The microCT consists of a microfocus tube which generates a cone-beam of X-rays, a rotating specimen stub on which the object to observe is put, and an electronic
detector system which acquires the images. The X-ray source is a 10 W microfocus tube (tungsten), which can respectively operate at currents and voltages up to 98mA and 100 kV. The stub can rotate and it usually use a 0.45rotation step for complete acquisition of 180for a total time of about 2 h.
Comparing different resolutions of CT systems, it’s possible to see that Medical- CT resolution is of about 100mm and laboratory micro-CT resolution range is from 5 to 10–20mm, depending to sample dimensions. This system is supplied with a 1 mm-thick aluminium plate, placed in front of the scintillator, in order to use it as hardware beam hardening minimiser filter.
MicroCT system used in this work, the Skyscan 1072 instrument (SkyScan, Kartuizersweg 3B, 2550 Kontich, Belgium),allows to obtain radiographic images as well as tomographic reconstruction ones, nevertheless all data acquiring and processing are software-driven.
From the frontal projection data it is then possible to reconstruct the cross- section images of the object, using the software “Cone_rec” (ver. 2.23, Skyscan, Belgium), which is based on the cone beam algorithm (14). These projection images in TIFF format were reconstructed in slice images in the BMP format.
These slices were also evaluated with “CTAnalyser” software (ver. 1.11, Skyscan, Belgium) in order to obtain the 3D structure and morphometric parameters.
To be processed in this way, images have to be binarized. The process of
“binarization” needs to choose a threshold value and has as a result an image composed only of black pixels (bone) and white pixels (non-bone).
After binarization, “CTAnalyser” software allows to calculate morphometric parameters and create the three-dimensional image of the internal structure of the analyzed sample from images’ slices in BMP format.
For each biomaterial sample was selected an internal Region Of Interest (ROI) and histomorphometric parameters for the corresponding Volume Of Interest (VOI) were calculated. The parameters analyzed, also called “primary morphometric indices”, were:
– Tissue Volume, TV, (mm3), total volume of the volume-of-interest (VOI). The 3D volume measurement is based on the marching cubes volume model of the VOI.
– Bone Volume, BV, (mm3), total volume of binarized objects within the VOI.
The 3D volume measurement is based on the marching cubes volume model of the binarized objects within the VOI.
– Tissue Surface, TS (mm2), the surface area of the volume of interest, measured in 3D (Marching cubes method).
– Bone Surface, BS, (mm2), the surface area of all the solid objects within the VOI, measured in 3D (Marching cubes method).
– Bone Volume Fraction, BV/TV, (%), the proportion of the VOI occupied by binarized solid objects.
– Specific Bone Surface, BS/BV, (1/mm), the ratio of solid surface to volume measured in 3D within the VOI. Surface to volume ratio or “specific surface” is a useful basic parameter for characterizing the thickness and complexity of structures.
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– Bone Surface Density, BS/TV, (1/mm), the ratio of surface area to total volume measured as described above in 3D, within the VOI, (Chiapasco and Romeo 2002; Cowan et al.2007; Urist1965; Mastrogiacomo et al.2005; Perilli et al.
2007; Gielkens et al.2008; Haı¨at et al.2007; Jones et al.2007; Kachelrieb2008;
Kamburoglu et al.2008; Kerckhofs et al.2008; Lin-Gibson et al.2007; Mare´chal et al.2005; Papadimitropoulos et al.2007; Parkinson et al.2008; Stauber and M€uller2008; van Lenthe et al.2007; Willems et al.2007).
There are also “secondary indices” for the quantification of bone architecture.
The model most widely used is the plate model, which assumes that all the trabecular bone is organized in infinite plates, with a certain thickness (Tb.Th), separation (Tb.Sp) and number per unit length (Tb.N) (Waarsing et al.2004).
The “secondary indices” are:
• Trabecular Thickness,Tb.Th, (mm), that represents the thickness of the trabecular bone
Tb:Th ¼ 2BV=BS
• Trabecular Number,Tb.N, (1/mm), that represents the trabecular density and it is the number of plates traversed by a line of unit length perpendicular to the plates.
