Histologic and histomorphometric evaluation of minicono abutment on implant surrounding tissue healing and bone resorption on implants placed in healed bone. An experimental study in dogs

: The objective of this evaluation was to measure the width and length of connective tissue (CT) and crestal bone resorption (CBR) related to minicono® abutment inserted in conical connection dental implants, which were placed crestal and subcrestally in a dog’s mandible. Materials and Methods: Forty-eight Top DM implants with the same coronal diameter were placed at the crestal level, 1 mm (


Introduction
Maxillary and mandibular bones undergo significant resorption due to several problems, including poor patient hygiene, periodontal issues, and the need for a tooth extraction. Most of the resorption processes occur in the soft bone, which is cortical, especially in the vestibular wall of the alveolus, maintaining the bone structure through the lingual wall [1][2][3][4]. The height of the alveolar ridges, both buccal and lingual, have resorption in two phases and are stimulated by the implant surface over the entire neck treated with the implant surface [5]. Numerous factors act in animal experiments, such as implant insertion time, the type of dental implant, the insertion toque and depth, the insertion torque, the biological width settlement, and the design of the implant, which will act by itself, further influencing bone resorption [6][7][8][9][10]. Clinical and radiographic studies have suggested that surface treatment of the healing abutment and the final abutment of the prosthesis does not influence crestal bone loss around implants [11]. It was shown that the abutment implant union plays a fundamental role due to its design, which, in some cases, can act through micro-movements, which entails a loss of crestal bone, both buccal and lingual, and an increase in soft tissue [12].This area can remodel itself in both thickness and height and is directly related to the size of the abutment, whether it is for healing or for the final abutment [12][13][14]. Placing a crestal implant between 1 and 2 mm restores the height and width dimensions of the gingival tissue around a healing abutment. The more coronal position of this soft tissue does not affect marginal bone loss; it maintains soft tissue thickness by acting as a protector of the alveolar ridges around the implant [12][13][14]. One of the fundamental factors is the design of the multiunit abutment, with a capacity for influencing the disposition of the collagen fibers in the perpendicular and oblique directions. Still, not all multiunit abutments have an ideal design. We previously published an article in which the use of wide healing abutments, that is, anatomical ones, increased the concentration of connective tissue, stimulating the orientation of the collagen fibers perpendicular and oblique to the soft tissue [15][16][17][18]. The best way to preserve the bundle bone is to determine the length and width stability of the connective tissue around the abutment, which is essential for long-term aesthetics in implants placed in the anterior sector [19,20]. Peri-implant bone remodeling occurs mainly in the initial phases between 91 and 98 days after the fist implant settlement, leading to subsequent alveolar bone preservation and enlargement of the soft tissue. As previously published by various authors, the platform switching technique stimulates bone protection by soft tissue establishment [20][21][22]. The main goal of this study is the evaluation of crestal bone loss when we connect a transepithelial abutment (minicono) screwed on implants located at different crestal levels (crestal, one and two mm under crestal bone). Our study aimed to assess if the implant position or the length of the transepithelial abutment (minicono) influences buccal or lingual plate bone loss.

Project design
The animals used in the study were 6 Fox Hound dogs of approximately 1.5 years with an approximate weight of 15 to 18 kg. These animals were pre-emptively caged prior to surgical treatment under the care of a university veterinarian. They were fed a soft diet with adequate nutritional support during the length of the project. The animals received antibiotics (Enrofloxacin 5 mg/kg two times a day) and analgesics (Meloxicam 0.2 mg/kg, 3 times a day) via the systemic route after surgery. The clinical veterinary examination determined that the dogs were in good general health [13][14][15][16]21]. Forty-eight TOP DM® implants (Bioner, Sistemas Implantológicos, Barcelona, Spain) with the exact dimensions 3.5 mm diameter by 8.5 mm and one 3 mm height minicono® (Bioner Sistemas Implantológicos, Barcelona, Spain) abutment were used in the study. Two important variables were obtained during this study, representing the overall clinical results: position of the implant and the length of the mincono abutment.

