Evaluation of Vertical Bone Augmentation Using a Titanium Ring Retained in Simultaneous Implants With Early Exposure: A Pilot Study in Dogs


 To evaluate the performance of titanium rings retained in implants placed simultaneously for vertical bone augmentation with early exposure. Twenty-four bone level tapered dental implants were inserted in the premolar areas of the mandible with standardized, 3.5 mm deep, vertical bone defects in four beagle dogs, and titanium rings containing a mixture of autogenous bone chips and Bio-oss were retained in the implants for vertical bone augmentation. Three weeks after the operation, all titanium rings were exposed, one titanium ring from each mandible of each dog was removed, and the other titanium ring was left in place with daily flushing. After 2 months, the dogs were sacrificed, the height of vertical bone augmentation was measured, and bone samples containing the implants were harvested and analyzed. The median (minimum-maximum) bone augmentation height (BAH) was 2.46 (2.18-2.62) mm on the buccal side and 2.80 (2.66-3.02) mm on the lingual side in the removal group; however, the BAH was 0.64 (0.32-0.92) mm and 0.70 (0.22-1.22) mm in the holding group. The bone volume/total volume (BV/TV) of the increased bone was 80.07% (72.64%-87.89%) with micro-CT analysis in the removal group, ideal bone osseointegration formed, and new bone was observed in the hard tissue sections. Within its limitations, the use of titanium rings retained in implants was found to be a reliable alternative method for vertical bone augmentation, and removing titanium rings is better for bone augmentation when early exposure occurs.


Introduction
Alveolar bone defects resulting from either periodontal disease or dental trauma will increase the di culty of implant placement operations 1 . To overcome unfavorable changes in the height and width of alveolar bone, a plethora of bone augmentation techniques have been adopted for implant placement either in a simultaneous or a consecutive approach 2,3 . Among them, vertical bone augmentation is a more di cult way to acquire adequate alveolar ridges for successful dental implant placement 4,5 . In most cases, a healing period of at least 6 months has been suggested to ensure successful augmentation and accurate implant positioning during the second surgery 6 .
A variety of techniques have been used for vertical bone augmentation in patients with atrophic alveolar ridges, such as distraction osteogenesis, autogenous block grafting (bone block or bone ring), and guided bone regeneration (GBR) using titanium mesh or expanded-polytetra uoroethylene (e-PTFE) membranes [7][8][9] . Resorption of the bone segment and inclination of the distractor will decrease the e cacy of distraction osteogenesis 10 , and the mobilized fragment should be more than 4 mm thick 11 . High technical sensitivity and potential risks, including nerve damage or hypoesthesia bone segment displacement, premature consolidation, distractor device instability, transport bone segment or mandible base fracture, and segment nonunion, will limit the popularity of distraction osteogenesis 12 . On the other hand, although autogenous block grafts are considered the gold standard for vertical bone augmentation, the drawbacks of autogenous block grafts are also obvious, including restricted donor sites, possible harvesting morbidity, reports of unpredictable resorption and poor patient acceptance 13 . GBR with titanium mesh is an effective alternative method for vertical bone augmentation 14 . The successful reconstruction of large discontinuity osseous defects by a titanium mesh was reported in 1985 15 . This metal has been used extensively in numerous surgical applications because it is highly reactive and has a low weight and can be easily passivated to form a protective oxide layer, which accounts for its high corrosion resistance 16 . A low density of titanium provides high strength and lightweight dental implant bone augmentation barrier membrane material 17 . Titanium mesh has a de nite strength that can guide the control of the contour pro le of regenerated bone and maintain a relatively stable space compared to conventional barrier membranes 18 . Moreover, the good biocompatibility of titanium mesh can avoid any immune response 14 . Previous studies have shown that when titanium mesh is applied as a barrier membrane, bone augmentation can reach up to 10 mm vertically and horizontally, and the amount of long-term bone resorption is small 19,20 .
During the early phase of healing, su cient xation of the graft and barrier membrane is essential to achieve early revascularization and graft integration. In addition, micromovement has been demonstrated to result in the development of brous tissue rather than bone tissue 21,22 . As a type of GBR membrane, titanium mesh adapts to various bone defects through bending and shaping, which may increase the time of surgery and generate sharp edges 14 . Moreover, the space maintenance capacity of titanium mesh is based on self-superior positional xation, which could be achieved by using a retentive pin, but the use of a retentive pin would make the implant surgery di cult and more cumbersome, regardless of whether it was implanted or removed 18 .
Based on several previous studies, the main complication of titanium mesh is postoperative exposure following wound dehiscence, at a rate of 14.8%-66% 20,23−27 . As another kind of nonresorbable barrier membrane of GBR, e-PTFE leads to a higher rate of infection once exposed 28 , but infection does not necessarily occur after titanium mesh exposure 19,29 . Furthermore, how to address postoperative exposure of titanium mesh is controversial 17,30 .
In view of these problems, we have developed an alternative titanium ring technique using a personalized titanium mesh printed by three-dimensional (3D) printing technology. The titanium ring was xed by the implant through a retaining screw and could be personalized by 3D printing according to the shape of the bone defect, including horizontal or vertical bone defects. The aim of the present study was to assess the bone augmentation effect of the titanium ring technique and to compare different treatment methods (removing the titanium and removing the titanium with ushing) after early exposure after treating standardized bone defects in beagle dog mandibles.

