Histologic and Micro-CT Analyses at Implants Placed ...

The International Journal of Oral & Maxillofacial Implants 739

The surgical procedure for sinus elevation includes the use of biomaterials of different composition,1

devices,2–5 or implants6–10 aiming to maintain the sub-antral volume that resulted after the elevation of the sinus membrane and to favor the growth of new bone.

The volume of the augmented space tends to de-crease during healing, as shown both in experimen-tal4,9,11–15 and clinical studies.10,16–19 Some grafting materials used for sinus floor elevation showed higher degrees of resorption1,20 compared with others.20,21 It also has to be considered that, for biomaterial scarcely resorbed, high remnants of that biomaterial could be

found at implant placement.22–25 In fact, after 4 to 6 months of healing, in clinical studies in which xenograft and allograft were applied for alveolar ridge preserva-tion after tooth extraction,22,23 the proportions of xe-nograft, allograft, or a mixture of the two biomaterials were 22.2%, 33.8%, and 33.2%, respectively. In another study, after 6 to 8 months from surgery, biopsy speci-mens were harvested from grafted sinuses performed with either a synthetic or a xenograft material. Pro-portions of 26.6% of synthetic and 37.7% of xenograft materials were found.24 Depending on the degree of resorbability of the biomaterial, different proportions of residues might be found. In a clinical study, biopsy specimens were collected 6 months after sinus floor augmentation performed with collagenated cortico-cancellous porcine bone. A proportion or residues of biomaterial of 14.7% was found.25

Clinical26–28 and experimental29 studies have been performed using granules of different sizes to deter-mine the most suitable dimension that might enhance bone formation and improve clinical outcomes af-ter sinus floor augmentation. However, these studies could not demonstrate differences in clinical and his-tologic outcomes between large and small particles of

Histologic and Micro-CT Analyses at Implants Placed Immediately After Maxillary Sinus Elevation

Using Large or Small Xenograft Granules: An Experimental Study in RabbitsKatsuhiko Masuda, DDS, PhD1/Erick Ricardo Silva, DDS, PhD2/

Karol Alí Apaza Alccayhuaman, DDS3/Daniele Botticelli, BMBS, PhD3/Samuel Porfirio Xavier, DDS, PhD2

Purpose: To compare the osseointegration at the portion of the implant within the elevated space after sinus elevation using different sizes of xenograft. Materials and Methods: Eighteen New Zealand rabbits were selected, antrostomies were prepared bilaterally through the nasal dorsum, and the sinus mucosa was elevated. Deproteinized bovine bone mineral with granules of either 1 to 2 mm (large sites) or 0.250 to 1.0 mm (small sites) were randomly used to fill the elevated space of the two sinuses. Subsequently, mini-implants were placed through the antrostomy, one in each sinus. The animals were euthanized 2, 4, and 8 weeks after surgery, six animals for each group. Microcomputed tomography (micro-CT) and histologic analyses were performed. Results: In the elevated space, at the histologic analysis after 2 weeks of healing, new bone formed on the implant surface was found in fractions of 18.8% ± 6.8% and 15.8% ± 9.6% in the large and small sites, respectively (P = .249). After 4 weeks, the respective fractions of new bone were 20.3% ± 3.5% and 23.3% ± 5.6% (P = .249). After 8 weeks, the proportions reached 33.9% ± 9.5% and 28.5% ± 10.3% (P = .173), respectively. At the micro-CT analysis, bone-to-implant contact percentage (BIC%) was 21.0% ± 2.3% and 21.2% ± 2.4% in the large and small sites, respectively (P = 1.000). The respective proportions of BIC% at the large and small sites were 20.5% ± 3.3% and 23.4% ± 5.4% after 4 weeks (P = .463), and 23.0% ± 2.7% and 25.8% ± 4.1% after 8 weeks (P = .249). Conclusion: The use of xenograft granules of different dimensions resulted in similar amounts of bone-to-implant contact at implants placed simultaneously with sinus floor augmentation. Int J Oral Maxillofac Implants 2020;35:739–748. doi: 10.11607/jomi.8067

Keywords: biomaterials, bone graft, histology

1Department of Pediatric Dentistry, Division of Oral Infection and Disease Control, Osaka University, Osaka, Japan; ARDEC Academy, Rimini, Italy.

