Artigo que será submetido à publicação, no periódico Clinical Implant Dentistry and
Related Research.
2D and 3D BIC analyses in human samples reconstructed with autogenous and xenogenous bone grafts
1Karla de Faria Vasconcelos, 2Lívia dos Santos Corpas, 3Bernardo Mattos da Silveira, 4Luis Eduardo Marques Padovan, 5Ivo Lambrichts, 6Reinhild Jacobs, 1Frab Norberto Bóscolo
1Department of Oral Diagnosis, Division of Oral Radiology, Piracicaba Dental School, State University of Campinas, Piracicaba, São Paulo, Brazil
2Department of Prosthetic Dentistry, Oral Health Sciences, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium
3Department of Prosthetic Dentistry, Brazilian Dentistry Association (ABO RJ), Rio de Janeiro, Brazil
4Department of Implant Dentistry, Latin American Institute of Research and Dental Training (ILAPEO), Curitiba, Brazil
5Laboratory of Histology, Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium
6Omfs impath research group, department imaging & pathology, faculty of medicine, university of Leuven and maxillofacial surgery, University Hospitals Leuven, Leuven, Belgium
Correspondence address: Karla de Faria Vasconcelos. University of Campinas, Piracicaba Dental School, Department of Oral Diagnosis, Av. Limeira, nº 901, Piracicaba, São Paulo, Brazil, 13414-903.
ABSTRACT
Information from histomorphometry, especially from bone-to-implant contact (BIC) calculations, are commonly used to evaluate osseointegration. Moreover, high BIC values are desired when seeking to achieve a more stable osseointegration. However, when this analysis is performed by a histological technician, it becomes limited to the calculation of the average found among specific histological sections and is therefore limited to information from only one portion of the sample. The present study was conducted in an attempt to provide a two-dimensional and a three- dimensional evaluation of the fixing screws’ surface bone formation. To accomplish this, seventeen samples containing fixing screws covered by 0.5 to 1.0mm of human bone were removed using a trephine drill, six months after the placement of the bone grafts in the maxilla. The samples were evaluated by means of SkyScan 1173 micro- computed tomography (micro-CT) scanner (Bruker, Kontich, Bélgica), while the histological cuts were obtained by means of the non-demineralization technique. The 3D BIC calculation was only possible in the images acquired through micro-CT. A segmentation protocol was created to be equally applied to all of the image data groups after their binarization. The structural indexes of the tissue surface and the intersection surface, as well as the relationship between the intersection surface and the tissue surface, were calculated in both the bone graft and native bone areas. The 3D values revealed differences between the native bone and bone graft areas (p=0.0021), with the highest values present in the native bone areas, whereas the 2D BIC analyses showed no statistically significant differences between the methods (p=0.78). Moreover, no statistically significant difference between the BIC values could be observed when the two types of graft (atugenous and xenogenous) were compared (p=0.43). Therefore, micro-CT proved to be a valuable tool for 2D and 3D BIC calculations, making it possible to both preserve the samples and evaluate the volume of the screw’s surface not only in specific cuts, but also in the histological technique as a whole. Keywords: Bone graft, Micro-computed tomography, Human bone, Bone-to-implant contact.
INTRODUCTION
The histological technique is considered to be the “gold standard” for the analysis of bone formation around dental implants, providing a high-resolution image.1 Information from histomorphometry, especially from the bone-to-implant contact (BIC) calculation, are commonly used in osseointegration evaluations. High BIC values represent a greater BIC and, consequently, a greater BIC stability. However, when this analysis is performed by a histological technician, it becomes limited to the calculation of the average found among specific histological sections and is therefore limited to information from only one portion of the sample.2
As the histological technique consists of microscopic observations of the region of interest, it requires a substantial preparation of the samples, including a series of stages for the preparation and analysis of the slices and, consequently, a prolonged execution time.3 As this method presents numerous stages, damage to the tissues during the preparation and the loss of information can occur, which reveal that this technique is quite limited. Two other distinct disadvantages to the histological technique include the loss of materials when making the cut, due to the thickness of the blade used (± 300 µm), and the limitation referent to 2D analyses, assuming that the selected cuts of the sample were representative of the entire specimen.4
Micro-computed tomography (micro-CT) has been widely used in BIC evaluations and has presented a good correlation when comparing images obtained using the histological technique and those from micro CT in these regions.5 Moreover, it allows for a three-dimensional analysis of the image, using a non- destructive technology.6-9 In this sense, the present study’s scientific justification is based on the three-dimensional and two-dimensional evaluations of the bone formation on the surface of fixing screws.
