A basic tenant of successful osseointegration is that the implant resides in a sufficient quality and quantity of bone to ensure bone contact and thus stabilization. A prospective, randomized controlled preclinical trial was conducted to evaluate the bone-to-implant contact (BIC) when placing implants in bone regenerated by 3 different combinations of biphasic calcium phosphate (BCP). Dental implants were placed into the regenerated ridges of 6 female foxhounds; the ridges were reconstructed with different formulations of BCP in combination with an hydroxyapatite collagen membrane. They were retrieved after 3 months to perform light microscopic and histomorphometric analyses. Implants in each group appeared to be stable and osseointegrated. Light microscopic evaluation revealed tight contacts between the implant threads with the surrounding bone for all 4 groups. The mean BIC ranged from 64.7% to 73.7%. This preclinical trial provided clinical and histologic evidence to support the efficacy of all 3 formulations of BCP to treat large alveolar ridge defects to receive osseointegrated dental implants.
Introduction
The basic tenant of successful osseointegration is that the implant resides in a sufficient quality and quantity of bone to ensure bone contact and stabilization. Over the years, dental implants demonstrated a high success rate.1
Biphasic calcium phosphate (BCP) has been investigated extensively and has emerged as a promising bone substitute material.2–10 This grafting material consists of fully synthetic hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP), mixed at various ratios. The slowly resorbing HA acts as stable volume maintainer for bone regeneration, whereas the fast dissolution of β-TCP releases calcium and phosphorous ions that stimulate new bone formation.
Several studies have shown that BCP is a biocompatible and effective bone replacement substitute in sinus augmentation, and implants placed in BCP grafted sites demonstrate a similar survival rate compared to other grafting materials.2,3,5,6 However, there are limited citations available for the application of BCP to treat large alveolar ridge defects.9,11 The goal of this study was to evaluate bone-to-implant contact (BIC) when implants were placed in bone regenerated by different combinations of BCP.
Materials and Methods
This prospective, randomized controlled preclinical trial utilized 6 female foxhounds to analyze the dental implants placed in bone regenerated with different formulations of BCP.
Study animals and biomaterials
The study protocol was approved by the Institutional Animal Care and Use Committee at PARF in Massachusetts. Six female hounds (age 2–3 years, weight 20–24 kg) which were bred exclusively for biomedical research purposes, were obtained from a licensed vendor. They were acclimated for 2 weeks prior to the research commencement, and were fed an appropriate diet with ad libitum access to water. The biomaterials utilized for this study were alloplastic BCPs in different formulations together with a barrier membrane:
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Group A (Osteon I, Dentium Co, Ltd, Seoul, South Korea): 70% HA and 30% β-TCP (particle size 0.5- to 1.0-mm, porosity 77%, macropore size 300–500 μm, crystallinity 97%, crystal size 0.059 μm); 7 sites.
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Group B (Osteon II, Dentium): 30% HA and 70% β-TCP (particle size 0.5- to 1.0-mm, porosity 70%, macropore size 250 μm, crystallinity 97%, crystal size 0.043 μm); 7 sites.
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Group C (Osteon II collagen, Dentium): a mixture of 30% HA, 70% β-TCP (particle size 0.5- to 1.0-mm, porosity 70%, macropore size 250 μm, crystallinity 97%, crystal size 0.043 μm), and collagen; 92% graft and 8% collagen by volume; 7 sites.
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Group D: negative control (no grafting).
The barrier membrane used in bone grafting was HA-collagen membrane (Genoss Co, Ltd, Suwon, South Korea).
General and local anesthesia
All surgical procedures were performed under general and local anesthesia in sterile conditions. Xylazine hydrochloride (2.2 mg/kg, intramuscularly) and tiletamine hydrochloride/zolazepam hydrochloride (10 mg/kg, intramuscularly) were administered initially, followed by inhalation of 1.5% to 2% isoflurane as a general anesthesia for the duration of the procedure. Local anesthesia (2% lidocaine with 1:100 000 epinephrine) was provided at the surgical sites.
Surgical extraction and defect creation
The bilateral mandibular first, second, third, and fourth premolars (P1–P4) were extracted. Standardized alveolar ridge defects (approximately 12 mm mesiodistally and 10 mm apicocoronally) were then created by removing the buccal plate and leaving the lingual wall intact. Periosteal releasing incisions were made, and the flaps were adapted to achieve tension-free, primary closure. There was a healing period of 2 months after the surgery to eliminate any spontaneous bone regeneration.
Ridge augmentation procedure
Two months after extraction and alveolar ridge defect creation, ridge augmentations were performed to repair the defects (Figure 1a). Each site was randomized to receive 1 of 4 treatment modalities (groups A–D). A crestal incision was made for surgical access, followed by mucoperiosteal flaps to expose the mental foraminae. The graft materials were condensed into the defects according to a randomized distribution pattern so that no two adjacent sites received the same material. HA-collagen membranes were used to cover all sites and were stabilized with stainless steel pins (Dentium). Tension-free primary flap closure was achieved by periosteal releasing incision and interrupted sutures. The animals received the standard postsurgical infection and pain control consisting of cefazolin sodium (20 mg/kg, intramuscularly) and buprenorphine HCl (0.02 mg/kg, intramuscularly). The sutures were removed after 14 days, and the animals were fed a soft diet during the entire healing period and treatment phase.
Figure 1a. A healing period of 2 months was allowed following the creation of chronic bony defects before each site of the jaw was randomized to receive 3 different bone grafting materials (groups A, B, and C), in addition to a negative control site (group D, defect only). Hydroxyapatite-collagen membranes were used to cover all grafted sites and were stabilized with stainless steel pins. Figure 1b. The surgical sites were allowed to heal for 3 months prior to the implant placement. Graft consolidation was noted in all 3 groups, but some grafted sites were distinguishable from the surrounding host bone due to the visible presence of remaining graft particles. Figure 1c. Trephine burs were used to harvest a bone core from each site, and the dental implants were then placed into the trephined sites. Figure 1d. Dental radiographs taken 3 months after the implant surgery revealed the presence of radiopaque granules surrounding the dental implants without noticeable bone loss. Figure 2a. Group D (negative control) specimen demonstrated the maintenance of the crestal bone level up to the cover screw and histologic features that were indicative of successful osseointegration. Figure 2b. A high magnification of the implant threads demonstrated tight contact with the surrounding bone.
Figure 1a. A healing period of 2 months was allowed following the creation of chronic bony defects before each site of the jaw was randomized to receive 3 different bone grafting materials (groups A, B, and C), in addition to a negative control site (group D, defect only). Hydroxyapatite-collagen membranes were used to cover all grafted sites and were stabilized with stainless steel pins. Figure 1b. The surgical sites were allowed to heal for 3 months prior to the implant placement. Graft consolidation was noted in all 3 groups, but some grafted sites were distinguishable from the surrounding host bone due to the visible presence of remaining graft particles. Figure 1c. Trephine burs were used to harvest a bone core from each site, and the dental implants were then placed into the trephined sites. Figure 1d. Dental radiographs taken 3 months after the implant surgery revealed the presence of radiopaque granules surrounding the dental implants without noticeable bone loss. Figure 2a. Group D (negative control) specimen demonstrated the maintenance of the crestal bone level up to the cover screw and histologic features that were indicative of successful osseointegration. Figure 2b. A high magnification of the implant threads demonstrated tight contact with the surrounding bone.
Implant placement
The surgical sites healed for 3 months at which time radiographs and photographs were taken. Full thickness flaps were elevated to expose the regenerated ridges (Figure 1b). The osteotomy was prepared by a 2.4-mm diameter trephine bur (Dentium) with the bone cores harvested for histologic analysis, and the results were presented in a previous report.11 The submerged implants had a sandblasted, large grit, and acid-etched surface (Dentium Superline) with a diameter of 3.6 mm and length of 10 mm (Figure 1c).
Histologic staining and histomorphometric analysis
The animals were sacrificed 3 months after the implant placement (Figure 1d), and the samples were fixed in 10% formalin and submitted for histologic analysis.
Fixed samples were dehydrated in a graded series of ethanol (60%, 80%, 96%, and absolute ethanol) using a dehydration system with agitation and vacuum. The blocks were infiltrated with Kulzer Technovit 7200 VLC-resin (Heraeus Kulzer, Wehrheim, Germany). Infiltrated specimens were placed into embedding molds, and polymerization was performed under blue and white light. Polymerized blocks were sectioned in a mesiodistal direction and parallel to the long axis of each implant. The slices were reduced by microgrinding and polishing using an Exakt grinding unit (EXAKT Advanced Technologies GmbH, Norderstedt, Germany) to an even thickness of 30–40 μm. Sections were stained with Sanderson's rapid bone strain and counter-stained with acid fuchsin and examined using both a Leica MZ16 stereomicroscope and a Leica 6000DRB light microscope (Leica Microsystems, Glattbrugg, Switzerland). Histomorphometric measurements were performed by using a software (ImageAcess, Imagic, Glattbrugg, Switzerland) to calculate the percentage of mineralized bone, soft tissue components (connective tissue and/or bone marrow), and residual graft particles along the bone-implant-contact surface.
Statistical analysis
Means and standard deviations were calculated for all quantitative data. Due to the sample size, collected data of each group were compared by Kruskal-Wallis test with Mann-Whitney U test for pairwise comparisons after Bonferroni correction to adjust for multiple statistic testings. P < .05 was considered to be statistically significant. The statistical analysis was performed using a commercially available software program (SPSS for Windows, version 19.0, IBM Corp, Somers, NY).
Results
No adverse events were observed during the implant healing stage. All implants appeared to be stable and osseointegrated (Figures 2a through 3f). They were surrounded by radiopaque granules without noticeable bone loss upon radiographic evaluation (Figure 3a through c). Light microscopic evaluation revealed tight contacts between the implant threads with the surrounding bone for all 4 groups, indicating successful osseointegration (Figures 2b and 3d through f).
Dental implants from tested groups (groups A, B, and C) demonstrated the maintenance of the crestal bone level up to the cover screw and histologic features that were indicative of successful osseointegration. High magnifications of implants threads (Figure 3d and f) revealed tight contact with the newly regenerated bone. Graft remnants can be noted in all 3 groups (Figure 3a,d = group A, Figure 3b,e = group B, Figure 3c,f = group C).
Dental implants from tested groups (groups A, B, and C) demonstrated the maintenance of the crestal bone level up to the cover screw and histologic features that were indicative of successful osseointegration. High magnifications of implants threads (Figure 3d and f) revealed tight contact with the newly regenerated bone. Graft remnants can be noted in all 3 groups (Figure 3a,d = group A, Figure 3b,e = group B, Figure 3c,f = group C).
Histologically, all implants demonstrated osseointegration. Light microscopy revealed excellent BIC in all groups. The mean BIC was 64.7% (group A), 73.7% (group B), 68.2% (group C), and 83.1% (group D) with some residual graft particles present away from the implant surface (Figure 4). No statistically significant difference was noted among groups A through D in terms of BIC (P = .173, Kruskal-Wallis test). New bone was found in contact with the implant surfaces with less BIC in the marrow space of the mandible. The newly formed bone was dense with normal bone marrow spaces and blood vessels.
Discussion
Previous studies have shown that BCP bone substitute material can be successfully utilized to reconstruct large alveolar bone defects.9,11 This study provided clinical and histologic evidence that regenerated bone can lead to dental implant osseointegration with survival of all implants.
BCP has been widely used as an augmentation material in sinus lift studies.2,3,5,6,12 Cha et al12 reported 95.56% cumulative survival rate in 45 implants, with the 80/20 HA/β-TCP (BCP) material. Lee et al3 used 50/50 HA/β-TCP (BCP) as bone substitute in the sinus lift and observed a 98.46% cumulative success rate after a mean period of 12 months. Tosta et al13 showed 100% implant survival rate in 15 patients using BCP. Taken together, our result demonstrated BCP is suitable and excellent material for large alveolar defect regeneration and excellent bone-to-implant contact resulting in osseointegration.
It has been shown that the ratios between HA and β-TCP significantly affect the resorption rate of BCP in vivo. The degradation rate of β-TCP is about 3–12 times faster than HA. Therefore, a higher HA ratio will result in a longer degradation time and more residual graft particles.14 A ratio about 20/80 HA/β-TCP stimulated earlier and more new bone formation.14,15 Our observation indicates more new bone formation with reduced residual matrices in group B, which is consistent with the above investigation.11
Although more new bone formation is related to a higher β-TCP ratio in BCP material, it was not clear how it will translate into improved BIC. All 3 experimental groups (groups A through C) demonstrated a high BIC (more than 60%), but without a statistical difference among different HA/β-TCP formulations. Albrektsson16 suggested a minimum of 50% BIC to ensure successful long-term loading of osseointegrated implants. All 3 groups demonstrated higher results and therefore meet the criterion.
Conclusion
This preclinical trial provides clinical and histologic evidence to support 3 formulations of HA/β-TCP to be efficacious bone substitutes for regenerating large alveolar ridge defects to receive osseointegrated dental implants.
Acknowledgment
This study was sponsored by a grant from Dentium and Genoss.
