**1. Introduction**

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> One alternative to addressing the overly narrow residual ridges of the mandible is the use of dental mini implants (with a diameter below 3 mm) [1], which represent an important therapeutic option in cases in which reconstructive surgeries are contraindicated or as an alternative to other surgical procedures that present greater morbidity [2]. For these cases, the use of alloys with improved mechanical properties is desired for use in dental implants [3]. In this context, grade V titanium mini transitional implants (MTIs) can be a better alternative in these clinical cases because of their high mechanical resistance. These procedures allow the clinician to perform restoration and maintenance work in the vertical dimension with the use of mini implants in conjunction with implant surgical therapy,

**Citation:** Beltrán, V.; Weber, B.; Lillo, R.; Manzanares, M.; Sanzana, C.; Fuentes, N.; Acuña-Mardones, P.; Valdivia-Gandur, I. Histomorphometric Analysis of Osseointegrated Grade V Titanium Mini Transitional Implants in Edentulous Mandible by Backscattered Scanning Electron Microscopy (BS-SEM). *Metals* **2021**, *11*, 2. https://dx.doi.org/10.3390/met1101 0002

Received: 30 October 2020 Accepted: 27 November 2020 Published: 22 December 2020

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which is considered to be an effective method to provide the patient with an immediate and comfortable transitional appliance [4].

Grade V titanium alloy is widely used to fabricate medical and dental implants due to its superior physical, mechanical [5,6], and biological properties compared to other available biomedical metals and alloys. Specifically, its resistance to corrosion, excellent biocompatibility, lightweight, and superior tribological properties make this material far superior to other metals and alloys and highly useful for dental application [7,8]. Moreover, various authors have demonstrated its osseointegration [9–11]. Complications related to biocompatibility [12] or toxicity [13] of titanium alloys have been reported, including a mildly increased inflammatory response in direct contact to skeletal muscle [14]. Still, the evidence that includes human biopsies is scant.

Ultrastructural bone analysis has been performed by backscattered scanning electron microscopy (BS-SEM). This is an effective technique for studying the osseointegration of dental implants [15]; however, there is scarce evidence regarding the osseointegration of grade V titanium dental implants in humans when considering transitional implants immediately loaded and associated with overdenture. The purpose of this study is to assess the use of grade V titanium MTIs as immediately loaded implants for temporary overdenture, performing the analysis of the bone tissue close to the implant surface in human biopsies.

#### **2. Materials and Methods**

#### *2.1. Patients and Surgical Procedure*

The study was approved by the Ethics Committee of the University Mayor, Chile (Protocol Nº246/006). Four female patients (60–68 years of age, health compatible with treatment) who were non-smokers, users of acrylic removable dentures over the completely edentulous mandible, and with horizontal atrophy of the anterior area were selected for treatment using an overdenture which was fixed temporally by MTIs. Prior to implant placement, all patients signed for informed consent regarding description of the placement and removal of the MTIs. The surgical protocol involved the simultaneous placement of submerged regular implants (4.0 mm Titamax EX or 4.3 mm Alvim Morse Taper) and MTIs for immediate loading in the edentulous lower mandible. The submerged regular implants that were not loaded were maintained and controlled for their osseointegration in the traditional way (6–8 months to implant osseointegration).

Each patient received two MTIs (2.9 mm in diameter and 12 mm long, Facility Neodent with Neoporos, Curitiba, Brazil) that were placed in the canine mandible region with O-rings for fixation of the overdentures. The MTIs were placed with a mandatory insertion torque of at least 35 Ncm and maximum 45 Ncm measured by a drill unit (W&H Implant Med). The suture was performed using polyglactin 910 4.0 (Vicryl®, Ethicon Endo-Surgery Inc., Greensboro, NC, USA). Immediately after the surgery, MTIs were loaded with a removable overdenture following the loading protocol under the Schnitman concept [16]. Then, patients were controlled at 2, 7, 30, 60, and 90 days.

After 8 months, the MTIs were extracted with a trephine bur of 3.5 mm in diameter, and the samples were conditioned for histomorphometric analysis by BS-SEM (Figure 1).

**Figure 1.** Bone sample with mini transitional implants (MTIs) obtained by trephine. A cut was made to obtain two samples (SA and SB) for backscattered scanning electron microscopy (BS-SEM) analysis. MTI = mini transitional implant; SA and SB = Segments A and B obtained after the cut.

#### *2.2. Histomorphometric Analysis of Samples by BS-SEM*

The MTIs were surgically removed using a trephine bur (implant plus its bone segment, Figure 1). They were subsequently fixed in 10% neutral buffered formaldehyde for 48 h. Later, the bone segments were prepared for their inclusion in light-curing resin (Technovit®). Posteriorly, the included samples were cut following the central longitudinal axis of the implant (Figure 1) using an Exact® diamond band saw (0.2 mm thick) and treated for BS-SEM analysis following the procedure described in the literature [17,18].

During BS-SEM analysis, several microphotographs were obtained (50× to 150×). The area of tissue between two threads of the implant surface was established as a study unit (Figure 2), referred to as the "bone area fraction occupancy" (BAFO). The following parameters were analyzed in the BAFOs:


In accordance with the variables described above, we performed quantitative valuation and qualitative analysis. Quantification of tissue areas was performed using the software ImageJ. The SPSS statistical software was used for the analysis of the quantitative results. The ANOVA or Kruskal–Wallis tests were applied and the *p*-value < 0.05 was considered significant.

**Figure 2.** In the upper part, the types of bone tissue studied in the samples are shown; in the lower part, the bone area fraction occupancy (BAFO) used as an analysis unit in this study is shown (delimited by the blue broken line between two implant threads). Samples of Bone to Implant Contact (BIC) area are shown in the boxes inside the inferior images. cl = cellular lacunae; ms = medullar space; vs = vascular space; MTI = mini transitional implant (surface).