MATERIALS AND METHODS
For the present study, samples were collected from maxillary edentulous patients who needed to undergo bone graft reconstruction to prepare for a later installation of bone implants. Excluded from this study were those who presented odontogenic infections or systemic disorders that could interfere in the result of the treatment and/or expose the patient to a possible surgical risk. Sample collection was performed by a single implant dentist after having been approved by the Research Ethics Committee (COEP) from the State University of Ponta Grossa (UEPG) in Paraná, Brazil, logged under protocol number 14558/11. The collected samples were sent to the Catholic University of Leuven (KULeuven) in Belgium for analysis.
This study used Orthogem® xenogenous bone blocks (Baumer, São Paulo, Brazil) of 10 x 15 x 5 cm and autogenous bone blocks extracted from the mandibular ramus. The bone blocks were fixed with Neodent® fixing screws (Neodent, Curitiba, Brazil), of 2 mm in diameter and received surface treatment prior to the installation of the bone implant in an attempt to favor osseointegration and make BIC calculations more feasible.
Seventeen bone samples, made up of a fixing screw covered with a 0.5 to 1mm thick bone tissue, were obtained using a trephine drill of 4.1mm in diameter, six months after the reconstructions had been performed. Seven samples came from the sites that had received an autogenous bone graft, while ten came from sites that had received a xenogenous bone graft. After the removal of the samples, these grafts were stored in a 10% formalin buffer solution. Before the histological process, micro- CT images were taken using a SkyScan 1172 micro-CT scanner (Bruker, Kontich, Bélgica), equipped with: 130kV, 60μA, 6μm voxel, and a 0.25mm thick aluminium filter. The reconstruction of the image was performed using the NRecon software (Bruker, Kontich, Belgium), while the quantitative analyses for the bone percentage calculation present on the screw surface and the 3D BIC calculation were performed using the CTAnalyzer software (Bruker, Kontich, Belgium).
The samples were fixed in formalin buffer solution and dehydrated in an ascending series of ethanol concentrations (60%, 70%, 80%, 90%, 96%, and 100%). The cuts were performed in the direction of the long axis of the fixing screw, using an ISOMET 1000 Precision Saw (Buehler, Lake Bluff, Illinois, USA). Next, each slice was polished, using a MINIMET Polisher (Buehler, Lake Bluff, Illinois, USA), resulting in a final thickness of approximately 70µm. After, the slices were stained with a combination of Stevenels blue to highlight the non-mineralized bone tissue and Van
Giesen red to highlight the mineralized bone tissue.
The histological exams were analyzed under a light microscope (Leitz Laborlux S, Wetzlar, Germany) at a magnification of 40X, 100X, and 400X. The evaluations of the histomorphometric proportions were carried out using the high sensitivity mode of a color video camera (AxioCam MRC5, Zeiss, Gottingen, Germany) mounted upon the light microscope and with a color image analysis from the software package (Axiovision 4.0, Zeiss, Gottingen, Germany).
The 3D BIC calculation was only possible in the images acquired through micro-CT. A segmentation protocol was created to be equally applied to all of the image data groups after their binarization. The structural indexes of the tissue surface and the intersection surface, as well as the relationship between the intersection surface and the tissue surface were calculated in both the graft and native bone areas.
