Tomaso Mainetti Niklaus P. Lang Franco Bengazi Vittorio Favero Luis Soto Cantero Daniele Botticelli
Sequential healing at implants installed immediately into extraction sockets. An experimental study in dogs
Authors’ affiliations: Tomaso Mainetti, Franco Bengazi, Vittorio Favero, Luis Soto Cantero, Daniele Botticelli, Faculty of Dentistry, University of Medical Science, La Habana, Cuba Niklaus P. Lang, Center for Dental Medicine, University of Zurich, Zurich, Switzerland; University of Bern, Bern, Switzerland Daniele Botticelli, ARDEC, Ariminum Odontologica, Rimini, Italy Daniele Botticelli, UNESP – Faculdade de Odontologia de Aracßatuba, UNESP – Universidade Estadual Paulista, S~ ao Paulo, Brasil
Key words: animal study, extraction sockets, histology, implants placed immediately into
Corresponding author: Dr Daniele Botticelli Avenida Salvador Allende y G Vedado, La Habana, Cuba Tel.: 537-879-3360 Fax: 537-870-3312 e-mail: [email protected]
extraction sockets (IPIES) of the fourth premolar and in the healed sites in the molar regions.
extraction sockets, sequential healing, wound healing Abstract Objective: To compare the sequential healing at implants installed in a healed alveolar bony ridge or immediately after tooth extraction without functional load. Material and methods: In the mandible of 12 dogs, the mesial roots of the first molars were endodontically treated, the tooth hemisected, and the distal roots extracted. After 3 months, the mesial roots of the fourth premolars were endodontically treated, the tooth hemisected, and the distal roots extracted in one side of the mandible. Implants were placed immediately into Healing abutments were placed, and the flaps were sutured to allow a non-submerged healing. The time of surgery and of sacrifices were planned in such a way to obtain biopsies representing the healing after 1 and 2 weeks and 1 and 3 months, respectively. Ground sections were prepared for histological evaluation of tissues components on the implant surface and the coronal termination level of osseointegration (M-B). Results: New bone apposition on the implant surface was slightly higher at the healed compared to the IPIES sites, being 7.4% and 4.1% after 1 week, and 67.3% and 65.3% after 3 months, respectively. Old bone was progressively resorbed, from 27.0% and 21.9% after 1 week, to 2.5% and 2.0% after 3 months, at healed and IPIES sites, respectively. M-B was 1.4 mm and 2.6 mm after 1 week, 1.2 mm and 1.2 mm after 3 months, at healed and IPIES sites, respectively. Conclusions: Similar patterns of sequential osseointegration were found at implants installed in healed alveolar bone or in alveolar sockets immediately after tooth extraction. The coronal termination level of osseointegration, that was different after 1 week, was found similar at the 3-month observation.
Date: Accepted 21 November 2014 To cite this article: Mainetti T, Lang NP, Bengazi F, Favero V, Soto Cantero L, Botticelli D. Sequential healing at implants installed immediately into extraction sockets. An experimental study in dogs. Clin. Oral Impl. Res. 00, 2014, 1–9 doi: 10.1111/clr.12533
Implants placed immediately into extraction sockets (IPIES, Sicilia & Botticelli 2012) have been shown to have a high rate of clinical success (for review, see Quirynen et al. 2007; Lang et al. 2012). A large number of animal experiments have shown, however, that the immediate installation of implants does not prevent the resorption of the alveolar ridges (e.g., Ara ujo et al. 2005; Botticelli et al. 2006; Caneva et al. 2010). The sequential healing at IPIES has been described in animal experiments (e.g., Ara ujo et al. 2006a,b; Vignoletti et al. 2009a,b; Blanco et al. 2013; Mainetti et al. 2014). In an experiment in dogs (Ara ujo et al. 2006b), implants were placed in the distal alveoli of the third premolars and of the first molars immediately after tooth extraction. In the molar region, a large gap resulted
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
after installation. It was observed that, the level of osseointegration was located at 1.5 mm apically of the coronal termination of the rough implant surface after 4 weeks while, after 12 weeks, the level was located at 0.8 mm of that landmark. This pattern of healing was also confirmed in animal studies (Botticelli et al. 2003a,b) in which marginal defects, 5 mm deep and 1.25 mm wide, were prepared in the region of implants installed in the alveolar bony ridge in the mandible of dogs. It was shown that bone apposition onto the implant surface started from the bottom of the defects and gradually developed coronally. A progressive gain in the level of osseointegration from 1.8 mm after 1 month, 3.1 mm after 2 months, and 4.5 mm after 4 months of healing was observed.
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Mainetti et al Sequential healing at implants
The sequential processes of osseointegration has been described both in humans (Bosshardt et al. 2011; Donos et al. 2011; Ivanovski et al. 2011; Lang et al. 2011) and animal studies (Mainetti et al. 2014; Rossi et al. 2014). In an animal study (Mainetti et al. 2014), the sequential healing at IPIES was compared with that of control implants installed in healed sites. All implants were immediately loaded. It was concluded that the two sites presented different patterns of healing during the early phases, but no statistically significant differences were revealed for the hard and soft tissues dimensions after 3 months following implant installation. However, there is a lack of comparative studies of healing at IPIES and control implants installed in healed alveolar ridges without loading. Hence, the aim of this study was to compare the sequential healing at implants installed in healed alveolar bony ridges or immediately after tooth extraction without functional loading.
Material and methods The research protocol was submitted to and approved by the Ethical Committee of the University of Medical Sciences, School of Dentistry, La Habana, Cuba. Twelve beagle dogs, with a mean body weight of 11 kg and a mean age of 1 year, were provided by Centro Nacional para la Producci on de Animales de Laboratorio (CENPALAB) of La Habana, Cuba. The animals were kept during the experimental period in kennels and on concrete runs at the university’s field laboratory with free access to water and feed of moistened balanced dog’s chow. Clinical procedures
Before the surgical sessions, the animals received atropine 0.02 mg/kg i.v. (Mayne Pharma, Napoli, Italia), metedomidine 0.04 mg/kg (Medetorâ; Virbac, Glattbrugg, Switzerland), and ketamine 50 5 mg/kg (Liorad, La Habana, Cuba) mixed in a syringe. The anesthesia was maintained with 2–3% Isoflurane-Vetâ (Merial, Merial Tolosa, France) and O2 at 95%. An intravenous infusion of 0.9% saline solution 10 ml/kg/h was kept during the surgeries, while blood pressure and O2 perfusion were constantly monitored. Local anesthesia was also provided. At the first surgical session, in both sides of the mandible, the mesial root of the first
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molar was endodontically treated, and subsequently, the tooth was hemisected and the distal root extracted. After 3 months of healing, in one randomly selected side of the mandible, the endodontic treatment of the mesial root of the fourth premolar was performed (Mtwoâ, Endopocketâ, Epfillâ; Sweden & Martina, Due Carrare, Padova, Italy), and the crown restored with composite. Full thickness flaps were elevated, and the buccal and lingual alveolar bony plates were exposed. The forth premolar was hemisected, and the distal root removed including the corresponding portion of the crown. Two customized implants, 8.5 mm long and 3.3 mm in diameter (Sweden & Martina) with a ZirTiâ surface were installed, one in the position of M1 (healed site), and one in the distal alveolus of P4 (IPIES site; Fig. 1a,b). All implants were placed with the coronal termination of the rough surface flush with the alveolar buccal bony crest. Subsequently, healing abutments were affixed. The flaps were carefully adapted around the healing abutments using single interrupted resorbable sutures (Vicrylâ 4–0; Johnson & Johnson, Medical S.p.A., Pomezia, Roma; Fig. 1c). No
occlusal contact with the maxillary teeth was allowed (Fig. 1d). The treatment in the other side of the mouth and sacrifices were scheduled in such a way as to collect biopsies representing the healing after 1 and 2 weeks, and 1 and 3 months. Antibiotics (Conveniaâ 852 mg.; Pfizer, New York, NY, USA) and anti-inflammatory/ analgesics 2 mg/kg tramadol (Altadolâ; Formevet, Milan, Italy) were administered for 5 days after surgery. An inspection of the wounds for clinical signs of complications and healing abutments cleaning were performed three times per week for the entire duration of the experiment. At the sacrifice, the animals were first anesthetized with a dose of 1 mg/kg of xylazine (Rompunâ, Kiel, Germany) and 10 mg/ kg of ketamine (Lioradâ, La Habana, Cuba) mixed in a syringe and administered i.m. and maintained with 2–3% Isoflurane-Vetâ (Merial, Merial Tolosa, France) and O2 95%. Eparin 12 i.u./kg i.v. (Athena Pharma, Pomezia, Italy) was injected before the heart was arrested with 25 meq of potassium chloride i.v. (Aica, La Habana, Cuba). The carotid arteries were perfused with a fixative (4% of formaldehyde solution).
(a)
(b)
(c)
(d)
Fig. 1. Clinical procedures. (a) Implants installed in a healed site in the first molar region (yellow arrow) and in the distal alveolus of the fourth premolar (light blue arrow). (b) A marginal defect resulted around the implant placed into the extraction socket. (c) The flaps were sutured to allow a non-submerged healing. (d) No occlusal contacts were allowed between the abutments and the teeth of the opposing jaw.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mainetti et al Sequential healing at implants
Histological preparation
The mandibles were removed and maintained in 4% formaldehyde solution. The histological procedures were performed in the laboratory of histology at the Faculty of Odontology of the University of La Habana. Block sections, each containing one implant, were obtained, dehydrated in a series of graded ethanol and subsequently embedded in resin (Technovitâ 7200 VLC; Kulzer, Friedrichsdorf, Germany). The blocks were cut in a bucco-lingual plane using a diamond band saw fitted in a precision slicing machine (Exaktâ; Apparatebau, Norderstedt, Germany). One central section was selected and
reduced to a thickness of about 50–60 lm using a cutting–grinding device (Exaktâ; Apparatebau). The histological slides were stained with Stevenel’s blue and alizarin red and examined under a standard light microscope for histometric analysis. Histological evaluations
The histological measurements were performed in an Eclipse Ci microscope (Nikon Corporation, Tokyo, Japan), equipped with a digital video camera (Digital Sight DS-2Mv; Nikon Corporation) connected to a computer, and using the software NIS-Elements D 4.10 (Laboratory Imaging; Nikon Corporation). The following landmarks were identified (Fig. 2): (IS) the shoulder of the implant, (B) the most coronal bone-to-implant contact. The apical termination of the implant was identified as well (A). The vertical distances between IS and B (IS-B) were assessed at 9100 magnification and, subsequently, the distances between the coronal termination of the rough surface (M) and B (distance M-B) were obtained by subtracting the height of the neck of the implant to IS-B. The percentages of new bone, old bone, soft tissues (connective matrix, bone marrow at various stages of maturation, etc.), clot, and bone debris/particles in contact with the implant surface were measured between B and A at a magnification 9200. The total amount of mineralized bone-to-implant contact percentage (MBIC%) was calculated (new + old bone). Data analysis
Fig. 2. Diagram illustrating the landmarks for the histological evaluation. (IS) the shoulder of the implant; (M) coronal end of the rough surface; (B) the most coronal bone-to-implant contact.
Six animals represented each period of healing (n = 6). Mean values and standard deviations as well as 25th, 50th (median), and 75th percentiles were calculated for each
outcome variable. The main outcome variables were percentage of new bone and the distance M-B. For statistical analyses, the IBM SPSS Statistics 19 software was used (IBM Inc., Chicago, IL, USA). The Wilcoxon signed rank test for dependent variables was used to assess differences between healed and IPIES sites. Moreover, as explorative investigation, the Mann–Whitney U-test for independent variables was used to analyze differences between periods. The level of significance was set at a = 0.05.
Results No major signs of inflammation were seen during the healing period, and at the time of sacrifice, all implants were still in function and stable. Histological evaluation
No artifacts were generated during histological processing nor were any tissue blocks destroyed. Hence, IPIES and healed sites each yielded an n = 6. IPIES sites
Mean values, standard deviations, medians and 25th–75th percentiles are reported in Table 1, for the percentage of tissues in contact with the implant surface, and in Table 2, for the distance between the coronal termination of the rough surface (M) and the most coronal contact of bone to the implant (B). Mean values are also illustrated in Fig. 3 for tissues in contact with the implant surface and in Fig. 4 for M-B. In the text, only mean values and standard deviations are reported. At the IPIES sites, after 1 week of healing, bone defects were still present between the
Table 1. Tissues in contact with the implant surface in percentage (%) at the IPIES sites New bone 1 week Mean (SD) Median (25th–75th) 2 weeks Mean (SD) Median (25th–75th) 1 month Mean (SD) Median (25th–75th) 3 months Mean (SD) Median (25th–75th)
Old bone
MBIC
Bone debris/particles
SOFT
CLOT
21.9 (9.8) 19.4 (18.6–23.8)‡
26.0 (10.3) 24.3 (22.0–29.5)†,‡
9.8 (5.0) 10.0 (6.8–13.7)
50.5 (14.9) 51.5 (46.4–57.8)
13.7 (5.7) 15.4 (12.4–17.4)‡
16.3 (6.2) 17.4 (13.1–18.6)†
21.6 (6.8) 23.0 (20.2–26.6)
38.0 (10.1) 38.9 (35.7–41.3)†
11.6 (2.4) 11.5 (9.8–13.5)*,†
40.4 (9.9) 35.2 (34.8–47.1)
9.9 (3.5) 10.7 (7.9–12.9)†
46.1 (10.8) 50.2 (36.9–53.2)
19.8 (6.9) 21.3 (18.3–24.5)†
65.8 (12.3) 63.5 (58.1–74.3)
2.8 (2.6) 1.6 (1.2–3.7)†
31.4 (11.6) 35.5 (22.2–40.3)
0.0 (0.0) 0.0 (0.0–0.0)
65.3 (22.8) 74.8 (53.7–80.2)‡
2.0 (0.7) 2.1 (1.9–2.2)‡
67.3 (23.0) 77.0 (56.4–82.3)‡
0.0 (0.0) 0.0 (0.0–0.0)‡
32.7 (23.0) 23.0 (17.7–43.6)
0.0 (0.0) 0.0 (0.0–0.0)‡
4.1 (1.5) 3.7 (3.4–5.1) *,†,‡
IPIES, implants placed immediately into extraction sockets; MBIC, total amount of mineralized bone-to-implant contact percentage, calculated as new + old bone; SOFT, soft tissue (connective matrix, bone marrow, etc.); CLOT, clot residues. *P < 0.05 between test and control. †P < 0.05 between one period of healing and the subsequent. ‡P < 0.05 between 1 week and 3 months of healing. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Table 2. Distance (M-B) between the coronal margin of the rough surface (M) and the coronal level of osseointegration (B) at the buccal (b) and lingual (l) aspects IPIES sites
1 week Mean (SD) Median (25th–75th) 2 weeks Mean (SD) Median (25th–75th) 1 month Mean (SD) Median (25th–75th) 3 months Mean (SD) Median (25th–75th)
Healed sites
b
l
b
l
2.6 (1.0) 2.6* (2.0–2.9)†
2.4 (1.6) 2.0 (1.3–3.6)
1.4 (0.5) 1.4* (1.2–1.8)
1.2 (0.7) 1.0 (0.8–1.1)
1.9 (0.3) 2.0 (1.7–2.1)
1.2 (0.4) 1.0 (0.9–1.5)
1.1 (0.7) 0.9 (0.6–1.3)
1.0 (0.9) 0.7 (0.6–1.4)
1.4 (0.7) 1.1 (1.0–2.0)
0.6 (0.6) 0.6 (0.1–1.0)
0.8 (0.3) 0.9 (0.7–1.0)
0.5 (0.5) 0.8 (0.1–0.9)
1.2 (0.5) 1.2 (0.9–1.3)†
0.8 (0.7) 0.6 (0.5–0.7)
1.2 (0.5) 1.2 (1.1–1.5)
1.1 (0.7) 0.8 (0.7–1.0)
*P < 0.05 between test and control. †P < 0.05 between 1 week and 3 months of healing.
Fig. 3. Composition of the tissues in contact with the implant surface at the implants placed immediately into extraction sockets (IPIES) sites. NEW, new bone; OLD, old bone; DEB/PART, bone debris and bone particles; SOFT, soft tissues (connective matrix, bone marrow, etc.); CLOT, clot residues.
Fig. 4. Graphic representing the mean values of the distances M-B at the buccal (b) and lingual (l) aspects at the various periods of healing. M = coronal end of the rough surface; B = most coronal bone-to-implant contact.
walls of the parent bone and the implant surface (Fig. 5a). The implants presented threads inserted into the parent old bone (Fig. 5b). New bone formation was found within the bottom of the remaining defect forming woven bone struts, partially calcified and partially as connective tissue matrix and osteoid
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tissue, bridging the bony walls of the extraction socket and the implant surface (Fig. 5c). Bone particles and bone debris were often found on the implant surface and incorporated into the struts. Similar struts were found in regions of the implant originally installed into the parent bone (Fig. 5d). A
percentage of 4.1 1.5% of newly formed bone was found on the implant surface, while old bone was still present at a higher quantity (21.9 9.8%). A total amount of 26.0 10.3% of mineralized bone (MBIC%) was observed. Bone particles and bone debris were found on the implant surface at a proportion of about 10%. Soft tissues were present at a percentage of about 51%. Approximately, 14% of the surfaces were still covered by a clot in various stages of degeneration. Signs of bone resorption were seen on bundle bone and in parent bone, however, rarely in vicinity of the implant surface. M-B at the buccal aspect was 2.6 1.0 mm. After 2 weeks of healing, the marginal defects were still present. However, a larger amount of struts of woven bone, connective provisional matrix, and osteoid compared to the previous period of healing were identified (Fig. 6a). The struts within the defects were connecting the parent bone, also represented by partially resorbed bundle bone, to the newly formed bone on the implant surface (Fig. 6b,c). A higher percentage of new bone was found in contact with the implant surface at this stage of healing (16.3 6.2%), the difference with the previous period of healing being statistically significant. The old bone was still present at a similar percentage of the previous period of healing (21.6 6.8%). A total amount of 38.0 10.1% of mineralized bone (MBIC%) was observed. About 12% of bone debris and bone particles, approximately 40% of soft tissues, and about 10% of residual tissue from disintegrating clot were detected at this stage of healing. Bone resorption was observed, particularly dynamic in the bony crestal region (Fig. 6d). The distance M-B at the buccal aspect was 1.9 0.3 mm, the difference with
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mainetti et al Sequential healing at implants
(a)
(b)
(c)
(d)
Fig. 5. Ground sections illustrating the healing at an implants placed immediately into extraction sockets (IPIES) site after 1 week. Stevenel’s blue and alizarin red stain. (a) Bone defects were still present between the walls of the parent bone and the implant surface. Image grabbed at 920 magnification. (b) Threads appeared to be inserted into the parent old bone. Original magnification 9100. (c) New bone formation was found within the bottom of the remaining defect forming, together the connective provisional matrix, bridges between the bony walls of the extraction socket and the implant surface. Original magnification 9100. (d) Similar bridges were also found in regions of the implant originally installed into the parent bone. Original magnification 9100.
(a)
(b)
(c)
(d)
Fig. 6. Ground sections illustrating the healing at an implants placed immediately into extraction sockets (IPIES) site after 2 weeks. Stevenel’s blue and alizarin red stain. (a) The marginal defects were still present, however, with a larger amount of bridges compared to the previous period of healing. Image grabbed at 920 magnification. (b) The bridges within the defects connected the parent bone, also represented by bundle bone partially resorbed, to the implant surface. Original magnification 9200. (c) The bridges gained attachment with the newly formed bone on the implant surface. Original magnification 9200. (d) Bone resorption was found to be dynamic in the bony crest region. Original magnification 9200.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
the previous period of healing not being statistically significant. After 1 month of healing, the new bone appeared to be more mature (parallel-fibered bone) compared to the previous stages of healing and presented with a percentage of 46.1 10.8%, the difference with the 2-week healing period being statistically significant (Fig. 7a,b). The percentages of old bone and MBIC% were 19.8 6.9% and 65.8 12.3%, respectively. The distance M-B at the buccal aspect was 1.4 0.7 mm, the difference with the previous period of healing not reaching statistical significance. After 3 months of healing, the surrounding bone at the implants appeared to be mature more mature compared to the previous periods of healing (Fig. 7c,d). New bone was found at a percentage of 65.3 22.8 and 6.1 10.8%, and very little old bone could be identified (2.0 0.7%). MBIC% was 67.3 23.0%. No statistically significant differences were found. The distance M-B at the buccal aspect was 1.2 0.5 mm. No statistically significant difference was found with the previous period. However, between 1week and 3-month periods, a statistically significant difference was verified. Healed sites
Mean values, standard deviations, medians and 25th–75th percentiles of the percentage of tissues in contact with the implant surface are reported in Table 3. Mean values are also illustrated in Fig. 8 for tissues in contact with the implant surface and in Fig. 4 for MB. In the text, only mean values and standard deviations are reported. At the healed sites, after 1 week of healing, newly formed bone was found on the surface of the implants connected to the parent bone by struts of newly formed calcified bone, connective tissue matrix, and osteoid tissue (Fig. 9a). These struts often included bone debris and bone particles that could be found within the connective tissue matrix and on the implant surface. Residues of disintegrating clot and signs of resorption were also observed (Fig. 9b). A percentage of 7.4 1.7% and 27.0 6.0% were measured of newly formed and old bone, respectively. A statistically significant difference was found between IPIES and healed sites for the new bone (P = 0.028). A total amount of 34.4 6.9% of MBIC% was observed. Bone debris and bone particles on the implant surface were present in a proportion of about 13%. M-B at the buccal aspect was 1.4 0.5 mm, the difference between IPIES and healed sites being statistically significant (P = 0.027).
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Mainetti et al Sequential healing at implants
(a)
(b)
(c)
(d)
significant at 1 and 3 months of healing, while, comparing the periods of healing longitudinally, only the difference between 2 weeks and 1 month was statistically significant. M-B was 0.8 0.3 mm and 1.2 0.5 mm after 1 and 3 months, respectively. No statistically significant differences were found between IPIES and healed sites at these observation periods (P = 0.068 and 0.916, respectively).
Discussion
Fig. 7. (a, b) Ground sections illustrating the healing at an implants placed immediately into extraction sockets (IPIES) site after 1 month. Stevenel’s blue and alizarin red stain. (a) The bottom of a marginal defect appeared filled with newly formed bone. Remnants of bundle bone were still identifiable (*). Original magnification 9100. (b) The spaces between threads were found to be filled with new bone originating from remnants of bundle bone (*). Original magnification 9100. (c, d) Ground sections illustrating the healing at an IPIES site after 3 months. Stevenel’s blue and alizarin red stain. The bone appeared to be more mature compared to the previous periods of healing. (c) Original magnification 9100. (d) Original magnification 9200.
At the second week of healing, a higher quantity of new bone (25.4 5.6%) compared to the previous period of healing was found on the surface of the implants (Fig. 9c). Struts composed of new bone, osteoid tissue lined by osteoblasts, connective tissue matrix, and bone debris/particles were also observed. Moreover, primary osteons were forming (Fig. 9d). No statistically significant difference was found between IPIES and
healed sites for the new bone. M-B at the buccal aspect was 1.1 0.7 mm, the difference between IPIES and healed sites not being statistically significant (P = 0.058). After 1 and 3 months of healing, bone maturation and remodeling processes were observed (Fig. 10a–d). New bone was found at a percentage of 50.5 8.3% and 67.3 9.4%, respectively. The differences between IPIES and healed sites were not statistically
The present study compared the sequential processes of osseointegration at implants placed immediately into extraction sockets (IPIES) and implants installed in healed sites without functional loading. The patterns of healing were very similar between the two sites. It was shown that new bone, osteoid tissue, and connective matrix were forming struts from the parent bone to the implant surface. These struts included also bone debris and bone particles that seemed to play an important role in the process of osseointegration. This is in agreement with the findings from a histological study in human (Bosshardt et al. 2011). In that study, 49 mini-implants were placed in the retromolar region of 28 human volunteers. After 1, 2, 4, and 6 weeks, biopsies containing the implants were collected and processed for histological analyses. It was concluded that bone debris originating from implant placement contributed to the processes of osseointegration. In the present study, tissue components were evaluated on the implant surface between the most coronal bone to implant contact (B) and the apex of the implant (A). This also means that the remaining defect
Table 3. Tissues in contact with the implant surface in percentage (%) at implants installed in healed sites New bone 1 week Mean (SD) Median (25th–75th) 2 weeks Mean (SD) Median (25th–75th) 1 month Mean (SD) Median (25th–75th) 3 months Mean (SD) Median (25th–75th)
Old bone
MBIC
Bone debris/particles
SOFT
CLOT
27.0 (6.0) 25.3 (22.5–28.8)†,‡
34.4 (6.9) 33.3 (29.2–38.1)†,‡
12.7 (5.4) 12.7 (10.4–14.6)‡
42.9 (7.5) 41.1 (37.9–46.4)‡
10.0 (5.3) 9.1 (5.5–15.0)
25.4 (5.6) 26.3 (23.2–27.1)†
23.4 (2.5) 23.2 (21.5–24.2)†
48.8 (7.1) 50.2 (44.9–53.0)†
6.4 (4.0) 6.3 (3.9–9.7)*
39.3 (6.0) 38.9 (38.1–40.3)†
5.5 (8.3) 2.6 (1.1–4.3)†
50.5 (8.3) 51.0 (47.8–54.8)†
19.1 (6.7) 21.2 (13.6–24.1)†
69.5 (10.0) 67.7 (64.1–72.8)
4.4 (3.2) 4.4 (3.1–5.3)†
26.0 (9.3) 27.4 (22.6–30.6)
0.0 (0.0) 0.0 (0.0–0.0)
67.3 (9.4) 66.8 (60.8–68.6)‡
2.5 (0.7) 2.3 (1.9–3.0)‡
69.8 (9.9) 69.2 (62.8–71.3)‡
0.0 (0.0) 0.0 (0.0–0.0)‡
30.2 (9.9) 30.8 (28.7–37.2)‡
0.0‡ (0.0) 0.0 (0.0–0.0)
7.4 (1.7) 7.7 (6.7–8.1) *,†,‡
MBIC, total amount of mineralized bone-to-implant contact percentage, calculated as new + old bone; SOFT, soft tissue (connective matrix, bone marrow, etc.); CLOT, clot residues. *P < 0.05 between test and control. †P < 0.05 between one period of healing and the subsequent. ‡P < 0.05 between 1 week and 3 months of healing.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mainetti et al Sequential healing at implants
Fig. 8. Composition of the tissues in contact with the implant surface at the healed sites. NEW, new bone; OLD, old bone; DEB/PART, bone debris and bone particles; SOFT, soft tissues (connective matrix, bone marrow, etc.); CLOT, clot residues.
(a)
(b)
(c)
(d)
Fig. 9. (a, b) Ground sections illustrating the healing at a healed site after 1 week. Stevenel’s blue and alizarin red stain. (a) Newly formed bone was found on the surface of the implant connected by bridges of bone, osteoid tissue, and connective matrix to the parent bone. These struts often included bone debris and bone particles that could be found within the connective tissue matrix and on the implant surface. Original magnification 9200. (b) Residues of clot and signs of resorption were also observed. Original magnification 9200. (c, d) Ground sections illustrating the healing at a healed site after 2 weeks. Stevenel’s blue and alizarin red stain. (c) A higher percentage of new bone was found on the implant surface compared to the previous period of healing. Original magnification 9200. (d) Struts forming primary osteons. Original magnification 9200.
region, located coronally to B, was excluded from evaluation at all periods of observation. The percentages of tissue components found were similar between IPIES and healed sites for all tissues and all periods of observation. Already after 7 days, new bone formation developed to the implant surface and reached about 4% and 7% at IPIES and healed sites, respectively. This is in agreement with other studies that reported data
on early osseointegration (Bosshardt et al. 2011; Mainetti et al. 2014; Rossi et al. 2014). In a study in dogs (Rossi et al. 2014), implants were installed in healed sites, and biopsies were harvested after 5, 10, 20, and 30 days. After 10 days, 13% of new bone was found on the implant surface. In another study in dogs (Mainetti et al. 2014), a similar experimental design to that of the present study was used, however, applying
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
immediate functional loading. The sequential healing at IPIES and at implants placed in healed sites were compared, and about 7–8% of new bone was found after 7 days of healing at both sites. In analogy, in a human study (Bosshardt et al. 2011), about 6% of new bone was found at implants installed in healed sites after 7 days. In the present study, bone formation on the implant surface increased over time, with a similar pattern between healed and IPIES sites. After 3 months, very similar values were evident, 69.8% and 67.3% at the healed and IPIES sites, respectively. It is interesting to note that most new bone was formed between 1 week and 1 month at both sites. This is in agreement with other studies that reported a similar trend of new bone formation (Abrahamsson et al. 2004; Bosshardt et al. 2011; Mainetti et al. 2014; Rossi et al. 2014). Likewise, the results of the present study are in agreement with another experiment in dogs (Rossi et al. 2012) in which circumferential marginal defects, 5 mm deep and 1.25 mm wide, were prepared at implants. It was shown that most of the new bone was produced between 10 and 20 days, forming from the basis of the parent bone. While new bone was forming, old bone was under resorption in the present study. The resorptive processes proceeded slowly during the first month and became substantial thereafter. At the 3-month observation, a low amount of old bone could be identified. Similar patterns of old bone resorption were observed in other animal (Mainetti et al. 2014; Rossi et al. 2014) and human studies (Bosshardt et al. 2011; Lang et al. 2011). The struts of new bone and connective tissue matrix were also found at the IPIES sites, within the remaining defects, bridging the socket walls to the implant surface. Bundle bone served as a basis for new bone formation, however, in association with resorptive processes. Remnants of bundle bone were also found in the later periods of healing, incorporated between old and newly formed bone. A similar pattern of healing for the bundle bone was also described in a study in monkeys (Scala et al. 2014). The sequential healing of alveoli after tooth extraction showed new bone formation at 10 days on the lateral walls of the extraction socket, in direct contact with bundle bone. Bone formation increased with time, filling completely the alveoli after 1 month. Bone remodeling modified the newly formed bone into a mature bone during the following periods. Bone formation
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Mainetti et al Sequential healing at implants
(a)
(b)
(c)
(d)
Fig. 10. (a–d) Ground sections illustrating the healing at a healed site after 1 month (a, b) and 3 months (c, d). Stevenel’s blue and alizarin red stain. Original magnification 9200. Bone maturation and remodeling process were observed at these period of healing.
progressed in conjunction with resorptive processes. These contributed to the reduction of the dimensions of the bundle bone. The length of the remaining bundle bone was reduced to 56% after 30 days, to 10% after 90 days, and to about 8% after 180 days (Scala et al. 2014). The struts that formed within the marginal gaps at the IPIES sites reaching the implant surface contributed to the reduction of the distance between the coronal termination of the rough surface (M) and the coronal termination level of osseointegration (B), especially during the first month of healing. The distance M-B at the IPIES sites was 2.6 mm after 1 week and decreased to 1.4 mm after 1 month. At the 3-month observation period, the distance was 1.2 mm
and was similar to that observed at the implants installed in the healed sites. This pattern of healing has also been described in other animal studies (Botticelli et al. 2003a, b; Ara ujo et al. 2006b) that showed that marginal gaps were filled with new bone already after 1 month. However, osseointegration took longer time, and started from the bottom of the defect, and proceeded coronally to achieve a complete osseointegration within 3–4 months of healing. The marginal healing of the hard tissue at implants installed in the healed sites presented differences compared to that at IPIES sites. After 1 week of healing, the coronal level of osseointegration was located at 1.4 mm from M. This is in agreement with other studies that reported values of 1.2–1.3 mm
(Mainetti et al. 2014; Rossi et al. 2014). The small modifications observed in the subsequent periods of healing may be attributed to the resorptive and appositional processes that occurred in the marginal region of the hard tissue. In conclusion, the present experiment showed that similar patterns of osseointegration were observed at IPIES and implants placed in healed sites. The healing started either from pristine implant bed surfaces in the healed sites or from partially resorbed sites of bundle bone in IPIES sites. Developing struts of woven bone reached direct contact with the implant surfaces in both sites. Bony struts were surrounding bone debris and particles at early observation periods. Within the marginal gaps, the struts of woven bone were bridging the osseointegrated surface to the parent bone that was mainly made up of bundle bone. From a clinical point of view, the present experiment documented that implant placement into fresh extraction sockets resulted in a similar extent of osseointegration compared to implants installed in healed sites.
Acknowledgements: This study has been supported by a grant from Sweden & Martina SRL, Due Carrare, Padova, Italia, by ARDEC, Ariminum Odontologica SRL, Rimini, Italia and by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, Brienz, Switzerland. The support of TePe Munhygienprodukter AB, Malm€ o, Sweden for the oral hygiene products is appreciated.
Conflict of interest The authors declare no conflict of interest.
References Abrahamsson, I., Berglundh, T., Linder, E., Lang, N.P. & Lindhe, J. (2004) Early bone formation adjacent to rough and turned endosseous implant surfaces. An experimental study in the dog. Clinical Oral Implants Research 15: 381–392. Ara ujo, M.G., Sukekava, F., Wennstr€ om, J.L. & Lindhe, J. (2005) Ridge alterations following implant placement in fresh extraction sockets: an experimental study in the dog. Journal of Clinical Periodontology 32: 645–652. Ara ujo, M.G., Sukekava, F., Wennstr€ om, J.L. & Lindhe, J. (2006a) Tissue modeling following
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implant placement in fresh extraction sockets. Clinical Oral Implants Research 17: 615–624. Ara ujo, M.G., Wennstr€ om, J.L. & Lindhe, J. (2006b) Modeling of the buccal and lingual bone walls of fresh extraction sites following implant installation. Clinical Oral Implants Research 17: 606–614. Blanco, J., Mareque, S., Li~ nares, A., Perez, J., Mu~ noz, F. & Ramos, I. (2013) Impact of immediate loading on early bone healing at two-piece implants placed in fresh extraction sockets: an experimental study in the beagle dog. Journal of Clinical Periodontolology 40: 421–429.
Bosshardt, D.D., Salvi, G.E., Huynh-Ba, G., Ivanovski, S., Donos, N. & Lang, N.P. (2011) The role of bone debris in early healing adjacent to hydrophilic and hydrophobic implant surfaces in man. Clinical Oral Implants Research 22: 357–364. Botticelli, D., Berglundh, T., Buser, D. & Lindhe, J. (2003a) Appositional bone formation in marginal defects at implants. An experimental study in the dog. Clinical Oral Implants Research 14: 1–9. Botticelli, D., Berglundh, T., Buser, D. & Lindhe, J. (2003b) The jumping distance revisited. An exper-
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Mainetti et al Sequential healing at implants
imental study in the dog. Clinical Oral Implants Research 14: 35–42. Botticelli, D., Persson, L.G., Lindhe, J. & Berglundh, T. (2006) Bone tissue formation adjacent to implants placed in fresh extraction sockets. An experimental study in dogs. Clinical Oral Implants Research 17: 351–358. Caneva, M., Salata, L.A., Scombatti de Souza, S., Baffone, G., Lang, N.P. & Botticelli, D. (2010) Influence of implant positioning in extraction sockets on osseointegration. Clinical Oral Implants Research 21: 43–49. Donos, N., Hamlet, S., Lang, N.P., Salvi, G.E., Huynh-Ba, G., Bosshardt, D.D. & Ivanovski, S. (2011) Gene expression profile of osseointegration of a hydrophilic compared with a hydrophobic microrough implant surface. Clinical Oral Implants Research 22: 365–372. Ivanovski, S., Hamlet, S., Salvi, G.E., Huynh-Ba, G., Bosshardt, D.D., Lang, N.P. & Donos, N. (2011) Transcriptional profiling of osseointegration in humans. Clinical Oral Implants Research 22: 373–381. Lang, N.P., Pun, B.L., Lau, K.Y., Li, K.Y. & Wong, M.C.M. (2012) A systematic review of survival and success rates of implants placed immediately into
fresh extraction sockets after at least one year. Clinical Oral Implants Research 23(Suppl. 5): 42–69. Lang, N.P., Salvi, G.E., Huynh-Ba, G., Ivanovski, S., Donos, N. & Bosshardt, D.D. (2011) Early osseointegration to hydrophilic and hydrophobic implant surfaces in humans. Clinical Oral Implants Research 22: 349–356. Mainetti, T., Lang, N.-P., Bengazi, F., Sbricoli, L., Soto Cantero, L. & Botticelli, D. (2014) Immediate loading of implants installed in a healed alveolar bony ridge or immediately after tooth extraction. An experimental study in dogs. Clinical Oral Implants Research doi: 10.1111/clr.12389 [Epub ahead of print]. Quirynen, M., Van Assche, N., Botticelli, D. & Berglundh, T. (2007) How does the timing of implant placement to extraction affect outcome? The International Journal of Oral & Maxillofacial Implants 22(Suppl.): 203–223. Rossi, F., Botticelli, D., Pantani, F., Pereira, F.P., Salata, L.A. & Lang, N.P. (2012) Bone healing pattern in surgically created circumferential defects around submerged implants: an experimental study in the dog. Clinical Oral Implants Research 23: 41–48. Rossi, F., Lang, N.P., De Santis, E., Morelli, F., Favero, G. & Botticelli, D. (2014) Bone-healing
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
pattern at the surface of titanium implants: an experimental study in the dog. Clinical Oral Implants Research 25: 124–131. Scala, A., Lang, P.L., Schweikert, M.T., de Oliveira, J.A., Rangel-Garcia, I. Jr & Botticelli, B. (2014) Sequential healing of open extraction sockets. An experimental study in monkeys. Clinical Oral Implants Research 25: 288–295. Sicilia, A. & Botticelli, D. (2012) Working Group 3. Computer-guided implant therapy and soft- and hard-tissue aspects. The Third EAO Consensus Conference 2012. Clinical Oral Implants Research 23(Suppl.): 157–161. Vignoletti, F., de Sanctis, M., Berglundh, T., Abrahamsson, I. & Sanz, M. (2009a) Early healing of implants placed into fresh extraction sockets: an experimental study in the beagle dog. II: ridge alterations. Journal of Clinical Periodontology 36: 688–697. Vignoletti, F., Johansson, C., Albrektsson, T., De Sanctis, M., San Roman, F. & Sanz, M. (2009b) Early healing of implants placed into fresh extraction sockets: an experimental study in the beagle dog. De novo bone formation. Journal of Clinical Periodontology 36: 265–277.
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Sequential healing at implants installed immediately into extraction sockets. An experimental study in dogs
Authors’ affiliations: Tomaso Mainetti, Franco Bengazi, Vittorio Favero, Luis Soto Cantero, Daniele Botticelli, Faculty of Dentistry, University of Medical Science, La Habana, Cuba Niklaus P. Lang, Center for Dental Medicine, University of Zurich, Zurich, Switzerland; University of Bern, Bern, Switzerland Daniele Botticelli, ARDEC, Ariminum Odontologica, Rimini, Italy Daniele Botticelli, UNESP – Faculdade de Odontologia de Aracßatuba, UNESP – Universidade Estadual Paulista, S~ ao Paulo, Brasil
Key words: animal study, extraction sockets, histology, implants placed immediately into
Corresponding author: Dr Daniele Botticelli Avenida Salvador Allende y G Vedado, La Habana, Cuba Tel.: 537-879-3360 Fax: 537-870-3312 e-mail: [email protected]
extraction sockets (IPIES) of the fourth premolar and in the healed sites in the molar regions.
extraction sockets, sequential healing, wound healing Abstract Objective: To compare the sequential healing at implants installed in a healed alveolar bony ridge or immediately after tooth extraction without functional load. Material and methods: In the mandible of 12 dogs, the mesial roots of the first molars were endodontically treated, the tooth hemisected, and the distal roots extracted. After 3 months, the mesial roots of the fourth premolars were endodontically treated, the tooth hemisected, and the distal roots extracted in one side of the mandible. Implants were placed immediately into Healing abutments were placed, and the flaps were sutured to allow a non-submerged healing. The time of surgery and of sacrifices were planned in such a way to obtain biopsies representing the healing after 1 and 2 weeks and 1 and 3 months, respectively. Ground sections were prepared for histological evaluation of tissues components on the implant surface and the coronal termination level of osseointegration (M-B). Results: New bone apposition on the implant surface was slightly higher at the healed compared to the IPIES sites, being 7.4% and 4.1% after 1 week, and 67.3% and 65.3% after 3 months, respectively. Old bone was progressively resorbed, from 27.0% and 21.9% after 1 week, to 2.5% and 2.0% after 3 months, at healed and IPIES sites, respectively. M-B was 1.4 mm and 2.6 mm after 1 week, 1.2 mm and 1.2 mm after 3 months, at healed and IPIES sites, respectively. Conclusions: Similar patterns of sequential osseointegration were found at implants installed in healed alveolar bone or in alveolar sockets immediately after tooth extraction. The coronal termination level of osseointegration, that was different after 1 week, was found similar at the 3-month observation.
Date: Accepted 21 November 2014 To cite this article: Mainetti T, Lang NP, Bengazi F, Favero V, Soto Cantero L, Botticelli D. Sequential healing at implants installed immediately into extraction sockets. An experimental study in dogs. Clin. Oral Impl. Res. 00, 2014, 1–9 doi: 10.1111/clr.12533
Implants placed immediately into extraction sockets (IPIES, Sicilia & Botticelli 2012) have been shown to have a high rate of clinical success (for review, see Quirynen et al. 2007; Lang et al. 2012). A large number of animal experiments have shown, however, that the immediate installation of implants does not prevent the resorption of the alveolar ridges (e.g., Ara ujo et al. 2005; Botticelli et al. 2006; Caneva et al. 2010). The sequential healing at IPIES has been described in animal experiments (e.g., Ara ujo et al. 2006a,b; Vignoletti et al. 2009a,b; Blanco et al. 2013; Mainetti et al. 2014). In an experiment in dogs (Ara ujo et al. 2006b), implants were placed in the distal alveoli of the third premolars and of the first molars immediately after tooth extraction. In the molar region, a large gap resulted
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
after installation. It was observed that, the level of osseointegration was located at 1.5 mm apically of the coronal termination of the rough implant surface after 4 weeks while, after 12 weeks, the level was located at 0.8 mm of that landmark. This pattern of healing was also confirmed in animal studies (Botticelli et al. 2003a,b) in which marginal defects, 5 mm deep and 1.25 mm wide, were prepared in the region of implants installed in the alveolar bony ridge in the mandible of dogs. It was shown that bone apposition onto the implant surface started from the bottom of the defects and gradually developed coronally. A progressive gain in the level of osseointegration from 1.8 mm after 1 month, 3.1 mm after 2 months, and 4.5 mm after 4 months of healing was observed.
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Mainetti et al Sequential healing at implants
The sequential processes of osseointegration has been described both in humans (Bosshardt et al. 2011; Donos et al. 2011; Ivanovski et al. 2011; Lang et al. 2011) and animal studies (Mainetti et al. 2014; Rossi et al. 2014). In an animal study (Mainetti et al. 2014), the sequential healing at IPIES was compared with that of control implants installed in healed sites. All implants were immediately loaded. It was concluded that the two sites presented different patterns of healing during the early phases, but no statistically significant differences were revealed for the hard and soft tissues dimensions after 3 months following implant installation. However, there is a lack of comparative studies of healing at IPIES and control implants installed in healed alveolar ridges without loading. Hence, the aim of this study was to compare the sequential healing at implants installed in healed alveolar bony ridges or immediately after tooth extraction without functional loading.
Material and methods The research protocol was submitted to and approved by the Ethical Committee of the University of Medical Sciences, School of Dentistry, La Habana, Cuba. Twelve beagle dogs, with a mean body weight of 11 kg and a mean age of 1 year, were provided by Centro Nacional para la Producci on de Animales de Laboratorio (CENPALAB) of La Habana, Cuba. The animals were kept during the experimental period in kennels and on concrete runs at the university’s field laboratory with free access to water and feed of moistened balanced dog’s chow. Clinical procedures
Before the surgical sessions, the animals received atropine 0.02 mg/kg i.v. (Mayne Pharma, Napoli, Italia), metedomidine 0.04 mg/kg (Medetorâ; Virbac, Glattbrugg, Switzerland), and ketamine 50 5 mg/kg (Liorad, La Habana, Cuba) mixed in a syringe. The anesthesia was maintained with 2–3% Isoflurane-Vetâ (Merial, Merial Tolosa, France) and O2 at 95%. An intravenous infusion of 0.9% saline solution 10 ml/kg/h was kept during the surgeries, while blood pressure and O2 perfusion were constantly monitored. Local anesthesia was also provided. At the first surgical session, in both sides of the mandible, the mesial root of the first
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molar was endodontically treated, and subsequently, the tooth was hemisected and the distal root extracted. After 3 months of healing, in one randomly selected side of the mandible, the endodontic treatment of the mesial root of the fourth premolar was performed (Mtwoâ, Endopocketâ, Epfillâ; Sweden & Martina, Due Carrare, Padova, Italy), and the crown restored with composite. Full thickness flaps were elevated, and the buccal and lingual alveolar bony plates were exposed. The forth premolar was hemisected, and the distal root removed including the corresponding portion of the crown. Two customized implants, 8.5 mm long and 3.3 mm in diameter (Sweden & Martina) with a ZirTiâ surface were installed, one in the position of M1 (healed site), and one in the distal alveolus of P4 (IPIES site; Fig. 1a,b). All implants were placed with the coronal termination of the rough surface flush with the alveolar buccal bony crest. Subsequently, healing abutments were affixed. The flaps were carefully adapted around the healing abutments using single interrupted resorbable sutures (Vicrylâ 4–0; Johnson & Johnson, Medical S.p.A., Pomezia, Roma; Fig. 1c). No
occlusal contact with the maxillary teeth was allowed (Fig. 1d). The treatment in the other side of the mouth and sacrifices were scheduled in such a way as to collect biopsies representing the healing after 1 and 2 weeks, and 1 and 3 months. Antibiotics (Conveniaâ 852 mg.; Pfizer, New York, NY, USA) and anti-inflammatory/ analgesics 2 mg/kg tramadol (Altadolâ; Formevet, Milan, Italy) were administered for 5 days after surgery. An inspection of the wounds for clinical signs of complications and healing abutments cleaning were performed three times per week for the entire duration of the experiment. At the sacrifice, the animals were first anesthetized with a dose of 1 mg/kg of xylazine (Rompunâ, Kiel, Germany) and 10 mg/ kg of ketamine (Lioradâ, La Habana, Cuba) mixed in a syringe and administered i.m. and maintained with 2–3% Isoflurane-Vetâ (Merial, Merial Tolosa, France) and O2 95%. Eparin 12 i.u./kg i.v. (Athena Pharma, Pomezia, Italy) was injected before the heart was arrested with 25 meq of potassium chloride i.v. (Aica, La Habana, Cuba). The carotid arteries were perfused with a fixative (4% of formaldehyde solution).
(a)
(b)
(c)
(d)
Fig. 1. Clinical procedures. (a) Implants installed in a healed site in the first molar region (yellow arrow) and in the distal alveolus of the fourth premolar (light blue arrow). (b) A marginal defect resulted around the implant placed into the extraction socket. (c) The flaps were sutured to allow a non-submerged healing. (d) No occlusal contacts were allowed between the abutments and the teeth of the opposing jaw.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mainetti et al Sequential healing at implants
Histological preparation
The mandibles were removed and maintained in 4% formaldehyde solution. The histological procedures were performed in the laboratory of histology at the Faculty of Odontology of the University of La Habana. Block sections, each containing one implant, were obtained, dehydrated in a series of graded ethanol and subsequently embedded in resin (Technovitâ 7200 VLC; Kulzer, Friedrichsdorf, Germany). The blocks were cut in a bucco-lingual plane using a diamond band saw fitted in a precision slicing machine (Exaktâ; Apparatebau, Norderstedt, Germany). One central section was selected and
reduced to a thickness of about 50–60 lm using a cutting–grinding device (Exaktâ; Apparatebau). The histological slides were stained with Stevenel’s blue and alizarin red and examined under a standard light microscope for histometric analysis. Histological evaluations
The histological measurements were performed in an Eclipse Ci microscope (Nikon Corporation, Tokyo, Japan), equipped with a digital video camera (Digital Sight DS-2Mv; Nikon Corporation) connected to a computer, and using the software NIS-Elements D 4.10 (Laboratory Imaging; Nikon Corporation). The following landmarks were identified (Fig. 2): (IS) the shoulder of the implant, (B) the most coronal bone-to-implant contact. The apical termination of the implant was identified as well (A). The vertical distances between IS and B (IS-B) were assessed at 9100 magnification and, subsequently, the distances between the coronal termination of the rough surface (M) and B (distance M-B) were obtained by subtracting the height of the neck of the implant to IS-B. The percentages of new bone, old bone, soft tissues (connective matrix, bone marrow at various stages of maturation, etc.), clot, and bone debris/particles in contact with the implant surface were measured between B and A at a magnification 9200. The total amount of mineralized bone-to-implant contact percentage (MBIC%) was calculated (new + old bone). Data analysis
Fig. 2. Diagram illustrating the landmarks for the histological evaluation. (IS) the shoulder of the implant; (M) coronal end of the rough surface; (B) the most coronal bone-to-implant contact.
Six animals represented each period of healing (n = 6). Mean values and standard deviations as well as 25th, 50th (median), and 75th percentiles were calculated for each
outcome variable. The main outcome variables were percentage of new bone and the distance M-B. For statistical analyses, the IBM SPSS Statistics 19 software was used (IBM Inc., Chicago, IL, USA). The Wilcoxon signed rank test for dependent variables was used to assess differences between healed and IPIES sites. Moreover, as explorative investigation, the Mann–Whitney U-test for independent variables was used to analyze differences between periods. The level of significance was set at a = 0.05.
Results No major signs of inflammation were seen during the healing period, and at the time of sacrifice, all implants were still in function and stable. Histological evaluation
No artifacts were generated during histological processing nor were any tissue blocks destroyed. Hence, IPIES and healed sites each yielded an n = 6. IPIES sites
Mean values, standard deviations, medians and 25th–75th percentiles are reported in Table 1, for the percentage of tissues in contact with the implant surface, and in Table 2, for the distance between the coronal termination of the rough surface (M) and the most coronal contact of bone to the implant (B). Mean values are also illustrated in Fig. 3 for tissues in contact with the implant surface and in Fig. 4 for M-B. In the text, only mean values and standard deviations are reported. At the IPIES sites, after 1 week of healing, bone defects were still present between the
Table 1. Tissues in contact with the implant surface in percentage (%) at the IPIES sites New bone 1 week Mean (SD) Median (25th–75th) 2 weeks Mean (SD) Median (25th–75th) 1 month Mean (SD) Median (25th–75th) 3 months Mean (SD) Median (25th–75th)
Old bone
MBIC
Bone debris/particles
SOFT
CLOT
21.9 (9.8) 19.4 (18.6–23.8)‡
26.0 (10.3) 24.3 (22.0–29.5)†,‡
9.8 (5.0) 10.0 (6.8–13.7)
50.5 (14.9) 51.5 (46.4–57.8)
13.7 (5.7) 15.4 (12.4–17.4)‡
16.3 (6.2) 17.4 (13.1–18.6)†
21.6 (6.8) 23.0 (20.2–26.6)
38.0 (10.1) 38.9 (35.7–41.3)†
11.6 (2.4) 11.5 (9.8–13.5)*,†
40.4 (9.9) 35.2 (34.8–47.1)
9.9 (3.5) 10.7 (7.9–12.9)†
46.1 (10.8) 50.2 (36.9–53.2)
19.8 (6.9) 21.3 (18.3–24.5)†
65.8 (12.3) 63.5 (58.1–74.3)
2.8 (2.6) 1.6 (1.2–3.7)†
31.4 (11.6) 35.5 (22.2–40.3)
0.0 (0.0) 0.0 (0.0–0.0)
65.3 (22.8) 74.8 (53.7–80.2)‡
2.0 (0.7) 2.1 (1.9–2.2)‡
67.3 (23.0) 77.0 (56.4–82.3)‡
0.0 (0.0) 0.0 (0.0–0.0)‡
32.7 (23.0) 23.0 (17.7–43.6)
0.0 (0.0) 0.0 (0.0–0.0)‡
4.1 (1.5) 3.7 (3.4–5.1) *,†,‡
IPIES, implants placed immediately into extraction sockets; MBIC, total amount of mineralized bone-to-implant contact percentage, calculated as new + old bone; SOFT, soft tissue (connective matrix, bone marrow, etc.); CLOT, clot residues. *P < 0.05 between test and control. †P < 0.05 between one period of healing and the subsequent. ‡P < 0.05 between 1 week and 3 months of healing. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Table 2. Distance (M-B) between the coronal margin of the rough surface (M) and the coronal level of osseointegration (B) at the buccal (b) and lingual (l) aspects IPIES sites
1 week Mean (SD) Median (25th–75th) 2 weeks Mean (SD) Median (25th–75th) 1 month Mean (SD) Median (25th–75th) 3 months Mean (SD) Median (25th–75th)
Healed sites
b
l
b
l
2.6 (1.0) 2.6* (2.0–2.9)†
2.4 (1.6) 2.0 (1.3–3.6)
1.4 (0.5) 1.4* (1.2–1.8)
1.2 (0.7) 1.0 (0.8–1.1)
1.9 (0.3) 2.0 (1.7–2.1)
1.2 (0.4) 1.0 (0.9–1.5)
1.1 (0.7) 0.9 (0.6–1.3)
1.0 (0.9) 0.7 (0.6–1.4)
1.4 (0.7) 1.1 (1.0–2.0)
0.6 (0.6) 0.6 (0.1–1.0)
0.8 (0.3) 0.9 (0.7–1.0)
0.5 (0.5) 0.8 (0.1–0.9)
1.2 (0.5) 1.2 (0.9–1.3)†
0.8 (0.7) 0.6 (0.5–0.7)
1.2 (0.5) 1.2 (1.1–1.5)
1.1 (0.7) 0.8 (0.7–1.0)
*P < 0.05 between test and control. †P < 0.05 between 1 week and 3 months of healing.