2Depto CTBMF e Periodontia FORP-USP- Faculty of Ribeirão Preto (SP), Brazil.

3ARDEC Academy, Rimini, Italy.

Correspondence to: Dr Karol Alí Apaza Alccayhuaman, ARDEC Academy, Viale Pascoli 67 – 47923 Rimini, Italy. Fax: +39 0541 393444. Email: [email protected]

Submitted September 20, 2019; accepted April 1, 2020. ©2020 by Quintessence Publishing Co Inc.

© 2020 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.

740 Volume 35, Number 4, 2020

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biomaterials. An experiment on sinus elevation in rab-bits30 reported higher bone density at the small gran-ule sites compared with the large granule sites, while a clinical study31 reported statistically significantly higher proportions of vital bone in the sinuses augmented with large granules compared to those filled with small granules. Consequently, data that might allow the se-lection of a biomaterial with the most suitable dimen-sions for bone formation are not yet conclusive. The hypothesis was that granules of different sizes might influence the osseointegration of the implant within the elevated space. Hence, the aim of the present study was to compare the osseointegration at the portion of the implant within the elevated space after sinus eleva-tion using different sizes of xenograft.

MATERIALS AND METHODS

Ethical StatementThe approval of the experiment was obtained from the Ethical Committee, Faculty of Dentistry of Ribeirão Pre-to, University of São Paulo, Brazil (2017.1.278.58.9). The animal care regulations of Brazil were rigorously fol-lowed. All the experiments and the maintenance care of the animals were carried out at the animal facility of the same university. The ARRIVE checklist and SYRCLE’s risk of bias tool for animal studies were followed.

Study DesignThe study was designed as an experimental split-mouth study. Maxillary sinus floor elevation was performed on both sides, and the elevated space was filled with either 1 to 2 mm (large sites; test) or 0.250 to 1.0 mm (small sites; control) xenograft granules. Eighteen rab-bits were used, divided into three groups of six animals, with each group allocated in a different period of heal-ing, ie, 2, 4, and 8 weeks.

Experimental ProceduresBefore surgery, the animals were anesthetized with 1.0 mg/kg of acepromazine (Acepran, Vetnil, Louveira) subcutaneously and a blend of 3.0 mg/kg xylazine (Dopaser, Hertape Calier) and 50 mg/kg of ketamine hydrochloride (Ketamin Agener, União Química Farmacêutica Nacional) intramuscularly. Local anes-thetic was also added.

A maxillofacial surgeon specialist (E.R.S.) performed all surgeries. A sagittal incision of ~2.5 cm was per-formed following the midline of the nasal dorsum to expose the nasal bone. Round antrostomies were pre-pared laterally to the nasal-incisal suture using twist drills of 2.8/3.2 mm in diameter to perforate the corti-cal layer of the nasal bone, and then, a drill 3.8 mm in diameter was applied to widen the most exterior edge

of the osteotomy, aiming to adapt the implant neck (Fig 1a). The mucosa of the sinus was pushed inward with small elevators (Bontempi) at both sites, and the space created was filled with similar volumes of either 1.0 to 2.0 (large) mm or 0.250 to 1.0 mm (small) granules of deproteinized bovine bone mineral (DBBM; Bio-Oss, Geistlich Biomaterials) (Fig 1b). Custom-made coni-cal implants (Emfils), with a moderately rough surface, blasted with aluminum oxide sand and acid-etched, were used. Details of the dimensions of the implants are shown in Fig 1c. The primary stability was obtained in the cortical layer (Fig 1d).

The periosteum was sutured with Polyglactin 910 5-0 (Ethicon, Johnson & Johnson) and the skin with Ethilon 4-0 (Ethicon, Johnson & Johnson).

EuthanasiaAnesthesia similar to that used for the surgeries was used, and euthanasia was obtained by a lethal dose of sodium thiopental (1.0 g, 2 mL, Thiopentax, Cristália Produtos Químicos Farmacêuticos). Biopsy specimens were retrieved in blocks and were fixed in 10% buffered formalin.