Methods
Six male beagle dogs with a weight between 10 and 13 kg and a mean age of 14 months were used in the present study. The animals were housed in the Animal Facility for Oral and Maxillofacial Reconstruction and Regeneration Experiments, School of Stomatology, Southwest Medical University at Luzhou, China. The appearance, behavior, reactivity and social interactions of the dogs were monitored during the entire treatment period. Each animal was kept in a separate cage with a light:dark cycle of 12:12 h and without excessive or potentially startling noises. This study was approved by the Animal Experiment Ethics Committee of Southwest Medical University, Luzhou, China (No. 2021-0528-1), and it is reported according to The ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All methods were performed in accordance with the relevant guidelines and regulations.

Titanium Ring and Retaining Screw
The titanium ring of the present study was rst designed by 3-matic Research (Release 11 for Windows, Materialise, Leuven, Belgium) following the creation of the original STL format le. A unique design feature of the titanium ring was that its inner ring was designed as a frustum of a cone with a central hole, which matched with the nonhex implant with a morse taper (Dentium Superline, Ltd, Gyeonggi-do, Korea) and retaining screw (Fig. 6). A pore size of 500 µm was created at the top of the titanium ring, with no pores on the sides.
The 3D printer (M290, EOS, Munich, Germany) used a high-energy laser to melt the titanium metal powder into slices of the required three-dimensional shape, and then the sintering machine accumulated these slices layer by layer to obtain the required titanium ring. The retaining screw stemmed from the abutment screw (Dentium Superline, Ltd., Gyeonggi-do, Korea) and could easily secure the titanium ring to the implant after machining (Fig. 7). Subsequently, the nal titanium ring and retaining screw were checked and polished. Before grafting in beagle dogs, all products were sterilized by high temperature and high pressure, reaching the requirements of a surveillance standard for sterilization (ISO 15883:2006).

Surgical Procedures
The study was designed as a pilot experimental study. All surgeries were performed under general anesthesia, and surgical interventions were performed under systemic anesthesia using pentobarbital sodium (i.m. 30 mg/kg). In the rst surgery, three premolars (P2-P4 per hemimandible) were atraumatically extracted by root separation from both sides of the mandible. All animals received 125 mg Amoclav (clavulanic acid and amoxicillin, Hanvet Pet Health, Shanghai, China) as antibiotics intraoperatively and twice a day for 7 days after the surgery. Appropriate therapy was administered when the behavior of the animals indicated adverse reactions or pain. The sutures were removed 7 days after the surgery.
After 3 months of healing, the second surgery involved a midcrestal incision and full-thickness mucoperiosteal ap elevation. Two standard three-dimensional ridge defects of 7.2 mm (mesio-distal and buccal-lingual) to 3.5 mm (apico-coronal) were created on P2 and P4 of each side of the mandible.
Following the drill protocol of the manufacturer, the rst guide drill was navigated and xed 5 mm below the bone defect, and then the nal drill was used to enlarge the diameter of the hole to 2.8 mm at 50 rpm while the autogenous bone particles were harvested. Bone level implants (Ø 3.6 mm, length 8 mm) were inserted in the hole to 5 mm to achieve adequate primary stability, with 3 mm exposed. A titanium ring was secured to the implant with a retaining screw, and a combination of deproteinized natural bovine bone mineral (DBBM) (Bio-oss, geistlish Pharma, Wolhusen, Switzerland) and autogenous bone particles was used to ll the void between the implant and the titanium ring. The ap was relaxed by releasing the periosteum and then sutured to allow for submerged healing of all implants.
Normal saline was used to irrigate the wound once a day for 3 weeks after surgery without applying anesthesia. Unfortunately, all titanium rings were exposed 3 weeks after the operation due to the dehiscence of the ap (Fig. 8). Subsequently, the experimental sites were randomly divided into two groups, and each group included p2 of one mandible and p4 on the other in the same beagle dog. In the titanium ring removal group, the small ap of soft tissue was elevated to remove the titanium ring, and then the soft tissue was sutured again after normal saline irrigation at 4 weeks. No surgical treatments were carried out in the titanium ring nonremoval group (holding group). However, daily ushing was still performed with normal saline.

Sample Preparation
After an osseointegration period of 3 months, the animals were euthanized by an intramuscular overdose of pentobarbital sodium (100 mg/kg). Block specimens of the entire implant and surrounding bone (mesiodistal 3 mm) were harvested except for loose implants after elevating the full-thickness mucoperiosteal ap. Furthermore, the trimmed mandible bone blocks containing the implants were xed in 4% paraformaldehyde and analyzed by means of micro-CT and histology.

Micro-CT Analysis
The biopsies were nondestructively examined rst with a radiographic computed microtomography (micro-CT) system. The samples were placed in a vertical position to the direction of the radiation and scanned using a micro-CT system (Venus, VNC-102; PINGSENG Health care, Kunshan, China) as follows: X-ray source, 80 kV, 80 µA; distance from the source to the object, 90 mm; distance from the source to the detector, 410 mm: slice thickness, 0.08 mm; voxel size, 0.049×0.049×0.08 mm. The micro-CT images (270 slices) were then reconstructed using 3D structural analysis software (Avatar; PINGSENG Health care, Kunshan, China). Threshold values were set for the segmentation between 5400 and 22800 for the implant and 2500 and 5400 for the bone tissue. The region of interest (ROI) was selected corresponding to the dimensions of the titanium ring, with a diameter of 7.2 mm and the base at 3 mm from the implant shoulder (V1). Bone directly under V1 with a diameter of 7.2 mm and a height of 5 mm was measured separately (V2). Then, the bone volume (BV, mm 3 ), BV/total volume (BV/TV), bone surface/TV (BS/TV) and bone mineral density (BMD, g/mm 3 ) were determined in V1. Only BMD was calculated in V2.

Histological Preparation
First, the bone samples were xed for one week, and the xation liquid was replaced overnight. Following rinsing in running tap water for 24 hours, trimming, and dehydrating in ascending concentrations of ethanol from 70-100%, the bone samples were made transparent by using ETC-3 (Combine, Changsha, China) for less than 4 hours, and then the bone samples were processed for the production of undecalci ed ground sections and embedded in a mixture of methylmethacrylate, dibutyl violet phthalate and benzoylperoxide. The embedded tissue blocks were cut buccolingually along the implant axis using a slow-speed rotating diamond serrated blade (LEICA1600 VC-50, Frankfurt, Germany) into approximately 0.08 mm thick ground sections. The sections were stained with toluidine blue and photographed under a light microscope equipped with a digital imaging system (CellSens; OLYMPUS, Tokyo, Japan).

Statistical Analysis
To quantify the effects of the titanium rings on the outcomes, absolute values were used. Total bone was de ned as newly formed bone (including osteoid and woven bone) and residual graft bone. All of the quantitative variables were characterized using descriptive statistics (median, minimum and maximum) instead of means and standard deviations, and a normal distribution was not assumed considering the low sample size. Kruskal-Wallis nonparametric analysis of variance was applied to estimate the differences in BAH over the buccalingual aspects of the implants in the two groups. In addition, the difference in BAH over the two groups was evaluated by the Wilcoxon rank-sum test. The Wilcoxon signed-rank test was used to estimate the differences in BMD over the V1 and V2 and in BAH over the buccolingual aspects of the implant between the removal group and the holding group, respectively. P values < 0.05 were considered statistically signi cant. The statistical analysis was conducted using SPSS for Windows Release 26.0, standard version (IBM SPSS, Chicago, IL, USA).

Clinical Observations
The titanium ring t perfectly into the prepared defective sockets without any need for recontouring or adjustment, and the implants were installed passively to gain initial stability. All six animals survived uneventfully. All titanium rings were exposed three weeks after surgery. Two implants were lost due to infection caused by exposure of the titanium ring in the holding group, but no implants were lost or loosened, and no signs of infection were observed in the removal group. The remaining six implants in the holding group were stable with titanium ring exposure, and the eight implants in the removal group were covered by gingival tissue.

Bone augmentation height (BAH) measurement
The distance between the implant shoulder and the bottom of the surrounding bone was assessed at the buccal and lingual regions by using Vernier calipers (accuracy 0.02 mm) (Fig. 1). Negative values were given if the top of the buccal and lingual bone was above the implant shoulder. This, in turn, meant that more bone quantity was acquired for higher negative values. In each implant, three repetitions were performed to calculate the mean value for reducing the deviation, and buccal and lingual BAH with the augmented areas of the circumferential defects was de ned as 3 mm minus the calculated mean value.
Median values of BAH were measured (in mm) to be 2.46 (2.18-2.62) mm in buccal of the R group, 2.8 (2.66-3.02) mm in lingual of the removal group, 0.64 (0.32-0.92) mm in buccal of the holding group, and 0.7 (0.22-1.22) mm in lingual of the holding group. The difference between BAH was statistically signi cant in the buccalingual aspects of the implants in the two groups (P < 0.001). The removal group exhibited a higher BAH than the holding group (P < 0.001). Lingual signi cantly increased BAH in contrast to buccal BAH in the R group (P = 0.002). Nevertheless, comparing the buccal and lingual regions in the holding group, the difference in BAH was not statistically signi cant (P = 0.374) ( Table 1) (Fig. 2).

Micro-CT analysis
Due to severe infection and poor bone augmentation in the holding group, only 8 bone sample blocks of the removal group were analyzed by micro-CT and histology.
The micro-CT image displayed obvious bone resorption in the buccal bone augmentation area, which led to a buccal bone width of less than 2 mm (Fig. 3). A high level of trabecular bone details was found in the reconstructed 3D CT image, which showed the distinct structures of the native and newly formed bone around the titanium implants. In the native bone region, the cancellous bone structure showed large, rodlike, connected trabeculae with fusiform or oval voids in between. However, there were also some areas of incompletely matured bone with a disorganized structure comprised of thin and incompact trabeculae, which were mostly observed near the implant shoulder (Fig. 4).
As seen in Table 2, the BV value of V1 was between 69.67 and 84.39 in the R group, with a median of 76.81. In addition, the BV/TV values were all more than 70% for all implants in the R group, showing a predictable vertical bone augmentation effect. In the analysis of the BS/TV, the median and range were 3.16 and 3.06-3.83, respectively, which indirectly re ects the bone augmentation effect of the titanium ring. Furthermore, the BMD results showed that there was a signi cant difference between V1 and V2 (P =0.004).

Histological observation
In general, all samples in the removal group showed ideal osseointegration over the surface of the implant and no signs of in ammation in the area of the implant. The structure of the graft material was either completely replaced by newly formed cancellous bone or in the process of intensive bone remodeling. Good bone healing and consolidation were observed in very dense bone. The new bone, which was near the superior border of the implant in the lingual region, showed signs of minor resorption in the removal group (Fig. 5).

Discussion
In the present study, 16 mandible alveolar surgical sites were vertically augmented using a titanium ring with a 1:1 mixture of autogenous bone particles and DBBM in beagle dogs. The titanium ring was derived from titanium mesh. The application of titanium mesh in terms of GBR techniques has received increasing attention due to reports that document predictable and consistent results with this material for 3-dimensional bone reconstruction, whether using delayed or simultaneous implantation 31 . Titanium mesh has good mechanical and biological properties. Its high strength and stiffness maintain a highly stable space support for regenerating bone and tissue integration 22 , and its low electrical conductivity and noncytotoxicity show high and persistent corrosion resistance and no effect on cell proliferation 32,17 .
Apparently, as a kind of barrier membrane, titanium mesh can satisfy the basic principle of GBR 14 .
The key factor for the success of GBR with titanium mesh is the stabilization of bone grafts beneath the membrane 25 . Speci cally, as blood clot formation is the start of the bone healing process and the successful osseointegration of titanium implants 33 , the interaction between the blood clot and the rough titanium surface induces early cell recruitment 34 . A stable blood clot between the broken bone ends is considered one of the most important factors for guaranteeing the integrity of the blood vessel and supporting subsequent granulation tissue formation after implantation 35 . The xation methods of titanium mesh mainly include suture xation and titanium screw xation 17 . However, the retention of sutures is relatively weak, and movement of the titanium mesh may occur due to surrounding stress [36]. In addition, the effect of titanium screw placement on implantation and additional incisions for removing the titanium mesh and screws in subsequent surgery will increase the patient's trauma and the di culty of the surgery 30,37 . In the present study, a new type of retaining screw was used to x the titanium ring on the implant, which exhibited acceptable osteogenic responses.
Vertical bone augmentation with simultaneous implant placement using a titanium ring results in a substantial shortening of the overall treatment but may produce complications in the form of postoperative wound dehiscence as in the present study. Similar results can be observed with titanium mesh. It is thought that when vertical ridge augmentation is performed, the risk of exposure of the titanium mesh is high 38-40 . Early exposure of the bone graft to the oral cavity and exposure occurring within 4 weeks after bone augmentation have been identi ed as reasons for infection and bone graft loss, and the soft tissue associated with in ammation remains in place and impedes the ingrowth of bone even after the resolution of the in ammation 17,41 . However, other studies have reported that premature exposure to titanium mesh did not result in either infection or decreased bone augmentation 19,42 . In the present study, all of the titanium rings were exposed to the oral cavity at 3 weeks, and no infection or purulence was detected, although wound dehiscence was detected within the rst month. Previous authors have reported similar results, that premature exposure did not result in infection of the treated sites or decreased bone formation 42,43 . Nevertheless, several other investigators have demonstrated that "premature" exposure had a more signi cant in uence on new bone formation than late exposure 18, 44,45 . In any case, tension-free sutures without any blanching in the suture line or over the graft, improvement of the barrier membrane and adequate periosteal scoring are the most effective solutions to prevent titanium mesh exposure 30,42,46 . In this study, the titanium ring produced by 3D printing had sharp edges, which might be the cause of the postoperative exposure 47 . In addition, the rough surface of the titanium ring was susceptible to bacterial contamination and could have caused mechanical irritation of the mucosal aps 48,49 .
Currently, the management of titanium mesh exposure varies. A clinical retrospective study reported that the titanium mesh should be removed as soon as possible, and subsequent anti-infection operations should be conducted if early exposure occurs 50 . In contrast, the mean values reported in a systematic review found that when titanium mesh exposure occurred, mesh removal was necessary in only 20% of the cases 51 . Hartmann et al. 52 , in a clinical study, rst classi ed the postexposure titanium mesh according to the area of mesh exposed and veri ed the effect of each treatment. The authors suggested premature removal of the mesh, and consequent soft-tissue healing was necessary when a large area of mesh exposure appeared. In the present study, the postexposure titanium rings were removed 4 weeks after the operation in the removal group.
The process of osseointegration can be summarized as follows: formation of a coagulum and granulation tissue and then bone formation and remodeling 53 . In in vivo studies, hard tissue is present around the Bio-Oss at 4 weeks because the bone grafts can be gradually replaced by newly formed bone 54,55 . The new bone includes woven bone often combined with both parallel-bered and lamellar bone at 4 weeks 56 . After 12 weeks of micro-CT analysis in this study, the percentage of new bone formation was observed to range from 72.64-87.98% in the removal group. Furthermore, pseudoperiosteum and ingrowth of soft tissue were not detected at the treated sites, which may be related to the removal of the titanium ring and needs more experimental evidence for further con rmation. The samples in this study were collected at 3 months postoperatively. However, the BMD of V1 was lower than that of V2, which showed that calcium deposition in osteoids requires additional time.
From the analysis of the BAH, interesting observations were made. The lingual alveolar bone height was acquired at an ideal level 57 . However, obvious bone resorption was found on the buccal form micro-CT. In our experience, titanium ring de ciency may cause bone grafts and immature bone to be affected by perioral buccal muscle pressure during the healing period, which causes stress bone resorption instead of in ammatory bone resorption in the removal group 58 . In the holding group, the treatment site without removal of the titanium ring was exposed to the oral cavity for a long time and caused severe in ammation. The implant was loose or lost as a result of osseointegration failure and peri-implant bone loss. Thus, similar to titanium mesh, titanium ring exposure did not directly result in signi cant bone resorption 59 . All pores of the titanium ring were designed on the crest in this study, which allowed bacteria to easily enter once it was exposed.
The data have to be explained with caution because the design of this study allowed only an explorative statistical analysis and a basis for future studies. Some limitations need to be taken into account regarding this animal study. Future studies should improve the design of the titanium rings and their surface preparation to prevent titanium exposure in order to evaluate the success of titanium rings.

Conclusions
Within the study limitations, the use of titanium rings retained in implants was found to be a reliable alternative method for vertical bone augmentation, and removal of the titanium rings is better for bone augmentation when early exposure of titanium rings occurs. Q.Q.L. and J.Y. contributed equally to this work.

Competing interests
The authors declare no competing interests.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics declarations
This animal experiment was approved by the Animal Ethics Committee of Southwest Medical University (reference number SWMU20210372).

Consent to publish
Manuscript is approved by all authors for publication