Fig. 3. Composition of the tissues in contact with the implant surface at the implants placed immediately into extraction sockets (IPIES) sites. NEW, new bone; OLD, old bone; DEB/PART, bone debris and bone particles; SOFT, soft tissues (connective matrix, bone marrow, etc.); CLOT, clot residues.
Fig. 4. Graphic representing the mean values of the distances M-B at the buccal (b) and lingual (l) aspects at the various periods of healing. M = coronal end of the rough surface; B = most coronal bone-to-implant contact.
walls of the parent bone and the implant surface (Fig. 5a). The implants presented threads inserted into the parent old bone (Fig. 5b). New bone formation was found within the bottom of the remaining defect forming woven bone struts, partially calcified and partially as connective tissue matrix and osteoid
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tissue, bridging the bony walls of the extraction socket and the implant surface (Fig. 5c). Bone particles and bone debris were often found on the implant surface and incorporated into the struts. Similar struts were found in regions of the implant originally installed into the parent bone (Fig. 5d). A
percentage of 4.1 1.5% of newly formed bone was found on the implant surface, while old bone was still present at a higher quantity (21.9 9.8%). A total amount of 26.0 10.3% of mineralized bone (MBIC%) was observed. Bone particles and bone debris were found on the implant surface at a proportion of about 10%. Soft tissues were present at a percentage of about 51%. Approximately, 14% of the surfaces were still covered by a clot in various stages of degeneration. Signs of bone resorption were seen on bundle bone and in parent bone, however, rarely in vicinity of the implant surface. M-B at the buccal aspect was 2.6 1.0 mm. After 2 weeks of healing, the marginal defects were still present. However, a larger amount of struts of woven bone, connective provisional matrix, and osteoid compared to the previous period of healing were identified (Fig. 6a). The struts within the defects were connecting the parent bone, also represented by partially resorbed bundle bone, to the newly formed bone on the implant surface (Fig. 6b,c). A higher percentage of new bone was found in contact with the implant surface at this stage of healing (16.3 6.2%), the difference with the previous period of healing being statistically significant. The old bone was still present at a similar percentage of the previous period of healing (21.6 6.8%). A total amount of 38.0 10.1% of mineralized bone (MBIC%) was observed. About 12% of bone debris and bone particles, approximately 40% of soft tissues, and about 10% of residual tissue from disintegrating clot were detected at this stage of healing. Bone resorption was observed, particularly dynamic in the bony crestal region (Fig. 6d). The distance M-B at the buccal aspect was 1.9 0.3 mm, the difference with
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mainetti et al Sequential healing at implants
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(b)
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Fig. 5. Ground sections illustrating the healing at an implants placed immediately into extraction sockets (IPIES) site after 1 week. Stevenel’s blue and alizarin red stain. (a) Bone defects were still present between the walls of the parent bone and the implant surface. Image grabbed at 920 magnification. (b) Threads appeared to be inserted into the parent old bone. Original magnification 9100. (c) New bone formation was found within the bottom of the remaining defect forming, together the connective provisional matrix, bridges between the bony walls of the extraction socket and the implant surface. Original magnification 9100. (d) Similar bridges were also found in regions of the implant originally installed into the parent bone. Original magnification 9100.
(a)
(b)
(c)
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Fig. 6. Ground sections illustrating the healing at an implants placed immediately into extraction sockets (IPIES) site after 2 weeks. Stevenel’s blue and alizarin red stain. (a) The marginal defects were still present, however, with a larger amount of bridges compared to the previous period of healing. Image grabbed at 920 magnification. (b) The bridges within the defects connected the parent bone, also represented by bundle bone partially resorbed, to the implant surface. Original magnification 9200. (c) The bridges gained attachment with the newly formed bone on the implant surface. Original magnification 9200. (d) Bone resorption was found to be dynamic in the bony crest region. Original magnification 9200.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
the previous period of healing not being statistically significant. After 1 month of healing, the new bone appeared to be more mature (parallel-fibered bone) compared to the previous stages of healing and presented with a percentage of 46.1 10.8%, the difference with the 2-week healing period being statistically significant (Fig. 7a,b). The percentages of old bone and MBIC% were 19.8 6.9% and 65.8 12.3%, respectively. The distance M-B at the buccal aspect was 1.4 0.7 mm, the difference with the previous period of healing not reaching statistical significance. After 3 months of healing, the surrounding bone at the implants appeared to be mature more mature compared to the previous periods of healing (Fig. 7c,d). New bone was found at a percentage of 65.3 22.8 and 6.1 10.8%, and very little old bone could be identified (2.0 0.7%). MBIC% was 67.3 23.0%. No statistically significant differences were found. The distance M-B at the buccal aspect was 1.2 0.5 mm. No statistically significant difference was found with the previous period. However, between 1week and 3-month periods, a statistically significant difference was verified. Healed sites
Mean values, standard deviations, medians and 25th–75th percentiles of the percentage of tissues in contact with the implant surface are reported in Table 3. Mean values are also illustrated in Fig. 8 for tissues in contact with the implant surface and in Fig. 4 for MB. In the text, only mean values and standard deviations are reported. At the healed sites, after 1 week of healing, newly formed bone was found on the surface of the implants connected to the parent bone by struts of newly formed calcified bone, connective tissue matrix, and osteoid tissue (Fig. 9a). These struts often included bone debris and bone particles that could be found within the connective tissue matrix and on the implant surface. Residues of disintegrating clot and signs of resorption were also observed (Fig. 9b). A percentage of 7.4 1.7% and 27.0 6.0% were measured of newly formed and old bone, respectively. A statistically significant difference was found between IPIES and healed sites for the new bone (P = 0.028). A total amount of 34.4 6.9% of MBIC% was observed. Bone debris and bone particles on the implant surface were present in a proportion of about 13%. M-B at the buccal aspect was 1.4 0.5 mm, the difference between IPIES and healed sites being statistically significant (P = 0.027).
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Mainetti et al Sequential healing at implants
(a)
(b)
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significant at 1 and 3 months of healing, while, comparing the periods of healing longitudinally, only the difference between 2 weeks and 1 month was statistically significant. M-B was 0.8 0.3 mm and 1.2 0.5 mm after 1 and 3 months, respectively. No statistically significant differences were found between IPIES and healed sites at these observation periods (P = 0.068 and 0.916, respectively).
Discussion
Fig. 7. (a, b) Ground sections illustrating the healing at an implants placed immediately into extraction sockets (IPIES) site after 1 month. Stevenel’s blue and alizarin red stain. (a) The bottom of a marginal defect appeared filled with newly formed bone. Remnants of bundle bone were still identifiable (*). Original magnification 9100. (b) The spaces between threads were found to be filled with new bone originating from remnants of bundle bone (*). Original magnification 9100. (c, d) Ground sections illustrating the healing at an IPIES site after 3 months. Stevenel’s blue and alizarin red stain. The bone appeared to be more mature compared to the previous periods of healing. (c) Original magnification 9100. (d) Original magnification 9200.
At the second week of healing, a higher quantity of new bone (25.4 5.6%) compared to the previous period of healing was found on the surface of the implants (Fig. 9c). Struts composed of new bone, osteoid tissue lined by osteoblasts, connective tissue matrix, and bone debris/particles were also observed. Moreover, primary osteons were forming (Fig. 9d). No statistically significant difference was found between IPIES and
healed sites for the new bone. M-B at the buccal aspect was 1.1 0.7 mm, the difference between IPIES and healed sites not being statistically significant (P = 0.058). After 1 and 3 months of healing, bone maturation and remodeling processes were observed (Fig. 10a–d). New bone was found at a percentage of 50.5 8.3% and 67.3 9.4%, respectively. The differences between IPIES and healed sites were not statistically
The present study compared the sequential processes of osseointegration at implants placed immediately into extraction sockets (IPIES) and implants installed in healed sites without functional loading. The patterns of healing were very similar between the two sites. It was shown that new bone, osteoid tissue, and connective matrix were forming struts from the parent bone to the implant surface. These struts included also bone debris and bone particles that seemed to play an important role in the process of osseointegration. This is in agreement with the findings from a histological study in human (Bosshardt et al. 2011). In that study, 49 mini-implants were placed in the retromolar region of 28 human volunteers. After 1, 2, 4, and 6 weeks, biopsies containing the implants were collected and processed for histological analyses. It was concluded that bone debris originating from implant placement contributed to the processes of osseointegration. In the present study, tissue components were evaluated on the implant surface between the most coronal bone to implant contact (B) and the apex of the implant (A). This also means that the remaining defect
Table 3. Tissues in contact with the implant surface in percentage (%) at implants installed in healed sites New bone 1 week Mean (SD) Median (25th–75th) 2 weeks Mean (SD) Median (25th–75th) 1 month Mean (SD) Median (25th–75th) 3 months Mean (SD) Median (25th–75th)
Old bone
MBIC
Bone debris/particles
SOFT
CLOT
27.0 (6.0) 25.3 (22.5–28.8)†,‡
34.4 (6.9) 33.3 (29.2–38.1)†,‡
12.7 (5.4) 12.7 (10.4–14.6)‡
42.9 (7.5) 41.1 (37.9–46.4)‡
10.0 (5.3) 9.1 (5.5–15.0)
25.4 (5.6) 26.3 (23.2–27.1)†
23.4 (2.5) 23.2 (21.5–24.2)†
48.8 (7.1) 50.2 (44.9–53.0)†
6.4 (4.0) 6.3 (3.9–9.7)*
39.3 (6.0) 38.9 (38.1–40.3)†
5.5 (8.3) 2.6 (1.1–4.3)†
50.5 (8.3) 51.0 (47.8–54.8)†
19.1 (6.7) 21.2 (13.6–24.1)†
69.5 (10.0) 67.7 (64.1–72.8)
4.4 (3.2) 4.4 (3.1–5.3)†
26.0 (9.3) 27.4 (22.6–30.6)
0.0 (0.0) 0.0 (0.0–0.0)
67.3 (9.4) 66.8 (60.8–68.6)‡
2.5 (0.7) 2.3 (1.9–3.0)‡
69.8 (9.9) 69.2 (62.8–71.3)‡
0.0 (0.0) 0.0 (0.0–0.0)‡
30.2 (9.9) 30.8 (28.7–37.2)‡
0.0‡ (0.0) 0.0 (0.0–0.0)
7.4 (1.7) 7.7 (6.7–8.1) *,†,‡
MBIC, total amount of mineralized bone-to-implant contact percentage, calculated as new + old bone; SOFT, soft tissue (connective matrix, bone marrow, etc.); CLOT, clot residues. *P < 0.05 between test and control. †P < 0.05 between one period of healing and the subsequent. ‡P < 0.05 between 1 week and 3 months of healing.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mainetti et al Sequential healing at implants
Fig. 8. Composition of the tissues in contact with the implant surface at the healed sites. NEW, new bone; OLD, old bone; DEB/PART, bone debris and bone particles; SOFT, soft tissues (connective matrix, bone marrow, etc.); CLOT, clot residues.
(a)
(b)
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Fig. 9. (a, b) Ground sections illustrating the healing at a healed site after 1 week. Stevenel’s blue and alizarin red stain. (a) Newly formed bone was found on the surface of the implant connected by bridges of bone, osteoid tissue, and connective matrix to the parent bone. These struts often included bone debris and bone particles that could be found within the connective tissue matrix and on the implant surface. Original magnification 9200. (b) Residues of clot and signs of resorption were also observed. Original magnification 9200. (c, d) Ground sections illustrating the healing at a healed site after 2 weeks. Stevenel’s blue and alizarin red stain. (c) A higher percentage of new bone was found on the implant surface compared to the previous period of healing. Original magnification 9200. (d) Struts forming primary osteons. Original magnification 9200.
region, located coronally to B, was excluded from evaluation at all periods of observation. The percentages of tissue components found were similar between IPIES and healed sites for all tissues and all periods of observation. Already after 7 days, new bone formation developed to the implant surface and reached about 4% and 7% at IPIES and healed sites, respectively. This is in agreement with other studies that reported data
on early osseointegration (Bosshardt et al. 2011; Mainetti et al. 2014; Rossi et al. 2014). In a study in dogs (Rossi et al. 2014), implants were installed in healed sites, and biopsies were harvested after 5, 10, 20, and 30 days. After 10 days, 13% of new bone was found on the implant surface. In another study in dogs (Mainetti et al. 2014), a similar experimental design to that of the present study was used, however, applying
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
immediate functional loading. The sequential healing at IPIES and at implants placed in healed sites were compared, and about 7–8% of new bone was found after 7 days of healing at both sites. In analogy, in a human study (Bosshardt et al. 2011), about 6% of new bone was found at implants installed in healed sites after 7 days. In the present study, bone formation on the implant surface increased over time, with a similar pattern between healed and IPIES sites. After 3 months, very similar values were evident, 69.8% and 67.3% at the healed and IPIES sites, respectively. It is interesting to note that most new bone was formed between 1 week and 1 month at both sites. This is in agreement with other studies that reported a similar trend of new bone formation (Abrahamsson et al. 2004; Bosshardt et al. 2011; Mainetti et al. 2014; Rossi et al. 2014). Likewise, the results of the present study are in agreement with another experiment in dogs (Rossi et al. 2012) in which circumferential marginal defects, 5 mm deep and 1.25 mm wide, were prepared at implants. It was shown that most of the new bone was produced between 10 and 20 days, forming from the basis of the parent bone. While new bone was forming, old bone was under resorption in the present study. The resorptive processes proceeded slowly during the first month and became substantial thereafter. At the 3-month observation, a low amount of old bone could be identified. Similar patterns of old bone resorption were observed in other animal (Mainetti et al. 2014; Rossi et al. 2014) and human studies (Bosshardt et al. 2011; Lang et al. 2011). The struts of new bone and connective tissue matrix were also found at the IPIES sites, within the remaining defects, bridging the socket walls to the implant surface. Bundle bone served as a basis for new bone formation, however, in association with resorptive processes. Remnants of bundle bone were also found in the later periods of healing, incorporated between old and newly formed bone. A similar pattern of healing for the bundle bone was also described in a study in monkeys (Scala et al. 2014). The sequential healing of alveoli after tooth extraction showed new bone formation at 10 days on the lateral walls of the extraction socket, in direct contact with bundle bone. Bone formation increased with time, filling completely the alveoli after 1 month. Bone remodeling modified the newly formed bone into a mature bone during the following periods. Bone formation
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Mainetti et al Sequential healing at implants
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Fig. 10. (a–d) Ground sections illustrating the healing at a healed site after 1 month (a, b) and 3 months (c, d). Stevenel’s blue and alizarin red stain. Original magnification 9200. Bone maturation and remodeling process were observed at these period of healing.
progressed in conjunction with resorptive processes. These contributed to the reduction of the dimensions of the bundle bone. The length of the remaining bundle bone was reduced to 56% after 30 days, to 10% after 90 days, and to about 8% after 180 days (Scala et al. 2014). The struts that formed within the marginal gaps at the IPIES sites reaching the implant surface contributed to the reduction of the distance between the coronal termination of the rough surface (M) and the coronal termination level of osseointegration (B), especially during the first month of healing. The distance M-B at the IPIES sites was 2.6 mm after 1 week and decreased to 1.4 mm after 1 month. At the 3-month observation period, the distance was 1.2 mm
and was similar to that observed at the implants installed in the healed sites. This pattern of healing has also been described in other animal studies (Botticelli et al. 2003a, b; Ara ujo et al. 2006b) that showed that marginal gaps were filled with new bone already after 1 month. However, osseointegration took longer time, and started from the bottom of the defect, and proceeded coronally to achieve a complete osseointegration within 3–4 months of healing. The marginal healing of the hard tissue at implants installed in the healed sites presented differences compared to that at IPIES sites. After 1 week of healing, the coronal level of osseointegration was located at 1.4 mm from M. This is in agreement with other studies that reported values of 1.2–1.3 mm
(Mainetti et al. 2014; Rossi et al. 2014). The small modifications observed in the subsequent periods of healing may be attributed to the resorptive and appositional processes that occurred in the marginal region of the hard tissue. In conclusion, the present experiment showed that similar patterns of osseointegration were observed at IPIES and implants placed in healed sites. The healing started either from pristine implant bed surfaces in the healed sites or from partially resorbed sites of bundle bone in IPIES sites. Developing struts of woven bone reached direct contact with the implant surfaces in both sites. Bony struts were surrounding bone debris and particles at early observation periods. Within the marginal gaps, the struts of woven bone were bridging the osseointegrated surface to the parent bone that was mainly made up of bundle bone. From a clinical point of view, the present experiment documented that implant placement into fresh extraction sockets resulted in a similar extent of osseointegration compared to implants installed in healed sites.
Acknowledgements: This study has been supported by a grant from Sweden & Martina SRL, Due Carrare, Padova, Italia, by ARDEC, Ariminum Odontologica SRL, Rimini, Italia and by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, Brienz, Switzerland. The support of TePe Munhygienprodukter AB, Malm€ o, Sweden for the oral hygiene products is appreciated.
Conflict of interest The authors declare no conflict of interest.
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