The 3D calculation was carried out in two different volumes of interest (VOI) per sample: one VOI was selected in the bone graft region and the other in the native bone region. The statistical test of multi-way ANOVA was performed in order to find possible correlations between the BIC values and the bone graft and native bone areas, as well as between the different bone grafts (autogenous and xenogenous).
The 2D BIC calculation was performed using the AxioVision software (Versão 4.7.1, Jena, Germany) (Figure 2). Corresponding representative cuts were selected in the two imaging modalities – histological and micro-CT (Figure 3). Software tools were used to analyze the entire surface of the screw in the two imaging modalities. The evaluations were performed by two trained examiners, at distinct times and on
the same computer, with the repetition of all measurements after 15 days. Initially, the entire surface of the screw was selected and at a second moment, only the contact areas of the mineralized bone (highlighted in red) with the screw’s surface were selected. These data were collected in an attempt to respond to another part of the second main purpose – to produce a two-dimensional evaluation of the bone percentage on the surface of the fixing screw.
The evaluations were performed by two trained examiners at distinct times and on the same computer, with the repletion of all measurements after 15 days. The Student’s t test was applied to compare the two techniques.
RESULTS
The 3D BIC calculation was carried out on 13 samples in two different VOIs per sample – one VOI was selected in the bone graft region and the other in the native bone region (Table 1). The statistical tests were conducted in an attempt to find possible correlations between the BIC and the bone graft and native bone areas, as well as between the BIC and the autogenous and xenogenous bone grafts. The multi-way ANOVA test showed a statistically significant difference in the BIC values in the different areas (p=0.0021), with the higher values being more present in the native bone areas. However, no statistically significant difference was observed between the types of bone grafts (p= 0.437).
The 2D BIC calculation was carried out on 14 samples, both in histological and micro-CT images (Table 2). When comparing the techniques, the multi-way ANOVA test showed no statistically significant differences between the 2D BIC values (p=0.802). This test also found no difference between the two types of bone grafts (p=0.097) (Table 3).
DISCUSSION
The methodology adopted in the present research was similar to that used in prior studies that validated the micro-CT technique, when compared to the
histological technique, for BIC evaluations.10 The analyses were carried out on micro- CT images with a resolution of 6μm, after having excluded 4 voxels from the fixing screw’s surface. The present study’s results corroborate with findings from Butz F et al. (2006), who found a significant correlation between histology and the micro-CT for the cortical bone (r=0.65; p<0.05), and bone marrow (r=0.92; p<0.05). These authors also emphasize the importance of the exclusion of the voxels, aimed at avoiding an overestimation of the calculated values. The non-exclusion of the artifact area can led to incorrect bone assessments in the peri-implant region.
As regards the 3D BIC calculation, the 3D micro-CT evaluation presented a statistically significant difference in the BIC values when comparing the native bone and bone graft areas, with the highest values found in the native bone areas. The method used in this study allowed for the calculation of the tissue surface, which corresponds to the surface of the implant, as well as for the intersection surface, which represents the amount of bone tissue on the surface of the dental implant. To compare the 3D BIC values in the bone graft and native bone areas, the intersection surface/tissue surface relationship was calculated in the two regions, which resulted in the percentage of the surface covered by bone. This result, higher in the native bone area, was expected, since the healing process and bone remodeling is a dynamic process and is directly dependent on the presence of cells and blood vessels to begin and maintain the healing process.11 In this sense, the greater supply of blood and cells in the native bone regions, rather than the areas that had received a bone graft, make the bone formation quicker and, consequently, presented higher BIC values.
Concerning the 2D BIC calculation, conducted in both the histological technique and the micro-CT imaging, the results showed an equivalence between the techniques (p=0.78), which has been quite refuted in the literature. 10,12,13 However, the limitation of the 2D technique for the two modalities is quite clear as well. In the histology technique, the destruction of the sample is the main disadvantage, whereas for the micro-CT, the 2D calculations represent an suboptimal use of this modality,
given that this same technique can be performed using a three-dimensional evaluation.