2.1.1.
Surgical procedure All animals in the study underwent a prior anesthesia technique in the femoral quadriceps with acepromazine 0.13%-0.26 mg/kg, metomidine clorhydrate (35 μg/kg), and tolfelanic acid (1.5 mg/ml). Then, an intravenous catheter was inserted (diameter 22 G) into the cephalic vein of the animals and propofol at a concentration of 19.5 mg/kg (1,95 ml/kg) was administered. Dental infiltration anaesthesia was administred directly to premolar and molar areas (lidocaine 40 mg), as previously published [13][14][15][16]21]. The study design can be seen in Figure 2. The surgical technique used to operate on the animals was supervised by the Chief Veterinarian of the University of Murcia. The extracted teeth were the lower premolars and the first molar bilaterally by odontosection (Figure 3a). Extraction areas were allowed to heal for 12 weeks before implant placement (Figure 3b). After three months of healing, 48 TOP DM implants 3.5 × 8.5 mm were randomly assigned crestally and subcrestally at 1-and 2-mm positions, placed with an insertion torque of ≥35 Ncm (Figure 3c). Dental implants were placed in the middle of the alveolus of extracted teeth, crestally and subcrestally at 1-and 2-mm positions (Figure 3d). Abutments at 3-mm in height were connected to the implants and screwed at 20 Ncm on each implant placed at the crestal and subcrestal levels ( Figure 3e). Four implants were placed in each hemi-mandible with a cover screw on top of each minicono abutment ( Figure 3f  During the first week after surgery, the animals received the following antibiotics and analgesics via an oral route: clindamycin (300 mg, two times a day) and naproxen (600 mg, three times a day). The sutures were removed after two weeks by a veterinarian [13][14][15][16]21]. Three animals were sacrificed after eight weeks and the other three were scarified at 12 weeks (n = 3) with an overdose of pentothal natrium (Abbot Laboratories, Madrid, Spain) [13][14][15][16]21].

Histological sample preparation
Each dog's implant with hard and soft tissues were divided into blocks and included in a fixing solution composed of 5% formalin. The samples were dehydrated with 1% ethanol and stored under vacuum [21]. Each of the samples were infiltrated with a special resin, called Technovit 7200® resin (Kulzer Heraeus, Germany), to fix the sample and to prepare the biopsy for analysis. The resin blocks were divided buccolingually parallel to the axis of the implant, making three different cuts. Each block was divided into four parts. Two fine blocks of 30 × 50 microns were produced for staining proposes with pricosirius-hematoxylin to distinguish between mature and immature bone. The other two thick blocks were later worn and polished for scanning electronic microscope. For a histomorphometry study, all images were enlarged at 20×, where eight different areas were analyzed using an Olympus DP 12 digital camera (Nagano, Japan) and the 4.0 micro-imaging software (Media Cybernetics, Silver Springs, USA) was used to analyze the samples. All samples were analyzed by the same lab technician to avoid bias [13][14][15][16]21].

Histomorphometric analysis
In all the biopsies performed, different marks were identified to evaluate the soft and hard tissue: implant shoulder (IS), buccal bone crest (BBC), lingual bone crest (LBC), Minicono shoulder (Mi), distance from first bone-to-implant contact-implant shoulder (A-IS), Buccal periimplant mucosa (B-PIM), Lingual peri-implant mucosa (L-PIM), and the apical portion of the junction epithelium (Je) ( Figure 5).

Digital radiographic evaluation
After the experiment was completed, each implant was individually and digitally radiographed in sections with two measurements: implant abutment junction (IAJ) and bone considering the lower level of crestal bone-to-implant contact. The same radiograph positioner was used in each radiograph and the images were repeated by the same surgeon three times. The radiographs were analyzed by the Newtom NTT Software (Newtom Cefla S.C., Imola Italia). All crestal bone measurements were performed in the mesial and distal directions to evaluate crestal bone loss between the minicono abutment and the implant connection. Radiographic measurements were made distally and mesially for each of the implants after an eight-week period and after 12 weeks for the implants placed crestally, both 1 and 2 mm below the crest level ( Figure 6). Measurements of implant stability were performed by an Osstell® Mentor device (W&H, Göteborg, Sweden) immediately on the day of surgery and at eight and 12 weeks after implant placement. Stability values were expressed in ISQ measurements six times in each implant and three times each on the buccolingual and mesiodistal surfaces.
Statistical analysis The statistical study was carried out using the SPSS 21.0 software (SPSS, Chicago, IL, USA). Differences between two tests and one control implant with 3 mm high MINICONO sites were evaluated with a Wilcoxon test. A descriptive analysis was performed (mean and standard deviation) for both groups. A Friedman test was applied to compare the means with a significance level of 95% (p < 0.05). Bruner and Langer and Mann-Whitney U tests were applied to analyze the differences between the evaluation distances. The differences between groups (n = 16 control group, n = 16 test 1 group, n = 16 test 2 group) were made by means of repeated studies, considering only the dog as the dependent mean. The height and resorption of the alveolar ridges of the bone, as well as the height of the peri-implant mucosa, were expressed in absolute and relative terms.