Experimental AnimalsEighteen New Zealand rabbits, 3.5 to 4 kg of weight and 4 to 5 months old, were used.

Housing and HusbandryThe rabbits were kept in rooms with controlled light and temperature, and each animal was in an individual cage with access to water and food. Professionals pro-vided for the care of the animals, monitoring wounds, biologic functions, and controlling pain and infection using appropriate drugs prior to, during, and after the experiment.

Sample SizeThe sample was calculated using the data from a study in minipigs in which the bone-to-implant contact per-centage (BIC%) was evaluated at implants placed im-mediately during sinus floor augmentation performed using either large or small DBBM particles.29 In that experiment, a difference of 5.9% in BIC% was assessed after 6 weeks of healing in favor of small compared with large granule sites. With a standard deviation of 3.6% (as reported as maximum value), a power of 0.8, and α = .05, six rabbits were estimated to be enough to re-ject the null hypothesis.

Randomization and Allocation ConcealmentThe website www.randomization.com was used to carry out the randomization by one author (D.B.) who was not involved in the surgery. Opaque sealed enve-lopes were arranged and opened after the preparation



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of both antrostomies by a masked author who was not involved in the surgery (S.P.X.) and communicated to the surgeon (E.R.S.).

Microcomputed Tomography EvaluationsAll biopsy specimens underwent a micro-CT analysis, carried out using micro-CT 1172 equipment (Bruker). The BIC% was evaluated in the full length of the im-plant contained within the subantral space. The volume of interest was evaluated in an anterior-posterior re-gion of 3.5 mm including the implant. The parameters were as follows: 9.92-µm isotropic pixel, 60 KV/165 µA, filter Al 0.5 mm, exposure time 596 ms, rotation step 0.4 degrees, frame average 4, and random movement 10. The software DataViewer (Bruker) was employed to re-position the cross-sectional images. The measurements were carried out with the software CTan (Bruker).

Histologic Preparation and AnalysesAfter the scanning at the micro-CT, all the specimens were dehydrated in alcohol, infiltrated in resin (LR White hard grid, London Resin), and then polymerized. Two ground sections were histologically processed us-ing a cutting and grinding system (Exakt, Apparatebau) and stained with either Stevenel’s blue and alizarin red or toluidine blue.

The implant surface was evaluated in two regions: the cortical region, delimited by the original cortical lay-er of the nasal bone; and the elevated space, which was the part of the implant included between the inner mar-gin of the original cortical layer and the apex of the im-plant. The following tissues on the implant surface were evaluated at ×100 magnification: new bone, old bone, and soft tissues (including marrow spaces, basic multi-cellular units [BMU], osteons, and vessels). The distance

from the inner profile of the cortical layer to the most apical contact of the bone to the implant surface (exten-sion of the osseointegration) and the distance from this point to the apical end of the implant, ie, the part of the implant that was not integrated, were also measured. Moreover, the area delimited by the implant, the sinus walls, and the sinus mucosa, was also measured.

Calibration for Histometric EvaluationsAll measurements were carried out by a well-trained assessor (K.A.A.A.) after calibration with another pro-fessional (D.B.). The interrater reliability in the identifi-cation of the tissues was Κ > 0.90.

Experimental OutcomesBoth histologic slides were evaluated for each implant, and mean values were used for further analyses. Per-centages were obtained for each tissue for the elevated regions and for the total length of the implant as well. The primary outcome was the new bone percentage in contact with the implant surface in the elevated region as evaluated in the histologic assessment. The second-ary outcome was the new bone percentage in contact with the implant surface in the elevated region as eval-uated in the micro-CT assessment.

Statistical MethodsDifferences between large (test) and small (control) sites were evaluated with the software IBM SPSS Statistics (IBM) applying the Wilcoxon rank sum test with α = .05. Differences between periods were evaluat-ed using the Mann-Whitney U test. With an explorative aim, the Pearson correlation coefficient was applied to new bone percentages evaluated at the histologic and micro-CT analyses.

Fig 1 Clinical view of the surgical proce-dures. (a) Round antrostomies were prepared laterally to nasal-incisal suture. (b) Xenograft granules placed within the elevated space. S = small granules; L = large granules. (c) Dimensions of the custom-made conical implants with a moderately rough surface. (d) Implants in position.

a

dc

5.9

∅ 4

.1

∅ 2

∅ 3

.5

4.70.4 × 45°

M2 × 0.4

b

S L



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RESULTS

All biopsy specimens were available for histologic and micro-CT analyses, so n = 6 was achieved for each pe-riod evaluated.

Histologic AnalysisAfter 2 weeks of healing, in both the large and small sites, new bone was found formed from the cortical layer of the nasal bone and from the sinus walls (Fig 2a). Apposition-al bone formation was observed on the implant surface, apically to the cortical region (Fig 3a), while a combina-tion of bone formation and resorptive processes were present in the cortical region, near the implant surface

(Figs 3b and 3c). A high number of xenograft granules exhibited zones of osseointegration on the surface (Fig 4a), while other granules were surrounded by a dense layer of cells (fibroblast-like cells) or had osteoclasts ad-hered on their surface (Fig 4b). Occasionally, the granules were observed in contact with the surface of the implant (Fig 4c). In some cases, new bone was also found above the apex of the implant, near the sinus mucosa (Fig 4d), while in other samples, the sinus mucosa was laying on the implant surface. At this stage of healing, the total new bone proportion on the implant surface (Table 1) was 23.3% ± 6.5% in the large sites and 19.7% ± 7.8% in the small sites (P = .249). After 4 weeks of healing (Figs 2b and 5a), the new bone was 25.7% ± 2.7% and 25.4%

Fig 2 Photomicrographs of ground sections illustrating the healing after (a) 2, (b) 4, and (c) 8 weeks of healing. S = small granules; L = large granules. Stevenel’s blue and alizarin red stain. Images originally grabbed with objective ×10.

Fig 3 Photomicrographs of ground sec-tions illustrating the healing after 2 weeks. (a) New bone formed from the base and the sinus wall. Osseointegration appeared to grow from the cortical layer. (b) Bone resorp-tion in the cortical layer in conjunction with new bone formation. (c) Woven bone formed in the cortical region, in contact with the im-plant surface and incorporating xenograft granules trapped in that region. Stevenel’s blue and alizarin red stain. Images originally grabbed with objective ×10.

a c

b

S S SL L

a b c



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± 6.1% in the large and small sites, respectively (P = .753). At the 8-week period (Figs 2c and 5b), new bone in-creased to 38.5% ± 8.2% in the large sites and 33.5% ± 8.7% in the small sites (P = .249).

In the elevated space (Fig 6; Table 2), in the 2-week period, new bone on the implant surface was repre-sented by 18.8% ± 6.8% in the large sites, while in the small sites, the per-centage was 15.8% ± 9.6% (P = .249). After 4 weeks of healing, new bone was 20.3% ± 3.5% and 23.3% ± 5.6% in the large and small sites, respec-tively (P = .249). At the 8-week pe-riod, new bone increased to 33.9% ± 9.5% in the large sites and to 28.5% ± 10.3% in the small sites (0.173).

In all periods, few particles of xenograft were noticed in contact with the implant surface, with pro-portions ranging from 1.7% to 3.6% and 0.8% to 2.3% in the large and small sites, respectively (Fig 4c).

In the cortical region, newly formed bone was present at high proportions in both groups, with 49.2% ± 18.3%, 64.5% ± 18.2%, and 58.0% ± 14.0% in the large group, and 53.4% ± 16.1%, 41.7% ± 15.8%, and 56.0% ± 20.2% in the small sites, after 2, 4, and 8 weeks (P = .753; .075; .600), respectively.

The height of the osseointegra-tion increased over time in both large and small sites, without pre-senting any statistically significant difference at any of the three peri-ods evaluated (Fig 7).

The area decreased between 2 and 8 weeks from 5.13% ± 1.52% mm2 to 4.87% ± 2.61% mm2 (5.1% reduction) in the large sites, and from 5.49% ± 1.63% mm2 to 4.73% ± 1.09% mm2 (13.7% reduction) in the small sites (Table 3). No differ-ences were disclosed between sites and periods.

Micro-CT AnalysisAfter 2 weeks of healing (Fig 8a), in the elevated space, BIC% was assessed at proportions of 21.0% ± 2.3% in the large sites, and

a

dc

b

Fig 4 Photomicrographs of ground sections illustrating the healing after 2 weeks. (a) New bone formed on the surface of the xenograft granules. (b) Granules of xenograft surrounded by a dense layer of cells (fibroblast-like cells) or with osteoclasts adhered on their surface (blue arrow). (c) Occasionally, the granules were found in contact with the implant surface. (d) New bone was also found above the apex of the implant, near the sinus mucosa. Stevenel’s blue and alizarin red stain. Images originally grabbed with objective ×10.

Table 1 Histologic Analysis: Tissues in Contact with Implant Surface (in %) at Various Periods of Healing

Total

New bone Soft tissue Old bone Xenograft

2 wk Large

Small

23.3 ± 6.5

23.1 (19.9; 24.9)19.7 ± 7.8

19.2 (13.8; 25.2)

73.2 ± 5.0

72.3 (72.0; 76.5)77.0 ± 6.7

77.5 (73.1; 82.2)

2.0 ± 1.4

1.7 (1.2; 2.7)2.3 ± 2.4

1.9 (0.7; 2.7)

1.5 ± 1.0

1.3 (1.0; 2.3)1.0 ± 0.4

1.0 (0.8; 1.0)

4 wk Large

Small

25.7 ± 2.7

25.6 (23.4; 27.5)25.4 ± 6.1

25.3 (23.3; 28.4)

70.8 ± 3.2

71.7 (70.1; 72.8)68.0 ± 3.5

69.1 (65.5; 69.2)

0.4 ± 0.7*

0.2 (0.0; 0.5)4.5 ± 2.5*

4.1 (3.4; 5.3)

3.2 ± 1.8

2.7 (2.4; 3.3)2.1 ± 0.7

2.2 (1.5; 2.5)

8 wk Large

Small

38.5 ± 8.2

41.1 (34.8; 44.1)33.5 ± 8.7

32.5 (28.5; 38.3)

58.2 ± 7.8

56.5 (52.9; 61.7)63.6 ± 8.6

62.6 (59.0; 67.4)

1.9 ± 0.9

1.8 (1.3; 2.59)2.3 ± 1.8

1.6 (1.2; 3.1)

1.4 ± 0.7

1.2 (0.9; 1.8)0.7 ± 0.8

0.5 (0.1; 0.8)

Mean values ± standard deviations. Medians (25th; 75th) percentiles. *P < .05



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21.2% ± 2.4% in the small sites (P = 1.000). After 4 weeks (Fig 8b), the respective proportions were 20.5% ± 3.3% and 23.4% ± 5.4% (P = .463), while after 8 weeks (Fig 8c), they were 23.0% ± 2.7% and 25.8% ± 4.1% (P = .249), respectively (Table 4).

The correlation coefficient r between histologic and micro-CT data was –0.4 (weak negative), –0.5 (moderate negative), and 0.4 (weak positive) for the large sites, and 0.0 (no linear relationship), 0.4 (weak positive), and 0.6 (moderate positive) for the small sites, after 2, 4, and 8 weeks of healing.

The volume of the grafted space (including the implant) de-creased between 2 and 8 weeks from 99.9 ± 9.2 mm3 to 79.1 ± 15.7 mm3 in the large sites (20.7% volume reduction), and from

98.6 ± 15.4 mm3 to 71.7 ± 15.5 mm3 in the small sites (Table 4; 27.2% volume reduc-tion). No differences were found between large and small sites. However, the differ-ence between 2 and 8 weeks was statisti-cally significant for both the large (P = .025) and the small (P = .016) sites.

Bone volume was 8.7% ± 1.2%, 10.6% ± 1.8%, and 12.2% ± 1.8% at the large sites, and 8.7% ± 1.4%, 11.7% ± 3.1%, and 13.9% ± 1.6% at the small sites, after 2, 4, and 8 weeks of healing (P = .917; .600; .116, respectively).

DISCUSSION

The aim of the present study was to com-pare the osseointegration at the portion of the implant within the elevated space after sinus elevation using different sizes of xenograft. After 2 weeks of healing, fractions of 18.8% and 15.8% of bone-to-implant contact (BIC%) were found in the large and small sites, respectively. These values increased over time, reaching frac-tions of 33.9% in the large sites and 28.5% in the small sites after 8 weeks of healing. No statistically significant differences were disclosed in any period.

These results agree with those from an-other experimental study in which implants were placed simultaneously with sinus floor augmentation in minipigs.29 In that study, similar to the present experiment, bilateral

Fig 5 Photomicrographs of ground sections. (a) Healing after 4 weeks. New bone in contact with the implant surface in the cortical region, showing a more advanced rate of maturation compared with the previ-ous period of healing. (b) Healing after 8 weeks. New bone in contact with the implant surface and incor-porating granules of biomaterial. Stevenel’s blue and alizarin red stain. Images originally grabbed with ob-jective ×10.

Table 2 Histologic Analysis: Tissues in Contact with Implant Surface (in %) at Various Periods of Healing Within the Elevated Space

Total

New bone Soft tissue Xenograft

2 wk Large

Small

18.8 ± 6.8

16.6 (14.9; 22.2)15.8 ± 9.6

14.7 (7.4; 21.9)

79.5 ± 5.9

81.2 (76.2; 82.4)83.1 ± 9.5

84.3 (76.9; 90.9)

1.7 ± 1.2

1.6 (1.1; 2.8)1.1 ± 0.5

1.1 (0.9; 1.1)

4 wk Large

Small

20.3 ± 3.5

20.4 (18.3; 21.7)23.3 ± 5.6

22.6 (21.1; 27.1)

76.1 ± 3.8

76.8 (73.8; 78.1)74.4 ± 5.1

75.3 (70.7; 75.8)

3.6 ± 2.1

3.2 (2.8; 3.7)2.3 ± 0.7

2.5 (1.8; 2.9)

8 wk Large

Small

33.9 ± 9.5

34.8 (29.2; 39.4)28.5 ± 10.3

28.1 (20.4; 36.0)

64.4 ± 9.4

63.0 (58.5; 69.8)70.7 ± 10.4

70.3 (63.8; 79.0)

1.8 ± 0.9

1.5 (1.2; 2.2)0.8 ± 1.0

0.6 (0.1; 1.0)

Mean values ± standard deviations. Medians (25th; 75th) percentiles. No statistically significant differences were found between large and small sites.

a b



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sinus floor elevation was performed using deprotein-ized xenograft composed of either large or small gran-ules. The healing was evaluated after 6 and 12 weeks, including five animals in each group. After 6 weeks,

within the elevated space, BIC% was 18.7% in the large sites and 15.4% in the small sites. This outcome showed a tendency to greater bone formation in the large com-pared with the small sites, similar to the results of the

Fig 6 Graphs illustrating the percentage of new bone in contact with the implant surface at the large and small sites, in the various periods evaluated.

Fig 7 Graphs illustrating the extension of new bone formation (bar in green) on the implant surface within the elevated space and the apical portion of the implant without new bone formation (bar in grey).

Fig 8 Micro-CT 3D images representing the healing in the grafted sinus after (a) 2, (b) 4, and (c) 8 weeks of healing. S = small granules; L = large granules.

Time (wk)

40

35

30

25

20

15

10

5

02

18.8

33.9

20.3

15.8

28.5

23.3

4 8

New

bon

e in

the

elev

ated

regi

on (%

)Large sitesSmall sites

2 wk 8 wk4 wk

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0Large LargeLargeSmall SmallSmall

Hei

ght o

f the

oss

eoin

tegr

atio

n (m

m)

2.18 1.86 1.772.16 0.74 0.97

1.59

1.97 1.99

1.60

2.962.79

S SSL L L

a b c

Table 3 Elevated Area (in mm2) at Various Periods of Healing

2 wk Large

Small

5.13 ± 1.52

5.27 (3.90; 6.29)5.49 ± 1.63

5.11 (4.23; 6.37)

4 wk Large

Small

5.36 ± 1.45

5.95 (4.09; 6.45)5.40 ± 0.90

5.24 (4.68; 5.87)

8 wk Large

Small

4.87 ± 2.61

4.24 (3.39; 5.83)4.73 ± 1.09

4.68 (4.19; 5.26)

Mean values in bold ± standard deviations. Medians (25th; 75th) percentiles. P < .05

Table 4 Micro-CT Analysis: Bone-to-Implant Contact in Percentage (BIC%) and Volume of the Elevated Space (mm3)

BIC% Volume (mm3) Bone volume (%)

2 wk Large

Small

21.0 ± 2.3

21.2 (20.0; 21.7)21.2 ± 2.4

20.3 (19.6; 22.5)

99.9 ± 9.2

99.4 (92.8; 104.7)98.6 ± 15.4

102.6 (88.1; 108.7)

8.7 ± 1.2

8.7 (7.9; 9.3)8.7 ± 1.4

9.0 (8.5; 9.5)

4 wk Large

Small

20.5 ± 3.3

20.2 (18.7; 23.0)23.4 ± 5.4

23.8 (21.1; 24.5)

86.5 ± 14.4

86.3 (78.1; 97.1)78.0 ± 11.5

78.6 (69.0; 81.8)

10.6 ± 1.8

10.8 (9.9; 11.9)11.7 ± 3.1

11.2 (9.6; 12.2)

8 wk Large

Small

23.0 ± 2.7

23.1 (22.8; 24.2)25.8 ± 4.1

27.7 (22.3; 28.1)

79.1 ± 15.7

79.3 (71.4; 88.9)71.7 ± 15.5

72.5 (64.5; 80.0)

12.2 ± 1.8

12.0 (10.7; 13.4)13.9 ± 1.6

13.9 (12.5; 14.5)



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present study after 8 weeks of healing. However, after 12 weeks, the respective proportions in that study29 became 32.2% and 35.3% for the large and small sites, respectively. No statistically significant differences were found in both periods examined.

DBBM granules of different dimensions also yielded similar results when bone density was evaluated.27,28 In a histologic study in humans, sinus floor elevation was carried out bilaterally in 10 subjects using DBBM composed of either large or small granules.27 After 8 months, hard tissue biopsy specimens were collected from the implant sites and examined under microscope. No statistically significant differences were disclosed in new bone density between the two grafted sinuses. In another histologic human study,28 a bilateral sinus floor elevation was performed in 10 patients using either large or small granules of DBBM. Again, no statistically significant differences were observed in biopsy speci-mens gathered after 6 to 9 months of healing.

However, in an experimental study in rabbits,30 in which particles of deproteinized rabbit bone of two dif-ferent dimensions were used to graft the sinuses, high-er bone density and bone-to-graft contact were seen at the small compared with the large particles.

Conversely, in a clinical study,31 opposite outcomes were observed. A sinus floor elevation was carried out bilaterally in 13 subjects. Biopsy specimens were taken from the lateral sinus walls after 6 to 8 months. Vital bone density was 26.8% for the large granules and 18.8% for the small granules, with the difference be-ing statistically significant. The outcome of that clinical study agrees with that of the present study.

Integration at implants placed in sinuses grafted with small DBBM granules was evaluated in an ani-mal experiment in rabbits.21 The elevated spaces were grafted with either DBBM of 0.250 to 1.0 mm or autog-enous bone, and implants with a moderately rough surface were placed simultaneously. The healing was evaluated after 7 and 40 days. After 7 days, BIC% was similar in both groups (7% to 8%). After 40 days, higher amounts of BIC% were found in both sites, with 56.7% at the autogenous sites and 40.3% in the DBBM sites.

In the present experiment, implants with a moder-ately rough surface were placed simultaneously with sinus elevation. Some human studies have evaluated the histologic healing at micro-implants placed simul-taneously with sinus floor elevation.32,33 The sinuses were shifted using either DBBM or biphasic calcium phosphate with different proportions of hydroxyapa-tite and beta-tricalcium phosphate. Micro-implants with a sandblasted, acid-etched (SLA) surface were placed through the alveolar crest in both studies, and biopsy specimens were gathered after 6 to 8 months of healing. BIC% values ranged from 35% to 38% in one study32 and from 55% to 65% in the other study.33

It is important to underline that the early stages of the healing at implants placed simultaneously with si-nus floor elevation9,21,32–38 differ from those at implants placed in healed grafted sinus.39 In the latter, multiple contacts with the regenerated bone can be achieved, and the healing process might be considered to be sim-ilar to that at implants placed in healed alveolar crest, as illustrated in human and animal studies.40–44

However, in the case of simultaneous implant placement, clot and xenograft surround the protrud-ing portion of the implants within the elevated space in the earliest period of healing. Under such condi-tions, the only contact with the preexisting bone at the time of placement is in the cortical region. In the present study, implants with a moderately rough sur-face were placed simultaneously with the sinus eleva-tion procedure, and osseointegration started from the portion of the implant close to the cortical layer, and subsequently bone grew toward the apex of the im-plant (Fig 7). This is in agreement with an experimental study in monkeys.9

In the present study, bone apposition did not reach the apical region of the implant (Fig 7). It can be argued that a longer healing time should have been allowed to complete the osseointegration of the implant up to the apex. However, in another similar study,21 integra-tion of the implants was also complete after 40 days of healing at sites grafted with DBBM xenograft. The differ-ence might be ascribed to the fact that some specimens presented the sinus mucosa in direct contact with the apical region of the implants. This loss of space-making effect might have hampered bone formation in this region.

In the case of sinus floor elevation with simultane-ous implant placement, the surface microgeometry plays an important role in osseointegration. As previ-ously described,32,33 a sandblasted, acid-etched surface resulted in a high degree of osseointegration at the portion of the implant protruding within the grafted si-nus. However, when a micro-implant with a turned sur-face was used, a very low degree of osseointegration was achieved.45

The micro-CT analysis on BIC% revealed small differ-ences compared with the histologic assessments. These differences might be because the histologic analysis was performed in only two central sections of the region, while the micro-CT analysis included the full surface of the implant protruding within the subantral region. Nevertheless, a weak uphill linear relationship was found when all data were compared. This might be due to the limitations of the micro-CT analysis to differentiate the density of the newly formed bone from the biomaterial possibly in contact with the implant.46 Moreover, the fact that new bone grows on the surface of the granules



Masuda et al

of biomaterial might have contributed to confound the discrimination between xenograft and bone.

As a limitation of the present study, it should be mentioned that the two ranges of granule sizes were not completely distinct, so the minimum differential in size between one xenograft and the other was zero, and the maximum was 1.75 mm. This, in turn, means that several granules of the two groups could have presented similar dimensions. This could have affected the possibility of finding statistically significant differ-ences between groups. Experiments that include a dis-tinct differential in size between graft granules should be performed. The chosen animal model has been shown to present faster osseointegration compared with humans,47 so the outcome should be generalized with caution for humans. Human studies with a large sample size should be conducted to get more clinically relevant information. The healing time used in the pres-ent study should be considered enough to allow com-plete osseointegration of the implants as shown by other studies, with similar or shorter periods of healing, in which proper osseointegration of the implants was reported.21,48 The beam hardening artifact might have affected the measurements at the micro-CT, resulting in an inaccurate outcome.49

The present study showed that bone apposition over time and percentages of osseointegration are not influenced by the size of the granules of the biomaterial used. This, in turn, means that the choice of the size of the biomaterial might be based on other factors, such as the dimensions of the sinus and of the ostium.50

CONCLUSIONS

The use of xenograft granules of different dimensions resulted in similar amounts of bone-to-implant con-tact at implants placed simultaneously with sinus floor augmentation.

ACKNOWLEDGMENTS

The cost of the experiment, including the biomaterial, was finan-cially sustained by ARDEC Academy, Rimini, Italy. The implants were provided free of charge by Emfils, Villa Nova, Itu, SP, Brazil. The au-thors disclosed no conflicts of interest.

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