Effect of Rough Surface Platforms on the Mucosal Attachment and the Marginal Bone Loss of Implants: A Dog Study

materials Article Effect of Rough Surface Platforms on the Mucosal Attachment and the Marginal Bone Loss of Implants: A Dog Study Javier Montero 1, Alberto Fernández-Ruiz 2, Beatriz Pardal-Peláez 1, Alvaro Jiménez-Guerra 3, Eugenio Velasco-Ortega 3,*, Ana I. Nicolás-Silvente 4and Loreto Monsalve-Guil 3 1School of Dentistry, Faculty of Medicine, University of Salamanca, 37007 Salamanca, Spain; [email protected] (J.M.); [email protected] (B.P.-P.) 2Private Practice Specialized in Oral Surgery and Implant Dentistry, 07800 Ibiza, Spain; [email protected] 3Faculty of Dentistry, University of Seville, 41009 Seville, Spain; [email protected] (A.J.-G.); [email protected] (L.M.-G.) 4School of Dentistry, University of Murcia, 30008 Murcia, Spain; [email protected] *Correspondence: [email protected] Received: 21 January 2020; Accepted: 9 February 2020; Published: 10 February 2020   Abstract: The preservation of peri-implant tissues is an important factor for implant success. This study aimed to assess the influence of the surface features of a butt-joint platform on soft-tissue attachment and bone resorption after immediate or delayed implant placement. All premolars and first molars of eight Beagle dogs were extracted on one mandible side. Twelve-weeks later, the same surgery was developed on the other side. Five implants with different platform surface configurations were randomly inserted into the post-extracted-sockets. On the healed side, the same five different implants were randomly placed. Implants were inserted 1 mm subcrestal to the buccal bony plate and were connected to abutments. The primary outcome variables were the supracrestal soft tissue (SST) adaptation and the bone resorption related to the implant shoulder. The SST height was significantly larger in immediate implants (IC95% 3.9–4.9 mm) compared to delayed implants (IC95% 3.1–3.5 mm). Marginal bone loss tended to be higher in immediate implants (IC95% 0.4–0.9 mm) than in delayed implants (IC95% 0.3–0.8 mm). Linear-regression analysis suggested that the SST height was significantly affected by the configuration of the platform (0.3–1.9 mm). Roughened surface platforms resulted in higher SST height when compared to machined surface platforms. Marginal bone loss was less pronounced in roughened designs. Keywords: implant platform; mucosal attachment; marginal bone loss 1. Introduction The biological phases of the healing dynamics of both hard and soft tissues after dental implant placement have been reported in animal experiments [ 1 , 2 ] and human studies [ 3 ]. Such dynamics have also been described for implants immediately placed into extraction sockets [4–6]. Marginal bone resorption occurs when an implant is inserted both in healed sites [ 7 , 8 ] and also in extraction sockets [ 4 , 7 ]. A systematic review reported that marginal bone resorption takes place irrespectively on the implant neck configuration [ 9 ]. Nevertheless, roughness and micro-threads at the implant neck contribute to marginal bone preservation [ 10 – 14 ]. Few studies have compared the effect of implant surface configurations and geometries on the peri-implant healing process at implants placed immediately into extraction sockets [ 15 , 16 ]. It seems that the presence of marginal micro-threads or grooves reduces marginal bone resorption and soft tissue recession. To date, most research regarding Materials 2020,13, 802; doi:10.3390/ma13030802 www.mdpi.com/journal/materials Materials 2020,13, 802 2 of 13 the clinical management of immediate implants is mainly focused on hard tissue integration and the effect of different implant designs or surgical approaches on clinical esthetic outcomes. However, there is insufficient evidence whether or not different implant designs and surface configurations enable the formation of an adequate soft-tissue seal at implants immediately placed into extraction sockets and compared to implants placed into fully-healed alveolar ridges. The objective of this experimental study was; therefore, to assess the influence of the surface roughness platform of butt-joint implants placed immediately into extraction sockets, with regards to the dimensions of the supracrestal peri-implant soft tissue seal and the preservation of marginal alveolar bone 12 weeks after implant placement. 2. Materials and Methods The experimental protocol was submitted and approved by the Ethical Committee of the University of Santiago of Compostela in Spain (Ref: 01/16-LU/001). The study was reported according to the ARRIVE guidelines. 2.1. Animal Sample The guidelines for animal care adopted in Spain were strictly followed. Eight 3-year-old male Beagle dogs supplied by Isoquimen SL (Sant Feli ú de Codines, Barcelona, Spain; RN ◦ B9900039) were included in the study. To minimize the number of dogs for ethical reasons (Three R requirements), but to maintain enough power of analysis, a split-mouth design was developed. The dogs were the unit of analysis. The sample size was estimated to be eight dogs for detecting differences in the supracrestal soft tissue height of 2 mm with a power of 80% and a probability of alpha =0.05. 