Bernhardt et al. (2012) compared the BIC and the Bone Volume (BV) obtained in the 2D images of the histological cuts and 2D and 3D images obtained from a Synchontron radiation micro-CT, using implants installed in mini-screws. Comparing 3-4 histological cuts per dental implant with the corresponding 3-4 micro-CT cuts, the authors found no statistically significant differences (1.95; p=0.703). When a 3D BIC analysis was performed and compared to the histological analysis, these authors still found no statistically significant differences (4.9%; p=0.171). Other authors have also observed a clear connection between the values obtained by the histomorphological technique and the micro-CT after having evaluated the BIC values at two different times (2 and 4 months) on the surface of titanium and hydroxyapatite implants.14 Such findings corroborate with the present study’s results, since, when the measurements taken in the two techniques were compared, no statistically significant difference between the techniques was observed (p= 0.78). However, the 2D and 3D correlation could not be performed in the present study due to the different types of measurements used. In the 2D technique, the bone graft and native bone regions were not shown, as the BIC was evaluated along the entire extension of the screw, whereas in the 3D technology, the BIC evaluations between the bone graft and native bone areas was carried out separately.
CONCLUSION
The 3D BIC calculation demonstrated that no statistically significant differences could be observed when comparing the two types of bone grafts, but could be identified when comparing the native bone and bone graft areas.
The 2D BIC evaluation showed no differences among the bone formations on the surface of the fixing screws when comparing the different types of bone grafts and the methods.
TABLES
Table 1 – Percent values of the 3D BIC calculated in the different samples and in the different areas. In the table, (-) represents the regions which the 3D BIC was unable to be calculated. Sample 3D BIC Native Bone (%) 3D BIC Bone Graft (%) A1 0.57 0.16 A2 - 0.34 A2 0.60 0.55 A3 0.37 - A4 - 0.45 X1 0.71 0.03 X2 0.66 0.02 X3 - 0.47 X4 0.57 - X5 0.66 0.03 X6 - 0.33 X7 0.49 0.03 X8 - 0.43
Table 2 – 2D BIC values calculated in micro-CT and histological cuts.
Sample 2D BIC (%) Micro-CT Histological 2D BIC (%)
A1 23.41 21.50 A2 17.21 15.12 A3 26.70 29.11 A4 25.81 25.65 A5 30.83 46.33 X1 26.07 27.11 X2 34.78 29.94 X3 24.25 23.12 X4 7.94 7.85 X5 1112 24.24 X6 34.24 30.45 X7 11.66 13.76 X8 14.56 19.50 X9 12.38 14.44
Table 3 – Average and standard deviation (sd) of 2D BIC values, comparing the two types of bone grafts and the two methods.
Histology Micro-CT p-values
Average sd Average sd Techniques Grafts
Autogenous 27.54 11.72 24.79 5.01 0.802 0.097
Xenogenous 18.93 10.78 19.66 10.34
FIGURES
Figure 1: Imaging binarization process, carried out by superimposing the real image (A) upon
Figure 2- 2D calculation in a histological sample. (A) the selection of the entire surface of the
fixing screw and (B) the selection of the areas in which the bone was in direct contact with the surface. The 2D BIC calculation is performed through the analysis of these two values in (%).
Figure 3: More representative cut of a sample that received an autogenous bone graft.
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CONCLUSÃO
1. Sítios implantares na maxila, reconstruídos com enxertos ósseos autógeno e xenógeno, após seis meses de remodelamento, apresentaram volume ósseo, distância e espessura entre as trabéculas distintos.
2. Segundo a Classificação de Lekholm e Zarb, os sítios analisados na presente pesquisa, foram classificados em tipo 3 e tipo 4 entretanto, apresentaram características microestruturais semelhantes, não havendo distinções entre os dois.
3. O cálculo do BIC 3D revelou não haver diferenças estatísticas entre os tipos de enxerto comparados e sim entre as áreas de osso nativo e enxerto.
4. A avaliação do BIC 2D revelou não haver diferença entre a formação óssea na superfície dos parafusos de fixação nos diferentes tipos de enxerto e entre as técnicas.
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