Results
In addition, the following outcomes related to the radiographic evaluation, clinical and histological observations of hard and soft tissue and implant stability were explained in detail.

Radiographic evaluation
Implant healing at crestal placement bone loss was 1.22 ± 0.8 mm at mesial aspect and 1.24 ± 0.3 mm at distal aspect. This resorption was more than the bone loss of implants placed at a 2 mm subcrestal position, and was measured as 0.83 ± 1.7 mm at the distal position and 0.80 ± 1.4 mm at the mesial aspect of the test implant (Table 1). There are significant differences in bone loss between the control and test implants at eight and 12 weeks (p = < 0.001) in distal and mesial aspect, as calculated by the Mann-Whitney test (Table 1).

Histological and clinical evaluation
Surgical sites healed without any problem, and no healing abutment and implants were lost. The extensive junction epithelium (Je) was specially displayed in the lingual preimplant connective tissue of all samples obtained, and in direct contact with the minicono abutment. The buccal preimplant mucosa was less wide than the lingual one for all minicono abutments. At eight weeks, new build bone was observed around blood vessels on top of the buccal crest in the premolar areas, as well as a wellformed epithelium and connective tissue with less inflammatory tissue around the preimplant minicono area. In the control group, the peri-implant mucosa was composed with the thin epithelium, with a short connective tissue facing the titanium portion of the minicono abutment. At 12 weeks, the lingual bone wall suffered less bone resorption, and the soft tissue was better attached at the coronal region of the minicono abutment, while the buccal crest suffered more resorption. The abutment region showed parallel-fibered soft tissue and new stablished stable bone. The test groups exhibited signs of ongoing remodeling better than the control group due to minocono abutment protection (Figure 7).  Table 2). 12 weeks: All results at week 12 were superior to those found at week 8 in all evaluation groups. The L-PIM-IS measurements in the lingual and buccal areas didn't show any differences between the test groups (p > 0.05). The L-PIM-IS and B-PIM-IS measurements showed higher values for the lingual side in both groups in comparison with the buccal side (p < 0.05); the Je-C distance did not display any differences between the groups ( Table 2).

3.2.2.
Hard tissue 8 weeks: The IS-B measurements in vestibular area were significantly higher in the control group when compared to both test groups (p < 0.05). No significant differences were observed in the lingual areas (p > 0.05) (Figure 8). The IS-C measurement was higher for the lingual and buccal areas of the control group when compared with the test 1 group (p > 0.05) ( Table 1). The crestal implants showed an increased formation of woven bone in the coronal area (Figure 8a). The test 1 group showed larger amounts of newly formed bone with native bone within the coronal and body parts of the implant (Figure 8c). The most significant value was found in the test 2 group, showing increased bone implant contacts (Figure 8e). The connective tissue was located laterally on top of the minicono abutment at 1-and 2-mm positions below the crest level ( Figure 8).
12 weeks: All measurements that were made at 12 weeks were increased in all groups ( Figure 8). The IS-B buccal side measurements were significantly higher in the control group compared with the test groups (p < 0.05), and no significant differences were observed at the lingual areas (p > 0.05). The IS-C distance was augmented in the lingual and buccal zones of the control group when compared with the test groups (p > 0.05) ( Table 1). After 12 weeks of healing, the entire implant surface was full surrounded with new bone in the test 1 and 2 groups when compared with the control group. The peri implant mucosa margin (PM) was located apically to the implant shoulder. At the lingual aspect of the implant, signs of the newly formed bone can be observed in the test 2 group (Figure 8f). The first bone-to implant contact (BIC) at the lingual aspect is located close to the marginal level of the rough surface. After 12 weeks of healing, the buccal-lingual section of the crestal group showed that the margin of the peri implant mucosa is 2 mm below crest.

Bone to implant contact (BIC)
The total BIC in the test 1 and 2 groups were higher when compared with the control group at eight and 12 weeks. New formed bone in the coronal region was augmented in the test 2 group after eight weeks, and much greater after 12 weeks when compared to the test 1 and control groups. After evaluation, the coronal area (first BIC) was far away at the implant shoulder in the control group when compared to the test 1 and 2 groups (Table 3).

Implant stability quotient measurement (ISQ values)
ISQ values for the immediate implants showed significant intragroup differences at eight and 12 weeks in all groups. The test 2 group showed higher ISQ values (mean, 77.62 ± 0.6) than the control group (mean, 72.09 ± 0.6); however, at 12 weeks for the immediate implants, the test 2 group showed higher values at the P3 area of 79.2 ± 0.9, which is different to control group value of 74.9 ± 0.6, indicating no statistically significant intergroup difference (Table 4). The clinical outcomes assessed in the study showed that using the 3 mm minicono abutment in subcrestal implants could protect the bundle bone and reduce crestal bone loss.

