to observe new bone formation under a fluorescence microscope.

2.3.2. Histomorphological analysis

Ectopic samples in muscle: Materials implanted in the femoral muscles were prepared as decalcified sections for observation of new bone formation.

In situ samples in alveolar cleft: Bone fragments and adjacent teeth were removed from the grafting region of the maxillary alveolar cleft, followed by conventional fixation, gradient alcohol dehydration and methylmethacrylate embedding. These samples were then made into undecalcified sections with the EXAKT cutting and grinding hard tissue system (E300CP/400CS/AW; Germany) at the Affiliated Stomatological Hospital of Guangzhou Medical University, China. These sections were dyed with Masson staining at the laboratory of Southern Medical University, China. Under a light microscope, bone formation in the grafting region and adjacent periodontal changes were observed.

All samples were photographed under 10× light microscopy; three sections from each sample and three consecutive fields of view from the edge to the center were used to calculate average values for each section [6]. All data were processed with ITK-SNAP version 3.0.0 at the Medical Imaging Center of the Chinese University of Hong Kong to calculate the ratio of new bone area to implant material area (b%) at different time points after implantation of OBCP and NBCP.

2.3.3. Spiral CT

Spiral CT were made immediately postoperatively and 4, 8, 12 weeks after surgery to observe alveolar ridge morphology, new bone height and width and bone resorption. Changes in maxillary volume were calculated with ITK-SNAP version 3.0.0 based on three-dimensional maxillary reconstruction.

2.4. Statistical analysis

Histomorphological and CT data were statistically analyzed with SPSS 18.0. All implants were analyzed with a paired t-test at an α level of 0.05, and results of this analysis indicated a significant difference ( $P < 0.05$ ).

3. Results

3.1. Physical and chemical properties of the materials

X-ray diffraction analysis showed that the BCP ceramics consisted of 60 hydroxyapatite/40β-tricalcium phosphate. The porosity of both ceramics was approximately 70%, as determined with the Archimedes water displacement method. Scanning electron microscopy (SEM) showed a pore size of 300 to 500 μm with interconnecting macropores in both ceramic, but the microstructure were significantly different. In OBCP group, the crystal was small and connected with each other just at the narrow neck, and there were a lot of micropores between the crystals on the macropore wall. Whereas, on the macropore wall in NBCP, due to the different mixing formula and sintering process, the crystal was big and connected with each other at all sides to become a total plate without any micropores. So, the images of SEM showed that OBCP and NBCP have the different microphysical structures despite the same chemical composition (Fig. 1).

Fig. 1.

XRD and SEM. ((a) XRD; (b) OBCP, a lot of micropores on the macropore wall; (c) NBCP, few micropores on the macropore wall.)

3.2. Gross observation

12 weeks after material implantation, the bilateral alveolar ridges showed increased fullness, a well-recovered appearance and arc shape, deepening vestibular sulcus, and no resorption or sinking of implanted materials (Fig. 2(c)). The wounds had healed well and no infection occurred in any of the experimental dogs.

Fig. 2.

Implantation procedure in alveolar defect. ((a) Preoperative, arrows showed the alveolar defect; (b) implant BCP in the alveolar defect; (c) 3 months postoperative, arrows showed arch shape the alveolar ridge.)

3.3. Fluorescent dye labeling

Fluorescent indicators deposit on the bone mineralization front through chelation with calcium ions. With this technique we can observe an obvious fluorescent marking on the trabecular bone surface of non-decalcified sections, which elucidates the status of new bone formation [15]. A confocal microscope (Leica TCS SP8, 81-1005) from South University of Technology was used to observe new bone formation after in situ repair. 12 weeks postoperation, a ring-shaped arrangement of red, yellow and green phosphorescent stripes was observed in both groups. Xylenol orange is red fluorescence, tetracycline is yellow fluorescence and calcein is green fluorescence (Fig. 3). The fluorescence results was in accord with histological examination findings. The red fluorescence was visible in both groups, indicating that there was new bone forming as early as 4 weeks after in situ repair and suggested that we may find more difference if setting the earlist observation time at 2 weeks or even 1 week postoperatively [16].

Fig. 3.

Sequential fluorescence of 12 weeks postoperative. (red: xylenol orange injected at 4 weeks; yellow: tetracycline at 8 weeks; green: calcein at 12 weeks.)

3.4. Histological examination

All sections were observed with a Nikon optical microscope at the Central Laboratory of Second People’s Hospital of Shenzhen.

3.4.1. In situ repair (alveolar cleft)

4 weeks after graft: New bone formation was significant difference between in OBCP and NBCP, both in mesial and distal material pores adjacent to autologous bone. In OBCP, calcified bone tissues tightly adhered to the material pores adjacent to autologous bone, and sesame seed-like osteocytes were present. A large amount of red-dyed collagen, which is non-calcified bone matrix, with marrow cavity-like shape and small vessels, was visible at the edge of the inner surface of the new bone (Fig. 4(a)). In the material pores distal to autologous bone, although no calcified bone tissue was found, large osteoclast-like cells adhered to the pore walls and columnar osteoblasts were arranged along the pore edge and red-dyed bone matrix deposition (Fig. 4(b)). Whereas, in NBCP group, the new bone just slowly grown from the mesial pores to the distal pores. So, just a small amount of new bone tissue had grown from the edge of the autologous bone into the material pores, and consequently in the distal pore to autologous bone, nearly no osteoclast-like cells were found with no presence of osteoblasts or osteocytes (Fig. 4(e), (f)). It was noteworthy that the initiation of new bone formation in OBCP was obviously earlier than in NBCP.

Fig. 4.

Histologic examination of the two groups of BCP in the alveolar defect. (OBCP: (a) 10×, new bone grown into the pore, arrow showed the marrow cavity-like shape; (b) 40×, red-dyed bone matrix deposition, arrow showed the large osteoclast-like cells; (c) 10×, arrow show thicker trabecular bone formation; (d) 40×, bone grown into the entire material, arrow showed the haversian canal structure; NBCP: (e) 10×, less new bone grown into the pore, arrow showed the new bone; (f) 40×, no bone in the pore, arrow showed few large osteoclast-like cells; (g) 10×, arrow showed the thin trabecular bone formation; (h) 40×, arrow showed the haversian canal structure.)

8 weeks and 12 week after graft: In OBCP group, 8 weeks after implantation, plentiful new bone tissue had grown into the entire material implant to form a streak structure similar to bone trabeculae, indicating active osteogenesis (Fig. 4(c)). 12 weeks after implantation, osteogenesis had slowed, bone remodeling became evident and the material gradually degraded. Plentiful mature bone tissue filled the gaps and was integrated tightly with the residual material, accompanied by a mass of Haversian canal structure (Fig. 4(d)). In NBCP group, at 8 weeks, a large amount of new bone tissue grew from the edge of autologous bone into the material pores (Fig. 4(g)). At 12 weeks, plentiful mature bone tissue was found filling the material pores (Fig. 4(h)) and some Haversian canals were also visible. There is not obviously difference between the two groups.

Fig. 5.

Percentage of new bone formation. ( $^{*}$ : $p < 0.05$ , NS: no significance.)

Statistical analysis: The SPSS v18.0 package was used to analyze the ratio of new bone area to material area (b%) at different time points using a paired t-test at an α level of 0.05 (Fig. 5). We found that there was significantly more new bone present in the OCP group than in the NBCP group 4 weeks after implantation ( $P < 0.05$ ), whereas there was no significant difference between the two groups at 8 or 12 weeks ( $P > 0.05$ ).

3.4.2. Ectopic repair (femoral muscle area)

OBCP group: All samples showed osteoinduction at each time point. At 4 weeks after implantation, large, nuclear-stained, tightly arranged osteoblasts were visible inside some pores close to the material edge, accompanied by non-calcified bone matrix deposition. A large number of multinuclear, nuclear-stained, polygonal osteoclast-like cells were seen in other pores. Only a small amount of calcified new bone tissue had formed in some individual material pores. The performance is very similar to that in the distal pores in situ repair. At 8 weeks, new bone had formed in all pores, with the thickness of new bone tissue accounting for approximately one-fourth and up to one-third of the pore radius. This new bone tissue was covered with one or more osteoblasts, which were tall, cube-shaped and nuclear stained. At 12 weeks, new bone tissues with lacunae had thickened and formed streak trabecular bones that connected adjacent pores. However, a typical Haversian canal structure was not yet present. It is showed that ectopic bone formation was a typical intramembranous ossification and had the osteogenic process similar to in situ bone formation. But due to the different environment comparing with in situ, the whole process was delayed bone initiation, less new bone mass and later bone tissue maturation (Fig. 6).

Fig. 6.

Histologic examination of the two group of BCP in the muscle (OBCP, 4 weeks: arrow showed the ringed osteoblast; 8 weeks: arrow showed the new bone formed in all pores, 12 weeks: arrow showed the marrow cavity-like shape and thicker bone.)

NBCP group: No heterotopic new bone tissues were observed in any of the control samples at any observation time point. Only connective tissue and osteoid were visible (Fig. 6), indicating that the NBCP was non-osteoinductive.

3.5. Spiral CT observation

CT images revealed the bone bridge formation, with new bone fully integrated with existing bone tissue and no obvious boundaries (Fig. 7(a)). The vertical height of the new bone bridge was close to that of the original alveolar bone (Fig. 7(b), (c)). Three-dimensional reconstruction showed good recovery of the alveolar ridge in both groups after implantation, with normal eruption of the permanent second incisors and canine teeth bilaterally, confirming that the BCP material did not affect tooth movement (Fig. 7(d)).

Fig. 7.

CT images at 12 weeks postoperative. ((a) arrow showed the bone bridge formation and no obvious boundaries; (b and c) arrow showed the vertical height of the new bone bridge was close to that of the original alveolar bone; (d) 3D reconstruction alveolar ridge.)

Imaging data were processed with ITK-SNAP (version 3.0.0) and used to calculate graft volume on both sides (Fig. 8). Alveolar ridge size at different stages were evaluated with one-way analysis of variance using SPSS 18.0; a value of $P > 0.05$ indicated homogeneity of variance. Analysis of variance showed no significant difference between groups in alveolar process size at different time points ( $P > 0.05$ ). Changes in alveolar ridge size at each time point after implantation were analyzed with a paired t-test, and indicated no significant differences between the groups ( $P > 0.05$ ). This finding indicates that the alveolar ridge had a good shape after implantation. The BCP material maintained its scaffold shape until new bone formed, avoiding the early collapse (Fig. 9).

Fig. 8.

CT images were processed with ITK-SNAP (version 3.0.0) software to calculate graft volume. ((a) red line showed range of premaxillary bone under the nasal floor and rostral to the nasal palatal hole; (b) the calculate model.)

Fig. 9.

Alveolar graft volume at different time.

4. Discussion

4.1. OBCP ceramics showed earlier, faster and more bone formation in situ

The histological examination showed the early new bone formation started both in pores mesial and distal adjacent to autologous bone in OBCP group. In contrast, there is no new bone formation in the distal pore in NBCP group. Osteoinductivity of OBCP undoubtedly is responsible for the difference. Although, the exact mechanism underlying osteoinduction by materials is still largely unknown, it was thought that the special surface of pore in OBCP affected the local microenvironment and promoted the adhesion and differentiation of autologous mesenchymal stem cells into osteoprogenitor, thereby triggering new bone formation as early as possible. The material distal edge was far away from the host bone and osteoprogenitor cells absented, but the pericytes and endothelial cells from the capillaries and connective tissue ingrowth may function as resting stem cells and progenitor cells; as a result, they may differentiate into osteoblasts to form new bone under the conditions provided by the osteoinductive ceramic [17–20]. So, OBCP is superior to nBCP in the early stages of in situ bone formation.

4.2. Histological processes of ectopic and in situ bone formation were similar

This study is the first to compare ectopic (in the muscle tissue) versus in situ (in the alveolar ridge) bone formation after implantation of BCP ceramics. In OBCP group, We found that the histological processes of ectopic and in situ bone formation were similar, both involving osteoblast and osteoclast adhesion and bone matrix production and mineralization. This finding indicated that ectopic bone formation induced by OBCP is not a pathological calcification, but is the typical intramembranous with induction of cell differentiation on the specific surface of the material. The only difference between ectopic and in situ bone formation was the initiation time and speed of bone formation, which depended on the implant environment. The increased cytokines and osteoblasts around the alveolar defect that provide a better environment for bone formation than ectopic implant [21–23].

4.3. The OBCP repaired the shape of alveolar ridge and permitted the tooth eruption

Due to the special biological properties of alveolar bone, the bone substitute material must fully reconstructs gaps in the alveolar bone cleft with the required height, ensuring normal tooth eruption in the region of the bone graft. In the present study, the OBCP ceramic that was suitable for closure of alveolar clefts by increasing the β-tricalcium phosphate content and porosity to reduce its mechanical strength. This porous material with less strength is suitable for intraoperative shaping and filling, and cannot excessively inhibit permanent tooth eruption. The autogenous iliac cancellous bone graft as the gold standard for alveolar cleft repair. [24], its most common complication is bone graft resorption, leading to bone mass deficiency in the recipient region and poor surgical results, such as poor shape of the alveolar bone [25–27]. In this study, postoperative CT images and statistical analysis demonstrated OBCP can maintain its scaffold shape until new bone forms, avoiding shape collapse of the alveolar ridge. According to previous studies, 80 to 96% of cleft palate patients need to wear orthodontic appliances following canine tooth eruption [28–30]. For patients who have difficulty with tooth eruption or who are missing teeth, dentures or dental implants are preferred. Thus, tooth movement and eruption is an important factor when assessing whether an implant material can be used for alveolar cleft repair. The postoperative CT examination revealed normal eruption of the permanent lateral incisors and canines bilaterally, confirming that the BCP material did not affect tooth movement. The advantage maybe also an excellent properties for implant tooth in alveolar ridge defect with teeth lost. In the future, we will try using dental implants at the bone-grafting region to repair the tooth lost in alveolar clefts and restore the occlusion function.

5. Conclusions

Intrinsically osteoinductive BCP ceramics have more advantages over non-osteoinductive BCP for in situ repair of alveolar cleft defects in dog model with the earlier, faster and more bone formation, and repair the shape of alveolar ridge without collapse and permit the tooth normal eruption.

Footnotes

Acknowledgements

All data were processed with ITK-SNAP version 3.0.0 at the Medical Imaging Center of the Chinese University of Hong Kong by Miss Lening-Li.

Conflict of interest

The authors have no conflict of interest to report.

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