2.2. Randomization and Allocation Concealment The randomization for the side of the mandible and the allocation of five different types of implants were performed by one of the authors that did not participate in the surgeries (AF). Sealed envelopes were used and opened at the time of surgery. The surgeon (EV) was informed about the selection of the hemimandible for implant placement before the first session of tooth extraction. The histological slides did not report indications about treatment allocation, nor did the examiner (JM) receive such information. 2.3. Surgical Preparations The animals were pre-sedated with an intramuscular injection using a combination of 25 µ g/kg medetomidine (Doctor Esteve, Barcelona, Spain) and 0.5 mg/kg morphine (Morfina Braun 2%, Braun, Barcelona, Spain). Then, general anesthesia was induced using 5 mg/kg/i.v. of propofol (Vetofol, Esteve, Barcelona, Spain) by intravenous infusion, followed by laryngeal intubation and the inhalation of a mix of 2.5%–4% isoflurane/sevoflurane and 30% oxygen from the ventilation tube. During the anesthesia, a veterinarian continuously recorded the electrocardiogram, arterial pressure, pulse oximetry, and capnography with non-invasive methods. Besides, submucous injections of articaine hydrochloride (Ultracain ® , Normon, Spain) were infiltrated locally for bleeding control. After the intra-sulcular incisions, full-thickness mucoperiosteal flaps were made, and all mandibular premolars and first molars were extracted at one randomly selected hemi-arcade. The flaps were closed with single interrupted 4-0 vicryl resorbable sutures. Twelve weeks after surgery, the same procedure was performed on the contralateral side, and five implants were installed into the extraction sockets in all premolars (P 1 –P 4 ) and molar (M 1 ) sites. Straight multi-unit screw-retained abutments were fixed with a closing screw onto all implants. Each multi-unit abutment was by the same manufacturer as its corresponding implant (Institute Straumann AG, Basel, Switzerland and Galimplant SLU, Sarria, Lugo, Spain). In the healed contralateral ridge, five delayed implants were inserted in the healed sites of P 1 –P 4 and M 1 . All implants received multi-unit abutments fixed with the closing screw. The implants were Materials 2020,13, 802 3 of 13 placed 1 mm subcrestal to the buccal plate level on both sides. Single 4-0 vicryl resorbable sutures were used to close the flaps, allowing non-submerged healing. 2.4. Implant Characteristics In each hemiarcade, five different types of implants of two different manufacturers were assessed (Figure 1): • Type I: Straumann BL (bone-level) implant with 3.3 mm diameter and 8 mm in length (Institute Straumann AG, Basel, Switzerland), with a body implant treated with SLA ® surface (mean Ra value of 1.66 µm) and a machined platform (mean Ravalue of 0.27 µm). • Type II: IPX-Std (standard) with 3.5 mm diameter and 8mm in length (Galimplant SLU, Sarria, Lugo, Spain), with an implant body and neck treated using a Nanoblast Plus ® technique (mean Ravalue of 1.69 µm) and a fully machined platform (mean Ravalue of 0.28 µm). • Type III: IPX-Half, with 3.5 mm diameter and 8 mm in length (Galimplant SLU, Sarria, Lugo, Spain) with an implant body, neck and the outer half of the platform surface treated with Nanoblast Plus ® technique (same Ravalue described in Type II treated surface) and the inner half of the platform machined (same Ravalue described in Type II machined surface). • Type IV: IPX-Full, with 3.5 mm diameter and 8 mm in length (Galimplant SLU, Sarria, Lugo, Spain) with the implant body, neck and the whole platform treated with the Nanoblast Plus ® technique (mean Ravalue of 1.69 µm). • Type V: IPX-Control, with 3.5 mm diameter and 8 mm in length (Galimplant SLU, Sarria, Lugo, Spain), with implant body, neck, and platform machined (mean Ravalue of 0.28 µm). Materials 2020, 13, x FOR PEER REVIEW 3 of 13 placed 1 mm subcrestal to the buccal plate level on both sides. Single 4-0 vicryl resorbable sutures were used to close the flaps, allowing non-submerged healing. 2.4. Implant Characteristics In each hemiarcade, five different types of implants of two different manufacturers were assessed (Figure 1):  Type I: Straumann BL (bone-level) implant with 3.3 mm diameter and 8 mm in length (Institute Straumann AG, Basel, Switzerland), with a body implant treated with SLA® surface (mean Ra value of 1.66 µm) and a machined platform (mean Ra value of 0.27 µm).  Type II: IPX-Std (standard) with 3.5 mm diameter and 8mm in length (Galimplant SLU, Sarria, Lugo, Spain), with an implant body and neck treated using a Nanoblast Plus® technique (mean Ra value of 1.69 µm) and a fully machined platform (mean Ra value of 0.28 µm).  Type III: IPX-Half, with 3.5 mm diameter and 8 mm in length (Galimplant SLU, Sarria, Lugo, Spain) with an implant body, neck and the outer half of the platform surface treated with Nanoblast Plus® technique (same Ra value described in Type II treated surface) and the inner half of the platform machined (same Ra value described in Type II machined surface).  Type IV: IPX-Full, with 3.5 mm diameter and 8 mm in length (Galimplant SLU, Sarria, Lugo, Spain) with the implant body, neck and the whole platform treated with the Nanoblast Plus® technique (mean Ra value of 1.69 µm).  Type V: IPX-Control, with 3.5 mm diameter and 8 mm in length (Galimplant SLU, Sarria, Lugo, Spain), with implant body, neck, and platform machined (mean Ra value of 0.28 µm). Figure 1. The topographic profiles and platform surfaces of the five implant designs evaluated: Straumann BL platform with SLA® surface platform; IPX-Std with a fully machined platform; IPX- Half with Nanoblast Plus® surface in the outer half of the platform and machined surface in the inner half of the platform; IPX-Full with the whole platform treated with Nanoblast Plus® and IPX-Control with full body and platform machined. The characteristics of each implant group are summarized in Table 1. The IPX implant surface received a surface conditioning treatment consisting of a mixed method (mechanical and chemical; The Nanoblast Plus® technique) for improving implant surface topography through two consecutive processes: aluminum oxide spraying and triple acid etching. This method was patented by the manufacturer Galimplant SLU (Sarria, Lugo, Spain). The SLA (Sandblast, Large-Grit, Acid-Etching) method is a method patented by Straumann (Straumann AG, Basel, Switzerland) that induces surface erosion by applying a strong acid onto the blasted surface. This treatment combines blasting with large-grit sand particles and acid etching sequentially to obtain macro roughness and micro pits. The roughness characteristics of each surface can be observed with a magnification of 1000× in Figure 2. Figure 1. The topographic profiles and platform surfaces of the five implant designs evaluated: Straumann BL platform with SLA ® surface platform; IPX-Std with a fully machined platform; IPX-Half with Nanoblast Plus ® surface in the outer half of the platform and machined surface in the inner half of the platform; IPX-Full with the whole platform treated with Nanoblast Plus ® and IPX-Control with full body and platform machined. The characteristics of each implant group are summarized in Table 1. The IPX implant surface received a surface conditioning treatment consisting of a mixed method (mechanical and chemical; The Nanoblast Plus ® technique) for improving implant surface topography through two consecutive processes: aluminum oxide spraying and triple acid etching. This method was patented by the manufacturer Galimplant SLU (Sarria, Lugo, Spain). The SLA (Sandblast, Large-Grit, Acid-Etching) method is a method patented by Straumann (Straumann AG, Basel, Switzerland) that induces surface erosion by applying a strong acid onto the blasted surface. This treatment combines blasting with large-grit sand particles and acid etching sequentially to obtain macro roughness and micro pits. The roughness characteristics of each surface can be observed with a magnification of 1000×in Figure 2. Materials 2020,13, 802 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and R a values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment RaValue Treated Surface RaValue Machined Surface Straumann BL Materials 2020, 13, x FOR PEER REVIEW 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and Ra values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment Ra Value Treated Surface Ra Value Machined Surface Straumann BL 3.3 × 8 SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std 3.5 × 8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half 3.5 × 8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full 3.5 × 8 Nanoblast Plus® Surface Nanoblast Plus® Surface 1.69 µm Not Apply IPX-Control 3.5 × 8 Machined Machined Not Apply 0.28 µm Figure 2. High magnification images (1000×) of the surfaces evaluated; (A) SLA® Surface, (B) Nanoblast Plus® Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M1), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). 3.3 ×8SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std Materials 2020, 13, x FOR PEER REVIEW 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and Ra values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment Ra Value Treated Surface Ra Value Machined Surface Straumann BL 3.3 × 8 SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std 3.5 × 8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half 3.5 × 8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full 3.5 × 8 Nanoblast Plus® Surface Nanoblast Plus® Surface 1.69 µm Not Apply IPX-Control 3.5 × 8 Machined Machined Not Apply 0.28 µm Figure 2. High magnification images (1000×) of the surfaces evaluated; (A) SLA® Surface, (B) Nanoblast Plus® Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M1), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). 