Discussion
The multiunit abutments that are used in surgeries allow for the establishment of soft tissue, as well as its remodeling around it [23]. The modifications around soft tissue were directly related to the establishment of the conditions offered by either the definitive abutment or the healing abutment [17]. In some cases, the micro-threads of the implants may be related to the loss of marginal bone because they can free a space between the soft tissue and the abutment, which further allows for the entry of bacteria leading to bone resorption [17]. This formation of the biological width will depend on the minicono or multiunit abutment that is placed in our previous studies [8,[13][14][15][16][17]21]. The use of healing multiunit abutments on top of the implants immediately after surgery didn't interfere into the connective tissue remodeling around them [23]. The biological width normally acted as a protector of the bone crests around the implant, as indicated mainly by the placement of the crystal of the implants, thus reducing the stress on the alveolar crests. However, the apical placement of the implants allows for a greater anchorage in the bone, improving the stability of the same increasing boneimplant contact [24]. Our studies on immediate post-extraction implants placed 1mm below the bone crest in experimental animals showed no influenced resorption of the surrounding bone walls after 12 weeks [25,26]. Many studies in which implants were placed at the coronal level with micro threads in the neck of the implant allowed for a restoration of the preimplant soft tissues under the neck of the implant [27]. The advantage of implants placed 1 mm under bone crest post extraction with a switched platform showed a reduction in the gap and the restoration of the biological width over the shoulder of the implants at 12 weeks, thereby protecting the crestal bone [28]. We defined placing the implants 2 mm under crest active implant surface, macro and microtopography, design and type of threads that help bone regeneration immediately and its apposition on the immediate implant [29][30][31]. However, the connective was influenced related to depth of the implant, thereby increasing its thickness and height, depending on the type of abutment that was placed [32,33]. Our study agreed with the data from Linkevicius et al., in 2020, where they showed that implant placement under the bone area considerably reduced bundle bone loss in a vertical direction, increasing the width of connective tissue to protect it [34]. The immediate placement of abutments, such as minicono on the implants, considerably reduced bone loss a month before the prosthesis was finished; however, after one year the difference between the groups was stable [35]. Madani et al. concluded that implants placed immediately or delayed with platform switching were the safest option for maintaining bone around the implants, placing the implants 1 mm below crestal bone, protecting and maintain the biological width [36]. It was shown that placing the 2 mm sub-crestal implants, where they find that the expanded platform implants lose more bone, is considerably reduced by increasing the soft thickness. In addition, implants located at the crestal level with platform switching considerably reduced the loss of bone and its surrounding structures such as soft tissue, thereby improving aesthetics [37]. They agreed with our work related to the placement of sub crestal implants that didn't influence the remodeling of soft and hard tissue. However, a decreased resorption in implants placed at crestal position led to an increase in bone implant contact (BIC), especially in cylindrical implants [6,7,38]. On the contrary, other studies spoke about the bone loss from the vestibular and lingual walls when the implants are under the crest; however, many of these studies were carried out in experimental animals, and its data can be cautiously transferred to humans [39][40][41][42]. In human studies, the main parameters to be considered are the height of the crestal bone and the implant location above or below the bone level [43]. The evaluation of those parameters must be supported by a radiographic clinical study and the inflammatory immune response related to implant type, macro design, length and position [43][44][45][46][47][48]. We concluded that implants with a minicono abutment placed 2 mm below crest in healed bone stimulated a soft tissue response to the width and height and protected bundle bone. Our findings guided clinical practitioners with the potential implications for implant placement location, especially sub-crestal and the minicono abutment for soft tissue and bone establishment.

Conclusions
In our conclusions, we can affirm that despite the limitations of an experimental study, bone resorption can be controlled if we place the implants 1 or 2 mm under the bone crest. The use of the minicono abutment minimizes bone loss, favors the restoration of the biological width by increasing the thickness and height of the connective tissue that sits directly on implant surface. The conformation of that soft tissue that we have found around a 3mm polished abutment were similar in both groups of implants placed 1 and 2mm below the crest. On the other hand, implants placed at crestal level soft tissue moved down minicono abutment reaching a new peri-implant mucosa establishment.

Use of AI tools declaration
The authors declare they have not used Artificial Intelligence (AI) tools in the creation of this article.

Conflict of interest
I am an editorial board and I was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests.