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Immediate implant placement in conjunction with guided bone regeneration and/or connective tissue grafts: an experimental study in canines

Purpose This study was conducted to assess the effect of hard and/or soft tissue grafting on immediate implants in a preclinical model. Methods In 5 mongrel dogs, the distal roots of P2 and P3 were extracted from the maxilla (4 sites in each animal), and immediate implant placement was performed. Each site was randomly assigned to 1 of the following 4 groups: i) gap filling with guided bone regeneration (the GBR group), ii) subepithelial connective tissue grafting (the SCTG group), iii) GBR and SCTG (the GBR/SCTG group), and iv) no further treatment (control). Non-submerged healing was provided for 4 months. Histological and histomorphometric analyses were performed. Results Peri-implant tissue height and thickness favored the SCTG group (height of peri-implant mucosa: 1.14 mm; tissue thickness at the implant shoulder and ±1 mm from the shoulder: 1.14 mm, 0.78 mm, and 1.57 mm, respectively; median value) over the other groups. Bone grafting was not effective at the level of the implant shoulder and on the coronal level of the shoulder. In addition, simultaneous soft and hard tissue augmentation (the GBR/SCTG group) led to a less favorable tissue contour compared to GBR or SCTG alone (height of peri-implant mucosa: 3.06 mm; thickness of peri-implant mucosa at the implant shoulder and ±1 mm from the shoulder: 0.72 mm, 0.3 mm, and 1.09 mm, respectively). Conclusion SCTG tended to have positive effects on the thickness and height of the peri-implant mucosa in immediate implant placement. However, simultaneous soft and hard tissue augmentation might not allow a satisfactory tissue contour in cases where the relationship between implant position and neighboring bone housing is unfavorable.


INTRODUCTION
Clinicians are confronted with several options when performing tooth extraction, ranging from ridge preservation techniques to a conventional protocol that awaits complete soft and hard tissue healing. Among these options, immediate implant placement (IIP) has been proposed to offer advantages to patients and clinicians by minimizing the number of required interventions and reducing the overall treatment time. IIP was initially introduced to prevent changes in the ridge profile following tooth extraction using extraction socket-sized implant dimensions [1]. However, evidence has demonstrated that IIP cannot prevent shrinkage [2,3]. As a result, numerous studies have analyzed various IIP protocols [4,5].
Considering that the esthetic zone is the main target area for the IIP protocol, a facial mucosal recession above a certain threshold (>1 mm) and/or a loss of mucosal volume can be detrimental and can even be considered as treatment failure [6,7]. To overcome potential volume loss, simultaneous grafting with connective tissue grafts, with or without concomitant bone augmentation, has been suggested. This resulted in a more stable facial mucosal margin, greater mucosal volume, and a higher pink esthetic score [8][9][10][11][12][13]. Unfortunately, the relevant parameters leading to a successful outcome have not been systematically evaluated, and the majority of the studies on this topic had a case series design. Moreover, there is a lack of histologic and histomorphometric data regarding soft tissue dimensions and composition after soft and/or hard tissue grafting.
Therefore, this preclinical study aimed to assess the effect of hard and/or soft tissue grafting on immediate implants in a preclinical canine model.

Animals
The present study was designed as a randomized controlled preclinical study that employed 5 mongrel dogs (>2 years old) weighing between 12 and 17 kg. All animals were kept in a purposedesigned room for experimental animals, provided a soft diet and access to water ad libitum, during the entire study period. The study protocol was approved by the Institutional Animal Care and Use Committee of Yonsei Medical Research Center, Seoul, South Korea (approval No. 2020-0085). This article was written in accordance with the ARRIVE guidelines [14].

Surgical procedures
All surgical procedures were performed under general anesthesia and sterile conditions in an operating room. General anesthesia was induced by an intravenous injection of atropine (0.04 mg/kg; Kwangmyung Pharmaceutical, Seoul, Korea) and an intramuscular injection of a combination of xylazine (Rompun ® ; Bayer Korea, Seoul, Korea) and ketamine (Ketara ® ; Yuhan Corporation, Seoul, Korea). Inhalation anesthesia (Gerolan ® ; Choongwae Pharmaceutical, Seoul, Korea) was maintained under monitoring of vital signs throughout the entire procedure. An intramuscular injection of antibiotics (20 mg/kg, cefazolin sodium; Yuhan, Seoul, Korea) was administered for 3 days post-surgery. Daily irrigation with 0.2% chlorhexidine solution (Hexamedin ® ; Bukwang Pharmaceutical, Seoul, Korea) was performed until suture removal. Sulcular incisions were made around the maxillary premolars (P2 and P3) and buccolingual flaps were reflected. Subsequently, P2 and P3 were hemi-sectioned and the distal roots were carefully extracted with no bucco-palatal luxating force, in order to preserve the buccal/ palatal bone plates. For the remaining mesial roots, pulpotomy was performed, and calcium hydroxide (Dycal ® ; Dentsply Sirona, York, PA, USA) was applied. https://doi.org/10.5051/jpis.2104040202

Contour augmentation at implant sites
https://jpis.org Subsequently, in the areas of the distal root of P2/P3, 2-piece dental implants (NR line Ø3.2×7 mm [diameter × length]; Dentium, Suwon, Korea) were placed, which led to the implant shoulder being at the level of the palatal bone crest and there being approximately 0.5-1.0 mm of space between the implant and the buccal bone wall. The healing abutments were then connected to the implants. The following 4 treatment modalities were then randomly assigned: i) guided bone regeneration (GBR) only (the GBR group), ii) subepithelial connective tissue grafting (SCTG) only (the SCTG group), iii) GBR + SCTG (the GBR/SCTG group), and iv) no other treatment (control). In the GBR group, biphasic calcium phosphate (Osteon™ III; Genoss, Suwon, Korea) was applied in the socket and on the outer surface of the buccal alveolus (width: between proximal areas of two adjacent teeth, height: 3-4 mm). The augmented area was covered with a cross-linked collagen membrane (collagen membrane P; Genoss). In the coronal part of the membrane, a hole was made using a punch and the membrane was secured by the healing abutment (Ø3.7×3.5 mm [diameter × length]; Dentium). The apical part of the membrane was fixed to the bone using 2 membrane tacks (Membrane Pin; Dentium). In the SCTG group, a subepithelial connective tissue graft was harvested from the palate. The width of the graft was approximately twice the diameter of the implant, with a height of 5 mm and a thickness of at least 1.5 mm. The graft was immobilized by suturing it onto the palatal flap. In the GBR/SCTG group, the subepithelial connective tissue graft was placed on top of the collagen membrane and secured to the palatal flap. In the control group, no additional tissue augmentation procedures were performed. Finally, the flaps were sutured and transmucosal healing was performed for all implants (Figure 1). The sutures were removed 14 days later.
At 4 months of healing, all animals were sacrificed with an overdose of anesthesia. Maxillary segments with augmented sites were then harvested.

Histologic processing
The dissected tissue sections were fixed in 10% neutral-buffered formalin. Subsequently, the specimens were dehydrated in a series of ethanol solutions and embedded in methyl methacrylate. A central bucco-oral section for each implant site was prepared at a thickness of 40-50 μm [15]. All sections were stained with Masson-Goldner trichrome staining.

Histomorphometric measurements
All histological slides were scanned (Panoramic 250 Flash III; 3DHISTECH, Budapest, Hungary) and analyzed using a specific software (CaseViewer ver. 2.1; 3DHISTECH). The following references were marked: margo mucosae (MM), implant shoulder (IS), first boneto-implant contact (fBIC), and buccal outline of the soft and hard tissue. Horizontal measurements were performed at 3 levels (at the level of the IS, 1 mm above and 1 mm below the IS).

Statistical analyses
Data are presented as mean ± standard deviation (SD), median, and quartiles. Due to the lack of HT in the majority of the specimens, statistical analysis was performed for MM-IS, MM-fBIC, and TT (SAS 9.4; SAS Institute, Cary, NC, USA). The sample size was too small for relevant statistical tests. The main goal was the description of the impact of the 4 treatments; however, to compare the parameters in the 4 treatment groups, parametric mixed linear models with the group as the impact factor were applied because of the clustered data within the dogs. Since all P values were quite large (P>0.5) and the sample size was small, we did not https://doi.org/10.5051/jpis.2104040202 add confidence intervals of the mean differences. The threshold for statistical significance was set at P<0.05.

Clinical healing
All dogs remained healthy, and no local infections occurred during the entire study period. However, 2 implants were lost during follow-up (1 in the GBR/SCTG group and 1 in the GBR group).

Histologic observations
Due to the proximity of the maxillary sinus, the apex of all implants protruded into the sinus cavity to various extents. All implants included in the histological processing (except for 2 implants lost during the healing period) were osseointegrated. Generally, the buccal bone plate tended to undergo moderate resorption. The majority of the specimens exhibited no bone-to-implant contact more coronal than the third thread of the implants. In the GBR and the GBR/SCTG groups, no remnants of the collagen membrane were observed. Newly formed bone was interconnected with bone substitute particles, but the space occupied by bone substitute particles appeared to be collapsed in the majority of the specimens. The fibrous connective tissue layer tended to be thicker in the SCTG and GBR/SCTG group than in the other groups (Figure 3).

Height of the peri-implant mucosa (vertical measurements)
On the buccal aspect, the median level of the MM was located coronally with respect to the implant platform, with distances ranging between 1.99 mm (Q1:  Figure 4A).
The median distance between the MM and the fBIC was in a similar range among the GBR, SCTG, and control groups (

Thickness of the peri-implant mucosa (horizontal measurements)
All specimens, except for 1 in the GBR group, exhibited no mineralized tissue at the 1-mm level above the IS. However, the observed mineralized tissue in the GBR group was scattered and consisted of non-integrated bone substitute particles.    Mineralized tissue was observed in only 5 specimens (2 in the GBR group, 1 in the SCTG group, 1 in the GBR/SCTG group, and 1 in the control group). The HT in specimens with mineralized tissue ranged between 0.28 mm (the GBR group) and 1.56 mm (the SCTG group) (Table 2, Figure 5C).
Despite some differences in the median values of the TT, there were no statistically significant differences between the mean values of the groups at any levels (P>0.05). Moreover, many specimens lacked hard tissue at the measured levels, making it impossible to conduct a proper-statistical analysis of the thickness of hard and soft tissues.

DISCUSSION
The present study investigated the effect of hard and/or soft tissue grafting on immediate implants and demonstrated that i) soft tissue augmentation alone (SCTG) resulted in a higher level of the MM and a greater overall tissue thickness compared to the control group and the groups with hard tissue augmentation, and was predominantly beneficial and more coronal than the IS; ii) hard tissue augmentation was predominantly effective below the IS; and iii) there was no benefit of simultaneous hard and soft tissue augmentation (SCTG/GBR) on the overall tissue thickness compared to hard tissue augmentation alone (GBR) or soft tissue grafting alone (SCTG).
The use of SCTG to improve the buccal contour and minimize mucosal recession when applying IIP protocols has gained considerable attention. In clinical studies, the addition of SCTG increased the mucosal thickness and width of keratinized tissue, and prevented mucosal recession and marginal bone loss [11,21,27]. In the present study, the SCTG group exhibited more favorable tissue thickness than the other groups, indicating that SCTG alone can establish a sufficient tissue profile at the soft tissue level. Partially in line with this, a few studies have demonstrated the possibility of using SCTG as an alternative to bone augmentation for specific indications with delayed implant placement [28,29]. These studies demonstrated favorable mucosal thickness, keratinized tissue height, and stable periodontal parameters. However, it should be noted that SCTG alone cannot compensate for the resorption of the buccal bone following IIP, which was also demonstrated in another preclinical study [17].
Bone grafting procedures with IIP generally refer to filling the gap between the implant and the socket wall. In the past, the necessity of filling this gap was controversial (depending on the size of the gap), but gap filling with a slowly resorbing bone substitute material is currently recommended in order to compensate for the loss of the buccal bone plate and to prevent collapse of the buccal contour. Clinically, the size of the gap should be considered in conjunction with the thickness of the buccal bone plate. In the present study, a relatively small gap (≤ 1 mm) was present after implant placement because of the small ridge width and the presence of the maxillary sinus. In this situation, it was suspected that mere filling of the gap would jeopardize the integrity of the buccal plate after remodeling. This concern was noted in a clinical study emphasizing that the distance between the implant surface and external buccal graft side should be ≥ 4 mm [30]. Thus, both gap filling and buccal overbuilding were performed in groups with bone augmentation in the present study. However, the effect of bone grafting was not as satisfactory as that of SCTG, especially at or above the IS. This unfavorable outcome might have been influenced by several factors, including: i) augmentation outside of the bony envelope, and ii) possible compression at the IS area due to flap tension and suturing.
Simultaneous soft and hard tissue grafting did not appear to be beneficial in the present study, especially considering the efforts undertaken during surgery. In the GBR/SCTG group, the SCTG was positioned on top of the GBR area, where overbuilding using particulate bone graft material and a collagen membrane was performed. After flap closure, the sites in the GBR/SCTG group might have exhibited more tension and pressure than those at other sites due to the increased thickness of the augmentation under the flap. A clinical study also demonstrated that SCTG did not lead to a decrease in mucosal volume loss compared to sites without SCTG [13]. The authors of that study suspected that vascular damage caused by the surgical technique when inserting the SCTG induced more bone loss. However, the exact reason for this finding is unknown.
The present findings should be interpreted with caution. It is a rule of thumb that resorption makes it necessary to perform over-augmentation with a resorbable barrier membrane for GBR [31]. The implant position in relation to bone housing must be chosen to provide stability to the augmented hard tissue. Moreover, if soft tissue augmentation is added to hard tissue augmentation, one should realize that additional room for the augmented tissue is https://doi.org/10.5051/jpis.2104040202

Contour augmentation at implant sites
https://jpis.org required. Additional care must be taken in situations where physiological bone remodeling is expected, such as IIP.
This study has several limitations, primarily its small sample size. Second, behavioral control was difficult in the experimental animals, unlike in human patients who became extremely careful after surgery. In a clinical prospective study, Chappuis and colleagues (2018) [32] performed simultaneous GBR and soft tissue volume augmentation using volume-stable collagen matrix, leading to 2.1 mm of tissue volume increase. Third, in the present canine study, the maxillary premolar region was chosen as the surgical site. This area provides less bone quantity and a shallower vestibulum on the oral side compared to a universal site for bone augmentation in dogs (i.e., the mandibular premolar region). These differences were due to the proximity of the maxillary sinus and location of the palate relative to the maxillary teeth. In the present study, implants with a narrow diameter were used to minimize the potential influence of the above factors.
In conclusion, careful case selection may be needed to implement soft and hard tissue augmentation when applying an IIP protocol. Preclinical studies using other defect models (such as box-type defects for favorable bone housing) and clinical studies are needed for further investigation.