3.5 ×8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half Materials 2020, 13, x FOR PEER REVIEW 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and Ra values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment Ra Value Treated Surface Ra Value Machined Surface Straumann BL 3.3 × 8 SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std 3.5 × 8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half 3.5 × 8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full 3.5 × 8 Nanoblast Plus® Surface Nanoblast Plus® Surface 1.69 µm Not Apply IPX-Control 3.5 × 8 Machined Machined Not Apply 0.28 µm Figure 2. High magnification images (1000×) of the surfaces evaluated; (A) SLA® Surface, (B) Nanoblast Plus® Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M1), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). 3.5 ×8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus ® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full Materials 2020, 13, x FOR PEER REVIEW 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and Ra values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment Ra Value Treated Surface Ra Value Machined Surface Straumann BL 3.3 × 8 SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std 3.5 × 8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half 3.5 × 8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full 3.5 × 8 Nanoblast Plus® Surface Nanoblast Plus® Surface 1.69 µm Not Apply IPX-Control 3.5 × 8 Machined Machined Not Apply 0.28 µm Figure 2. High magnification images (1000×) of the surfaces evaluated; (A) SLA® Surface, (B) Nanoblast Plus® Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M1), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). 3.5 ×8 Nanoblast Plus® Surface Nanoblast Plus ® Surface 1.69 µm Not Apply IPX-Control Materials 2020, 13, x FOR PEER REVIEW 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and Ra values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment Ra Value Treated Surface Ra Value Machined Surface Straumann BL 3.3 × 8 SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std 3.5 × 8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half 3.5 × 8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full 3.5 × 8 Nanoblast Plus® Surface Nanoblast Plus® Surface 1.69 µm Not Apply IPX-Control 3.5 × 8 Machined Machined Not Apply 0.28 µm Figure 2. High magnification images (1000×) of the surfaces evaluated; (A) SLA® Surface, (B) Nanoblast Plus® Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M1), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). 3.5 ×8 Machined Machined Not Apply 0.28 µm Materials 2020, 13, x FOR PEER REVIEW 4 of 13 Table 1. Summary of the main characteristics of each experimental group, including implant design, implant size, body treatment, platform treatment, and Ra values (mean and standard deviation) for both treated and machined surfaces. Experimental Group Implant Design Implant Size Body Treatment Platform Treatment Ra Value Treated Surface Ra Value Machined Surface Straumann BL 3.3 × 8 SLA® Surface Machined 1.66 µm 0.27 µm IPX-Std 3.5 × 8 Nanoblast Plus® Surface Machined 1.69 µm 0.28 µm IPX-Half 3.5 × 8 Nanoblast Plus® Surface Outer Half: Nanoblast Plus® Surface Inner Half: Machined 1.69 µm 0.28 µm IPX-Full 3.5 × 8 Nanoblast Plus® Surface Nanoblast Plus® Surface 1.69 µm Not Apply IPX-Control 3.5 × 8 Machined Machined Not Apply 0.28 µm Figure 2. High magnification images (1000×) of the surfaces evaluated; (A) SLA® Surface, (B) Nanoblast Plus® Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M1), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). Figure 2. High magnification images (1000 × ) of the surfaces evaluated; ( A ) SLA ® Surface, ( B ) Nanoblast Plus®Surface, and (C) IPX Machined Surface. Within each experimental group, half of the implants were placed in the healed ridge, and the counterparts were located into fresh extraction sockets. The IPX-Control implants were always inserted at the most distal site (M 1 ), but the rest of the implants were randomly assigned using closed envelopes to distinct premolar sites (P1–P4). 2.5. Maintenance Procedures The protocol for postoperative care included a daily dose of cefovecin sodium (8 mg/kg/ Convenia ® , Zoetis, Spain) and morphine (0.5 mg/Kg/day) injected intramuscularly during five days, as well as meloxicam (0.2 mg/Kg/p.o.), dispensed with meals. The sutures were removed one week post-surgery. Plaque control was performed by brushing the dogs’ teeth three times per week, with a 2% chlorhexidine gel. Materials 2020,13, 802 5 of 13 2.6. Euthanasia Twelve weeks after implant placement, all animals were euthanized by an overdose of pentobarbital (40–60 mg/Kg/IV), and mandibular biopsies were obtained for histological processing. 2.7. Histological Preparation The tissue blocks containing the implants and surrounding tissues were prepared for non-decalcified sectioning, where blocks were fixed in buffered 10% formaldehyde for at least four days at 4 ◦ C. Afterward, the specimens were dehydrated in a series of ascending concentrations of ethanol solutions for 72 h at each stage (80%, 96%, 100%), and finally embedded in polymethylmethacrylate resin during three weeks at room temperature (until the resin was hard to the touch). Final curing was achieved by placing the samples into a 37 ◦ C oven for 72 h. The blocks were then sectioned with a precision cut-offExakt machine (Exakt Aparatebau GMBH, Norderstedt, Germany) and polished using silicon carbide grit papers in a descending size-grain protocol (from #600 to #4000 grit papers). Using this approach, 2 to 4 slices of 100–200 µ m-thick sections were obtained buccolingually for the implants and subsequently analyzed. The specimens were stained using the Levai Laczko technique [ 17 ] (Figures 3and 4). Materials 2020, 13, x FOR PEER REVIEW 5 of 13 2.5. Maintenance Procedures The protocol for postoperative care included a daily dose of cefovecin sodium (8 mg/kg/ Convenia® , Zoetis, Spain) and morphine (0.5 mg/Kg/day) injected intramuscularly during five days, as well as meloxicam (0.2 mg/Kg/p.o.), dispensed with meals. The sutures were removed one week post-surgery. Plaque control was performed by brushing the dogs’ teeth three times per week, with a 2% chlorhexidine gel. 2.6. Euthanasia Twelve weeks after implant placement, all animals were euthanized by an overdose of pentobarbital (40–60 mg/Kg/IV), and mandibular biopsies were obtained for histological processing. 2.7. Histological Preparation The tissue blocks containing the implants and surrounding tissues were prepared for nondecalcified sectioning, where blocks were fixed in buffered 10% formaldehyde for at least four days at 4 °C. Afterward, the specimens were dehydrated in a series of ascending concentrations of ethanol solutions for 72 h at each stage (80%, 96%, 100%), and finally embedded in polymethylmethacrylate resin during three weeks at room temperature (until the resin was hard to the touch). Final curing was achieved by placing the samples into a 37 °C oven for 72 h. The blocks were then sectioned with a precision cut-off Exakt machine (Exakt Aparatebau GMBH, Norderstedt, Germany) and polished using silicon carbide grit papers in a descending size-grain protocol (from #600 to #4000 grit papers). Using this approach, 2 to 4 slices of 100–200 μm-thick sections were obtained buccolingually for the implants and subsequently analyzed. The specimens were stained using the Levai Laczko technique [17] (Figures 3 and 4). (A) (B) (C) (D) (E) Figure 3. Histological images of immediate implants. (A) Straumann BL; (B) IPX-Std; (C) IPX-Half; (D) IPX-Full; (E) IPX-Control. (A) (B) (C) (D) (E) Figure 4. Histological images of delayed implants. (A) Straumann BL; (B) IPX-Std; (C) IPX-Half; (D) IPX-Full; (E) IPX-Control. Photomicrographs were obtained using a Zeiss-Stemi 2000-C photomicroscope (Carl Zeiss Microimaging, Thornwood, NY, USA) at a magnification of 4×. An image analysis program (Image Pro Premier, Media Cybernetics, USA; CellSens, Olympus, Japan) was used to measure the peri- Figure 3. Histological images of immediate implants. ( A ) Straumann BL; ( B ) IPX-Std; ( C ) IPX-Half; ( D ) IPX-Full; (E) IPX-Control. Materials 2020, 13, x FOR PEER REVIEW 5 of 13 2.5. Maintenance Procedures The protocol for postoperative care included a daily dose of cefovecin sodium (8 mg/kg/ Convenia® , Zoetis, Spain) and morphine (0.5 mg/Kg/day) injected intramuscularly during five days, as well as meloxicam (0.2 mg/Kg/p.o.), dispensed with meals. The sutures were removed one week post-surgery. Plaque control was performed by brushing the dogs’ teeth three times per week, with a 2% chlorhexidine gel. 2.6. Euthanasia Twelve weeks after implant placement, all animals were euthanized by an overdose of pentobarbital (40–60 mg/Kg/IV), and mandibular biopsies were obtained for histological processing. 2.7. Histological Preparation The tissue blocks containing the implants and surrounding tissues were prepared for nondecalcified sectioning, where blocks were fixed in buffered 10% formaldehyde for at least four days at 4 °C. Afterward, the specimens were dehydrated in a series of ascending concentrations of ethanol solutions for 72 h at each stage (80%, 96%, 100%), and finally embedded in polymethylmethacrylate resin during three weeks at room temperature (until the resin was hard to the touch). Final curing was achieved by placing the samples into a 37 °C oven for 72 h. The blocks were then sectioned with a precision cut-off Exakt machine (Exakt Aparatebau GMBH, Norderstedt, Germany) and polished using silicon carbide grit papers in a descending size-grain protocol (from #600 to #4000 grit papers). Using this approach, 2 to 4 slices of 100–200 μm-thick sections were obtained buccolingually for the implants and subsequently analyzed. The specimens were stained using the Levai Laczko technique [17] (Figures 3 and 4). (A) (B) (C) (D) (E) Figure 3. Histological images of immediate implants. (A) Straumann BL; (B) IPX-Std; (C) IPX-Half; (D) IPX-Full; (E) IPX-Control. (A) (B) (C) (D) (E) Figure 4. Histological images of delayed implants. (A) Straumann BL; (B) IPX-Std; (C) IPX-Half; (D) IPX-Full; (E) IPX-Control. Photomicrographs were obtained using a Zeiss-Stemi 2000-C photomicroscope (Carl Zeiss Microimaging, Thornwood, NY, USA) at a magnification of 4×. An image analysis program (Image Pro Premier, Media Cybernetics, USA; CellSens, Olympus, Japan) was used to measure the peri- Figure 4. Histological images of delayed implants. ( A ) Straumann BL; ( B ) IPX-Std; ( C ) IPX-Half; ( D ) IPX-Full; (E) IPX-Control. Photomicrographs were obtained using a Zeiss-Stemi 2000-C photomicroscope (Carl Zeiss Microimaging, Thornwood, NY, USA) at a magnification of 4 × . An image analysis program (Image Pro Premier, Media Cybernetics, USA; CellSens, Olympus, Japan) was used to measure the peri-implant tissue healing based on the following landmarks: Implant shoulder (IS); the most coronal point of contact between bone and implant (B); the margin of the peri-implant mucosa (PM); and the apical border of the junctional epithelium (aJE). These topographic references (Figure 5) were used to measure the following linear parameters (mm): The supracrestal soft tissue height (the distance between PM and B); the epithelial attachment Materials 2020,13, 802 6 of 13 length (the distance between PM and aJE); the connective adaptation heights (the distance between aJE and B); and the marginal bone loss (the distance between IS and B). In addition, the percentage of bone-to-implant contact (BIC) was measured in both the buccal and lingual sides of the implants. Materials 2020, 13, x FOR PEER REVIEW 6 of 13 implant tissue healing based on the following landmarks: Implant shoulder (IS); the most coronal point of contact between bone and implant (B); the margin of the peri-implant mucosa (PM); and the apical border of the junctional epithelium (aJE). These topographic references (Figure 5) were used to measure the following linear parameters (mm): The supracrestal soft tissue height (the distance between PM and B); the epithelial attachment length (the distance between PM and aJE); the connective adaptation heights (the distance between aJE and B); and the marginal bone loss (the distance between IS and B). In addition, the percentage of bone-to-implant contact (BIC) was measured in both the buccal and lingual sides of the implants. Figure 5. Diagram illustrating the landmarks for histological evaluation. IS—implant shoulder; B— most coronal bone-to-implant contact location; C—top of the alveolar crest; aJE—the apical border of the junctional epithelium; PM—the top of the margin of the peri-implant mucosa. 2.8. Data Analysis Mean values and standard deviations (SD) were calculated for each outcome variable. Differences between the immediate and delayed implant sites were analyzed using Student’s t-test for paired observations. Differences between the distinct groups of implants were analyzed by ANOVA with post hoc Bonferroni corrections. Spearman correlation coefficients were used to evaluate the linear relationship between quantitative and ordinal variables. Finally, backward stepwise linear regression models were calculated for predicting both supracrestal soft tissue adaptation length and the average marginal bone loss. The level of significance was established at 0.05. All analyses were carried out with SPSS 18.0 (SPSS Inc., Chicago, IL, USA) software. Data were corrected with SUDAAN 11 Statistical Software (RTI International, SC, USA) for the autocorrelation of all variables derived from this split-mouth design study. 3. Results All implants healed uneventfully without clinical signs of inflammation in the peri-implant mucosa. The supracrestal soft tissues were comprised of a dense connective tissue and a mature junctional epithelium. The connective tissue was rich in collagen, poor in vessels, and in some cases, with only a few scattered inflammatory cells at the level of the implant shoulder. Adjacent to this area, collagen fibers ran parallel to the long axis of the implant. Longer connective and epithelial layers were identified among immediate implants as compared to the delayed implants. Figure 5. Diagram illustrating the landmarks for histological evaluation. IS—implant shoulder; B—most coronal bone-to-implant contact location; C—top of the alveolar crest; aJE—the apical border of the junctional epithelium; PM—the top of the margin of the peri-implant mucosa. 2.8. Data Analysis Mean values and standard deviations (SD) were calculated for each outcome variable. Differences between the immediate and delayed implant sites were analyzed using Student’s t-test for paired observations. Differences between the distinct groups of implants were analyzed by ANOVA with post hoc Bonferroni corrections. Spearman correlation coefficients were used to evaluate the linear relationship between quantitative and ordinal variables. Finally, backward stepwise linear regression models were calculated for predicting both supracrestal soft tissue adaptation length and the average marginal bone loss. The level of significance was established at 0.05. All analyses were carried out with SPSS 18.0 (SPSS Inc., Chicago, IL, USA) software. Data were corrected with SUDAAN 11 Statistical Software (RTI International, SC, USA) for the autocorrelation of all variables derived from this split-mouth design study. 3. Results All implants healed uneventfully without clinical signs of inflammation in the peri-implant mucosa. The supracrestal soft tissues were comprised of a dense connective tissue and a mature junctional epithelium. The connective tissue was rich in collagen, poor in vessels, and in some cases, with only a few scattered inflammatory cells at the level of the implant shoulder. Adjacent to this area, collagen fibers ran parallel to the long axis of the implant. Longer connective and epithelial layers were identified among immediate implants as compared to the delayed implants. The supracrestal soft tissue height (formerly: biological width) (PM-B; Table 2) was significantly smaller at the lingual than at the buccal bony plate; This difference was statistically significant for both immediately placed and delayed implants (p <0.001). Moreover, the supracrestal soft tissue height was significantly larger for implants immediately placed into extraction sockets (IC95% 3.9–4.9 mm) than Materials 2020,13, 802 7 of 13 for the delayed placed implants (IC95% 3.1–3.5 mm). Intergroup comparisons using ANOVA showed that the supracrestal soft tissue height at the lingual side was significantly shorter for Strauman BL (1.8 ±0.2 mm) implants than for IPX-Std (3.0 ±0.5 mm) or IPX-Control (2.9 ±1.1 mm) delayed implants. Table 2. Comparison of the biological width (the distance between PM and B) between deferred and immediate implants in both the buccal and lingual sides (mm) among the implant subgroups. * Statistically significant inter-group comparisons according to ANOVA test (p<0.05). The letters (a,b) next to the symbol * indicate the groups that are statistically different after Post Hoc Bonferroni correction. Biological Width Immediate Implants (n =40) Delayed Implants (n =40) Comparisons by Student T; p-Value Buccal Lingual Buccal Lingual Buccal Lingual Mean (sd) Mean (sd) Mean (sd) Mean (sd) Strauman BL 3.8(0.7) 3.1(1.7) * 3.2(0.6) 1.8(0.2) *,a T=1.6 p=0.13 T=2.2 p=0.04 IPX-Std 4.4(2.3) 3.9(1.0) * 4.0(0.7) 3.0(0.5) *,b T=0.4 p=0.68 T=2.1 p=0.05 IPX-Half 4.6(2.4) 4.4(2.3) 4.2(1.1) 2.9(0.5) T=0.4 p=0.72 T=1.8 p=0.13 IPX-Full 4.5(1.8) 3.9(1.1) * 4.1(0.3) 2.7(0.6) * T=0.6 p=0.56 T=2.5 p=0.03 IPX-Control 5.1(1.1) 4.7(1.3) * 4.3(0.6) 2.9(1.1) *,b T=1.5 p=0.18 T=2.8 p=0.02 Comparisons by ANOVA: p-value F=0.4 p=0.79 F=1.1 p=0.40 F=2.1 p=0.11 F=3.3 p=0.02 Regarding the width of the epithelial barrier (Table 3), this was significantly shorter for delayed (IC95% 1.5–1.8 mm) than for immediately placed implants (IC95% 1.9–2.5 mm). No statistically significant differences resulted from the inter-group comparison by ANOVA. Table 3. Comparison of the epithelial width (the distance between PM and aJE) between deferred and immediate implants in both the buccal and lingual sides (mm) among the implant subgroups. * Statistically significant inter-group comparisons according to ANOVA test (p<0.05). Epithelial Width Immediate Implants (n =40) Delayed Implants (n =40) Comparisons by Student T; p-Value Buccal Lingual Buccal Lingual Buccal Lingual Mean (sd) Mean (sd) Mean (sd) Mean (sd) Strauman BL 2.0(0.6) 2.1(1.7) 2.2(0.5) 0.9(0.3) T=0.5 p=0.65 T=1.8 p=0.11 IPX-Std 2.6(1.3) 2.0(1.1) 2.1(0.9) 1.4(0.5) T=0.9 p=0.39 T=1.3 p=0.22 IPX-Half 2.7(1.3) 2.1(1.1) 2.2(0.5) 1.2(0.7) T=1.1 p=0.30 T=1.9 p=0.08 IPX-Full 2.9(1.0) * 1.9(0.6) 2.1(0.5) * 1.3(0.7) T=2.1 p=0.05 T=1.8 p=0.10 IPX-Control 2.4(0.9) 1.8(0.5) 2.3(0.6) 1.3(0.5) T=0.2 p=0.82 T=1.9 p=0.08 Comparisons by ANOVA; F=0.7 p=0.60 F=0.1 p=0.97 F=0.1 p=0.97 F=0.8 p=0.53 p-value The “connective tissue attachment” was significantly wider at immediately placed (IC95% 1.8–2.6 mm) than at delayed implants (IC95% 1.5–1.8 mm) (Table 4). Moreover, for delayed implants, the Materials 2020,13, 802 8 of 13 buccal connective tissue seal was significantly thinner for Straumann BL (1.1 ± 0.5 mm) than for IPX-half (2.0 ±1.0 mm) implants. Table 4. Comparison of the connective width (the distance between aJE and B) between delayed and immediate implants in both the buccal and lingual sides (mm) among the implant subgroups. * Statistically significant inter-group comparisons according to ANOVA test (p<0.05). The letters (a,b) next to the symbol * indicate the groups that are statistically different after post hoc Bonferroni correction. Connective Width Immediate Implants (n =40) Delayed Implants (n =40) Comparisons by Student T; p-Value Buccal Lingual Buccal Lingual Buccal Lingual Mean (sd) Mean (sd) Mean (sd) Mean (sd) Strauman BL 1.8(0.6) * 1.1(0.5) a1.1(0.5) *,a 1.0(0.2) T=2.5 p=0.03 T=0.3 p=0.75 IPX-Std 2.1(1.5) 2.0(1.2) 1.9(0.5) 1.6(0.5) T=0.3 p=0.75 T=0.7 p=0.51 IPX-Half 2.2(1.4) 2.3(1.5) 2.0(1.0) b1.6(0.5) T=0.3 p=0.75 T=1.1 p=0.30 IPX-Full 1.6(1.0) 2.0(0.6) * 2.0(0.6) 1.5(0.2) * T=0.8 p=0.45 T=2.2 p=0.05 IPX-Control 2.7(1.2) 3.0(1.3) *,b 2.0(0.2) 1.7(0.8) * T=1.6 p=0.16 T=2.2 p=0.05 