Impact of simultaneous placement of implant and block bone graft substitute: an in vivo peri-implant defect model

Background Insufficient bone volume around an implant is a common obstacle when dental implant treatment is considered. Limited vertical or horizontal bone dimensions may lead to exposed implant threads following placement or a gap between the bone and implant. This is often addressed by bone augmentation procedures prior to or at the time of implant placement. This study evaluated bone healing when a synthetic TiO2 block scaffold was placed in circumferential peri-implant defects with buccal fenestrations. Methods The mandibular premolars were extracted and the alveolar bone left to heal for 4 weeks prior to implant placement in six minipigs. Two cylindrical defects were created in each hemi-mandible and were subsequent to implant placement allocated to treatment with either TiO2 scaffold or sham in a split mouth design. After 12 weeks of healing time, the samples were harvested. Microcomputed tomography (MicroCT) was used to investigate defect fill and integrity of the block scaffold. Distances from implant to bone in vertical and horizontal directions, percentage of bone to implant contact and defect fill were analysed by histology. Results MicroCT analysis demonstrated no differences between the groups for defect fill. Three of twelve scaffolds were partly fractured. At the buccal sites, histomorphometric analysis demonstrated higher bone fraction, higher percentage bone to implant contact and shorter distance from implant top to bone 0.5 mm lateral to implant surface in sham group as compared to the TiO2 group. Conclusions This study demonstrated less bone formation with the use of TiO2 scaffold block in combination with implant placement in cylindrical defects with buccal bone fenestrations, as compared to sham sites.


Background
Dental implants are commonly used to replace missing teeth. Implants were originally placed in healed alveolar ridges following physiologic bone resorption after tooth extraction [1]. With the change towards prosthetically-driven implant placement, the ideal implant position may present challenging bone architecture which may render exposure of implant threads or a gap between the alveolar bone and the implant. A peri implant gap of less than 2-2.25 mm often leads to spontaneous healing as demonstrated in several studies [2,3]. For gaps exceeding 2 mm or exposed implant threads, the use of a graft material has been recommended, but there is a controversy as to which material is superior [4].
Autologous bone grafts are considered the gold standard for bone augmentation, but are associated with donor site morbidity and unpredictable graft resorption [5][6][7]. Allografts and xenografts are widely used and well documented [8], but run the risk of disease transmission from graft to host [9]. Synthetic bone graft substitutes circumvent many disadvantages with auto-, allo-and xenografts, which may lead to its increased use also reflected by recent regulatory changes by the European Union [10].
Clinical procedures combining vertical bone augmentation and implant placement are often performed in two stages. Currently, only few studies have reported on the use of block grafts in circumferential peri-implant defects in conjunction with implant placement, such as the bone ring technique [11,12]. However the technique is scarcely reported in the literature, lacking control groups and only reporting short follow-up times [13]. Gaikwad et al. suggested that results from animal studies on autogenous bone rings were not sufficiently robust to support this technique in humans [14]. Previous studies on the bone ring technique are also widely heterogeneous with regard to graft materials, surgical protocols and peri-implant defect size and shape. As described by Botticelli et al., circumferential defects with a gap distance to the implant of up to 2.25 mm heal spontaneously, but if a concomitant buccal dehiscence was present, healing was incomplete [2].
This study evaluates a porous ceramic TiO 2 bone graft substitute in a peri-implant defect model with a buccal dehiscence. TiO 2 scaffolds have documented biocompatible properties with compressive strength similar to trabecular bone [15,16]. The average pore size close to 400 μm, high porosity and interconnectivity gives a favourable pore architecture for osteogenesis [17][18][19]. Studies in minipigs have demonstrated bone ingrowth and angiogenesis within the structures of the TiO 2 scaffolds in extraction sockets and in peri-implant dehiscence defects [18,19].
In a buccal dehiscence model by Botticelli et al. particulate graft materials were assessed [20]. Using a synthetic block material may be more advantageous in such a model as targeted porosity and interconnectivity can be designed, which is impossible with the use of particulate grafts. Therefore, in the present study a TiO 2 block scaffold was used simultaneous to implant placement for bone augmentation of 2.5 mm circumferential defects with buccal dehiscences in an experimental minipig model.
The aim of this experimental study was to evaluate the use of a TiO 2 block scaffold in the bone ring technique for peri-implant bone defects with buccal fenestrations, without the use of barrier membranes, to evaluate bone healing.

Methods
Ceramic TiO 2 scaffolds were fabricated by polymer foam replication as described by Tiainen et al. [16]. TiO 2 slurry was prepared by gradually dispersing 65 g of TiO 2 powder (Kronos 1171, Kronos Titan GmbH, Leverkusen, Germany) cleaned with 1 M NaOH into 25 ml of sterilised H 2 O and stirred at 5000 rpm for 2.5 h. The slurry was adjusted to pH 1.5 during the stirring with 1 M HCl. Cylindrical polyurethane foam templates (60 ppi, Bulbren S, Eurofoam GmbH, Wiesbaden, Germany) were soaked in the slurry and squeezed to remove excess slurry. The polymer sponge was burned out before sintering at 1500°C for 20 h. After sintering, the scaffolds were immersed with a slurry containing 40 g of powder dispersed in 25 ml of sterilised water. Excess slurry was removed by centrifugation and the second coating was sintered at 1500°C for 20 h. The finished scaffolds were 7.0-7.5 mm in diameter and 5 mm in height. Prior to use, the scaffolds were steam sterilised at 121°C for 20 min.

Animals
Six female minipigs (Göttingen minipig, Sus scrofa, Ellegaard AS, Dalmose, Denmark) aged 27-32 months and weighing 42-51 kg were used. The animals were kept in a centre for large experimental animals at the Veterinary Teaching Hospital Rof Codina in Lugo, Spain. The animals were kept on a soft diet and subjected to oral hygiene by mechanical cleaning once every 3 weeks during the experimental study. The Regional Ethics Committee for Animal Research of the University of Santiago de Compostela approved the protocol (Ref. AE-LU-001/ 002/14).

Experimental design
Mandibular premolars were extracted 4 weeks before implant placement. Mucoperiosteal flaps were bilaterally reflected and teeth carefully removed after tooth separation. Primary healing was accomplished by mattress sutures. Prophylactic administration of cefovecin sodium (8 mg/kg body weight S.C. S.I.D., Convenia®, Zoetis, Spain) was given postoperatively.
Four weeks after extraction, full thickness flaps were raised on the buccal and lingual side and the alveolar crest was levelled to create a flat surface for osteotomies. In each mandibular quadrant, two implant osteotomies were prepared. Thereafter, peri-implant defects were made with a trephine bur, 8 mm in diameter and 5 mm in height (Meisinger Bone Management® Systems, Hager & Meisinger GmbH, Neuss, Germany). The defects were placed with a minimum of 10 mm between the osteotomy centres, and at least 6 mm from neighbouring teeth. The defects were made so that the buccal portion of the defect wall was missing. Dental implants (OsseoSpeed TX 3.0 S 11 mm, Dentsply) were placed in the centre of the defect with the coronal 5 mm of the implants exposed.
The treatment allocation was assigned by a split mouth design where sham and TiO 2 alternated between anterior and posterior location for every other animal. For the sites allocated to treatment with the TiO 2 scaffold, a cylindrical block scaffold was perforated in the centre, creating a donut-shaped scaffold to fit around the implant and shaped to fit the defect with burs or scissors. The scaffold was secured in the defect by pressfit installation before the flap was passively adapted and primary wound closure was obtained (Fig. 1). After 12 weeks of healing, the animals were euthanized using a lethal dose of sodium Pentothal (40-60 mg/kg/i.v., Dolethal, Vetoquinol, France) and the mandibles were dissected and fixed in formalin.

Micro-computed tomography scanning
Microcomputed tomographic imaging (microCT) of the samples was performed (SkyScan 1172, Bruker microCT, Kontich, Belgium) with a resolution of 7 μm, using 100 kV and 100 μA, with 3 average images every 0.4 degrees for a 360-degree rotation with an aluminium and copper filter. The data were reconstructed using NRecon software (Bruker microCT, Kontich, Belgium) with ring artefact correction of 10%.
The defect fill volume was analysed for a volume of interest (VOI) slightly smaller than the scaffold, to avoid possible errors at the interfaces of the scaffold's outer and inner border (Fig. 2). The VOI was a cylindrical donut-shaped area with a height of approximately 2.3 mm (including the first ten upper threads of the implant) and a diameter of 6.0 mm concentric with the implant. Individual thresholds was set to measure the defect fill of bone and scaffold. Percentage of defect fill was analysed in CTan (Bruker microCT, Kontich, Belgium).

Histological preparation
The tissue blocks were dehydrated in different graded ethanol series (70-100%), and infiltrated with four different graded mixtures of ethanol and infiltrating resin, glycometacrylate (Technovit 7200® VLC, Heraus Kulzer GMBH, Werheim, Germany). The samples were then polymerised, first under low intensity UV light for 4 h, followed by high intensity UV light for 12 h. Finally, the samples were placed in an oven at 37°C for 24 h to assure a complete polymerisation. Longitudinal sections in bucco-lingual direction were obtained by cutting with a band saw and mechanically micropolished (Exakt Apparatebau, Norderstedt, Germany) with silicon carbide papers until a thickness for approximately 50 μm was obtained. The slides were stained with Levai Laczko for both histological examination and histomorphometric analysis.
All sections were observed using light microscopy and a PC-based image capture system (BX51, DP71, Olympus Corporation, Japan) and histometrically analysed. Quantitative histology was performed by a masked examiner using PC-based image analysis programs: Cellsens 1.13 (Olympus Corporation, Japan) and Image Pro-Premier 9.0 (Media cybernetics, Bethesda, MD, USA).

Histological analyses
The images were painted with a digital tablet (Cintiq, Wacom, Japan) and Photoshop CS (Adobe, USA). The region of interest was the buccal and lingual rectangles defined from the top of the implant to the bottom of the defect created and from the surface of the implant to the lateral portion of the defect. The fraction of possible bone fill within each rectangle was calculated, excluding scaffold material in the TiO 2 group (Fig. 3a).
Vertical linear measurements were made for two distances: 1) Implant shoulder to the first bone contact (first bone-to-implant contact, FBIC) and 2) Implant shoulder to the first bone contact 500 μm lateral to the implant (FBIC500) (Fig. 3b). Percentage of bone-toimplant contact (%BIC) was calculated for the first 5 mm from the implant top.
Horizontal bone growth within the defect was measured from the implant surface to the first bone contact in the buccal and lingual direction. Measurements were done in millimetre increments from the top of the implant to defect bottom 5 mm apically. The defect dimensions were set as reference points, hence when no bone was seen the value was set at a maximum 3 mm (Fig. 3c).
All measurements were done for both the buccal and lingual side of the implants using ImageJ.
(ImageJ 1.52a, National Institutes of Health, USA). Linear measurements of soft tissue dimensions were performed for the shortest distance from implant shoulder to the oral cavity using ImageJ (Fig. 4a). The area of soft tissue above the implants was measured in Photoshop CS6 (Fig. 4b). One sample was excluded from the analysis due to missing soft tissue following the histological preparation.

Statistics
Comparison across the two groups was performed using a paired t-test between contralaterals when normality was assumed and Wilcoxon Signed Rank Test when normality test failed. All statistical analyses were performed using SigmaPlot 14 (Systat Software, San Jose, CA, USA). Statistical significance was set at the 0.05 level.

Results
Post-operative healing following surgery was uneventful. Five implants presented small mucosal perforations to the oral cavity at the time of harvest (Fig. 3c), four in the TiO 2 scaffold sites and one in the sham sites.

MicroCT
The apical portion of the implants placed in sound bone showed osseointegration at all sites, but the defect areas showed large variation of bone fill. The largest variations were seen in the TiO 2 scaffold sites. One site was missing most of the scaffold and was subsequently excluded from further microCT analysis. Two sites demonstrated a fracture of approximately one fourth of the scaffold, one of which was missing while the other remained in the defect. The remaining nine scaffolds maintained their structural integrity. All but the excluded scaffold demonstrated bone growth within the porous scaffold structures in various degrees. As for the sham sites,

Histology
All implants were osseointegrated. New bone formation was clearly distinguishable from the original bone and the defects were readily identified. Except for one sham site (Fig. 5a), no buccal bone wall was completely reestablished to the level of the implant shoulder in the defects. The TiO 2 scaffold which was in part missing on MicroCT showed no contact between the TiO 2 scaffold and the bone in histology (Fig. 5d). The remaining 11 scaffolds were in intimate contact with the bone and demonstrated new bone formation in various degrees within the porous scaffold structures.

Defect fill
Results are presented in Fig. 6

Soft tissue measurements
Results are presented in Fig. 8. No statistically significant differences were found between sham and TiO 2 for either shortest soft tissue distance to oral cavity from implant shoulder or area of soft tissue above the implant.

Discussion
This study demonstrated less bone formation on the buccal side with the use of TiO 2 scaffold block along with simultaneous implant placement in circumferential defects with buccal bone fenestrations, as compared to sham sites.
In microCT, no significant difference was seen in comparing %DF. However, the region of interest analysed for %DF was different in histomorphometry and microCT. The microCT analysis set out to evaluate a 3D volume within the scaffolds at the upper part of the implants, excluding the interface between the scaffold and adjacent structures. This volume of interest was chosen to evaluate the space within the porous scaffold structures at the upper part of the implant. As previously demonstrated, bone growth occurs from the defect borders [21]. The 3D microCT analysis did not differentiate between buccal and lingual sites.
The fact that differences in defect fill, bone to implant contact and vertical FBIC500 were observed in the buccal but not lingual sites points to the impact of the lack of a buccal bone wall in this model. The TiO 2 scaffold did not improve bone healing at this aspect. A similar defect model in dogs by Botticelli et al. demonstrated an incomplete healing when the buccal bone wall was removed [2]. In comparison, they found a defect resolution in four wall defects for both gap distances evaluated (1 and 2.25 mm). This demonstrated buccal bone fenestrations as a challenging model. In contrast to the aforementioned study by Botticelli et al., no collagen membrane was used to cover the defects in the present study. Guided bone regeneration (GBR) is defined as the use of a barrier membrane to direct the growth of new bone and has become a predictable therapeutic method used routinely [22,23]. Barrier membranes should maintain the space for bone formation and exclude soft tissue invasion of the defect and are commonly used in augmentation procedures. In parallel with its barrier function, the membrane plays an active role in hosting and modulating the molecular activities during GBR [24]. However, their clinical efficacy is debated [25]. Several studies have shown defects healed both with and without membrane for immediate implant placement in four wall defects [26][27][28]. When used in the bone ring technique, studies have reported no benefits of a membrane [29,30]. As such, it was opted not to use a membrane in the present study to avoid confounding factors and to assess the bone regeneration capacity of the TiO 2 scaffolds.
Barrier membranes are also used to ensure the stability of bone graft materials and it can be argued that this aspect is not necessary in a block graft, which furthermore is non-resorbable. Additionally, comparison with the use of empty defects would be cumbersome with the use of membranes as the inadequate mechanical properties of a collagen membrane would likely render insufficient space maintenance in the relatively large defects, compared to the TiO 2 scaffolds. Closing the site without the use of a membrane covering the scaffold material resulted consequently in a direct contact between the gingival flap and the TiO 2 scaffold. Soft tissue measurements were performed on histological samples to evaluate any effect of the TiO 2 scaffold on soft tissue and no differences were found in neither buccal nor lingual sites. The results may indicate that the scaffolds were well tolerated by the soft tissue, even when suspended over the scaffold ridge in the buccal sites. A non-resorbable membrane with enhanced mechanical strength could have been considered and may have had a positive influence on the bone reconstruction and soft tissue dimensions. In the present study, nine out of twelve TiO 2 scaffolds were intact at the end of the study. The new bone formation within the porous structures in eleven out of twelve scaffolds may indicate sufficient mechanical stability of the scaffolds for bone healing. Fractured scaffold edges were not found as previously reported [31]. This can be explained by three-wall defects in the present study, which provide stability and shelters the scaffold from external forces in contrast to its use in lateral bone augmentation models. The aforementioned study also used a fixation screw to secure the block, which resulted in partial scaffold fracture around the screw due to stress concentration. The press-fit installation of the scaffold may have provided more even stress distribution for the brittle TiO 2 scaffolds, thereby maintained the augmented volume throughout the evaluated period. In both studies, the scaffold allowed easy chairside adaptation with dental burs or scissors. This enabled efficient shaping of the scaffold to fill the augmented space, which is advantageous for bone graft substitute materials [10]. In comparison, particulate graft materials often require a flexible membrane for mechanical stabilisation, which also limits the expandable space. Alternatively, a stiff barrier like titanium meshes needs to be manually bent and shaped in a time consuming process and ill adapted meshes increase the risk of mucosal rupture and exposure of the mesh [32].
Mucosal perforations to the oral cavity were found clinically at five sites, four of which in the TiO 2 scaffold group. An implant cover screw, which is temporarily used to close the implant lumen during osseointegration, could have potentially resulted in less mucosal perforations. However, bone growth was confirmed within the porous scaffold structures at all but one of the TiO 2 scaffold sites. As it was a challenging defect model, a longer healing time might show continued bone growth, and the single time point evaluated is a limiting factor of this study. In comparison, when GBR is performed in a two staged approach, implant placement is carried out from 6 to 12 months after GBR surgery [33].
The results suggest that the TiO 2 scaffolds hold the basic requirements for a bone substitute; osteoconductive, biocompatible, space-making capability and volume maintenance [34], also confirmed in previous in vivo studies [18,19,31]. However, the TiO 2 scaffolds are lacking osteoinductive properties as bone formation is seen only from the lateral borders of the grafts, originating from the parent bone. Different strategies have been proposed to improve new bone formation and in vitro studies applying various coatings to the scaffold have shown possibilities for increased osteogenic potential [35][36][37]. More recently, cationic doping of TiO 2 scaffolds with Ca, Sr or Mg have shown ion release in addition to the benefit of increased mechanical strength [38]. The released Mg resulted in significantly increased osteogenic differentiation. As the ions Ca, Sr and Mg influence bone biology [39][40][41], doping could be a prospect for further investigation.

Conclusions
Less bone formation was observed on the buccal side with the use of TiO 2 scaffold block in combination with simultaneous implant placement in circumferential defects with buccal bone dehiscences, as compared to sham sites.