Comparisons by ANOVA; p-value F=0.8 p=0.54 F=2.7 p=0.05 F=2.6 p=0.05 F=2.0 p=0.13 The marginal bone loss (IS-B) tended to be higher for immediately placed (IC95% 0.4–0.9 mm) than for delayed implants (IC95% 0.3–0.8 mm), although the difference did not reach statistical significance (Table 5). The inter-group comparison by ANOVA was not significant either, but it was observed that the marginal bone loss was inversely correlated (r s = − 0.46; p=0.008) with the extension of the modified platform (null: Strauman and IPX-Std; half: IPX-Half; Full: IPX-Full), but only for delayed implants. Table 5. Comparison of the marginal bone loss (the distance between IS and B) between delayed and immediate implants in both the buccal and lingual sides (mm) among the implant subgroups. * Statistically significant inter-group comparisons according to ANOVA test (p<0.05). Marginal Bone Loss Immediate Implants (n =40) Delayed Implants (n =40) Comparisons by Student T; p-Value Buccal Lingual Buccal Lingual Buccal Lingual Mean (sd) Mean (sd) Mean (sd) Mean (sd) Strauman BL 1.3(0.8) * 0.6(0.3) 0.6(0.4) * 0.4(0.3) T=2.5 p=0.03 T=1.6 p=0.13 IPX-Std 0.3(0.9) 0.3(0.4) 0.2(0.3) 0.4(0.5) T=0.4 p=0.70 T=0.2 p=0.83 IPX-Half 0.8(0.6) 0.5(0.4) * 0.7(1.3) 0.1(0.3) * T=0.2 p=0.81 T=2.3 p=0.04 IPX-Full 1.0(0.9) 0.3(0.4) 0.3(0.6) 0.2(0.4) T=1.8 p=0.10 T=0.4 p=0.68 IPX-Control 0.8(1.4) 0.9(0.8) 1.6(2.2) 0.9(1.1) T=0.9 p=0.31 T=0.0 p=0.99 Comparisons by ANOVA; p-value F=1.2 p=0.35 F=2.0 p=0.13 F=1.9 p=0.14 F=2.0 p=0.12 The differences in BIC% between delayed implants (IC95% 60.6%–72.6%) and immediately placed implants (IC95% 53.1%–65.5%) was statistically significant (Table 6). Materials 2020,13, 802 9 of 13 Table 6. Comparison of the BIC (bone-to-implant-contact) between delayed and immediate implants in both the buccal and lingual sides (mm) among the implant subgroups. The letters (a,b) indicate the groups that are statistically different after post hoc Bonferroni correction. BIC Immediate Implants (n =40) Delayed Implants (n =40) Comparisons by Student T; p-value Mean (sd) Mean (sd) Strauman BL 54.8(22.9) 64.6(19.1) T =0.9 p=0.37 IPX-Std 62.3(16.3) 68.6(21.3) T =0.7 p=0.52 IPX-Half 72.5(12.8) a72.7(9.8) T =0.0 p=0.97 IPX-Full 63.9(19.6) 74.2(15.6) T =1.2 p=0.26 IPX-Control 43.2(15.2) b52.9(21.3) T =1.1 p=0.31 Comparisons by ANOVA; p-value F=3.1 p=0.03 F =1.8 p=0.15 The results of the linear regression analyses (Table 7) show that the supracrestal soft tissue height around dental implants ranged, on average, from 2.9 to 4.1 mm. This height was significantly reduced (between 0.5–1.6 mm), as implants were placed into healed ridges, and significantly increased according to the extension of platform modification (0.3–1.9 mm), and compared to the healing of implants with non-modified platforms (IPX-Std and Straumann). Likewise, the average marginal bone loss ranged from 0.6 to 1.1 mm, and this loss could be minimized by 0.1–0.5 mm when the implants were inserted into healed bone or when the platform of the implants was modified (0.1–0.8 mm); in that, the greater the size of the area modified on the platform, the smaller the amount of bone loss. Table 7. Linear regression model (backward stepwise) for predicting the average biological width and the average marginal bone loss after including all the potential predictors (time of implant placement and grade of surface treatment). * For these analyses, the mechanized implants placed on the M 1 were excluded to assess the true effect of the platform configuration. † F=6.97: df:2; p-value =0.001. R 2 = 0.29. ‡F=3.17: df:2; p-value =0.031. R2=0.14. Dependent Variable/Parameters Standardized βp-Value βCI-95% β Lower Upper Average Biological Width † Constant (mm) <0.001 3.5 2.9 4.1 Time of placement (immediate as reference) −0.43 0.001 −1.0 −0.5 −1.6 Grade of surface treatment (none/half/full) 1.0 0.007 1.1 0.3 1.9 Average Marginal Bone Loss ‡ Constant (mm) <0.001 0.8 0.6 1.1 Time of placement (immediate as reference) −0.24 0.04 −0.3 −0.1 −0.5 Grade of surface treatment (none/half/full) −0.83 0.02 −0.4 −0.1 −0.8 4. Discussion This experimental study evaluated the influence of the surface roughness features at the implant platform on the peri-implant soft tissue and marginal bone loss, after 12 weeks of non-submerged healing, when implants were placed immediately or 12 weeks after tooth extraction. In the present study, three types of controls were used, the negative control (IPX-Control) providing an implant with a turned surface without modification (Figure 1). The positive control was a commercially available IPX implant (IPX-Std), with a non-modified platform, but an implant surface modified with a mechanochemical method (Nanoblast Plus ® ). The reference control was a Straumann BL implant, with the implant surface being modified with a different mechanochemical process (Sandblast, Large-Grit, Acid-Etching), but with a non-modified platform. The use of different types of IPX implants with the same dimensions (diameter and length), thread design, and surface treatment, but with different