Tb:N:¼1=2BS=TV
• Trabecular Separation,Tb.Sp, (mm), that represents the distance between edges of the bone trabeculae (Chiapasco and Romeo2002; Cowan et al.2007; Urist 1965; Mastrogiacomo et al.2005; Jones et al.2007; Kachelrieb2008; Kerckhofs et al.2008; Lin-Gibson et al.2007; Papadimitropoulos et al.2007; Stauber and M€uller2008; van Lenthe et al.2007).
Tb:Sp ¼1=Tb Nð Tb:ThÞ
By means of “3D Creator.exe” software it’s possible to obtain the 3D inside structure of a biomaterial sample.
This software includes all the functions that are necessary to work on obtained 3D images, i.e. allows to process them with complex algorithm to obtain a better display and understanding of observed ultrastructures. (Chiapasco and Romeo 2002; Guldberg et al.2008; Urist1965)
In this study three commercialised osteoconductor biomaterials of different origins have been selected and named according to the following list:
1. Biomaterial 1 – Bovine hydroxyapatite, (Bio-Oss, Geistlich Biomaterials, Italy);
2. Biomaterial 2 – Bioceramic hydroxyapatite, (ENGIpore, Fin-Ceramica Faenza S.p.A, Italy);
3. Biomaterial 3 – Poli Acid (D, L – lactic – co-glicolic), (SINTBONE, GhimasS.p.
A, Italy)
Bovine hydroxyapatite (biomaterial 1) is a bone substitute material for natural bone regeneration. This medical device is obtained from the mineral portion of bovine bone and is used by dental and oral surgeons in order to build up a new bone.
This material is used in implantology (teeth replacement by means of implants), periodontology (for diseases affecting the tissue supporting the teeth) and for large bone defects in oral and maxillofacial surgery. Bio-Oss® supports the body’s natural healing processes because of its close resemblance to human tissue. More than 700 scientific studies, in fact, demonstrated a long-term success with this material, as the product has been used in bone regeneration for 24 years.
Bioceramic hydroxyapatite (biomaterial 2) is an innovative synthetic porous hydroxyapatite biomaterial with a trabecular structure similar to natural bone. It is a ready available bone graft, manufactured to a highly controlled specification and free of disease transmission risk. Different shapes have been designed to provide surgeons with a full range of products to meet their bone grafting needs. Its particular structure gives this material a porosity of almost 90%, and this guarantees an easy access to cells, biological fluids, and signaling molecules throughout the bone substitute. Despite the highly porous structure, this material is able to resist compression forces like natural cancellous bone. Once applied in situ, the material rapidly absorbs all bio-active proteins, growth factors and bone precursor cells contained in the physiological fluids. This is the starting point of the biological cascade leading to effective bone regeneration.
Poli Acid (D, L – lactic – co-glicolic) synthetic osteoconductive biomaterial 3 is a synthetic osteoconductor biomaterial used in dental and maxilla-facial surgery, obtained from sintering process of co-polymer micro-particles (PLA/PGA). This biomaterial behaves like a rigid micro-porous texture keeping space among granules, making channels ready for vascularisation and tissue penetration. As a result of the presence of a large amount of channels in this biomaterial, blood, plasma and serum can easily get through it, causing the immediate formation of a stable blood clot making both inside and outside the biomaterial.
Each biomaterial as shaped block, in preliminary phase, has been subjected to microtomographic analysis to evaluate the most important morphometric chara- cteristics, before clinical use. Bovine hydroxyapatite was cancellous bone block of (13.396.5) mm dimensions, Bioceramic hydroxyapatite was block of (105 5) mm dimensions, Poli Acid was block of (165.56) mm dimensions.
Then, the same biomaterials shaped in granular version have been glued in an ideal human bone defect.
Three healthy patients, aged 18–36, needing for a surgical dental extraction of lower third molars, have been selected.
Each patient was informed and has signed an informed consent form according to the declaration of Helsinki that requires the patient adhesion to the clinical experimental study.
In the post-extractive side of each patient has been implanted one of the biomaterials (test sample) used in this study, while the opposite side has been treated only with blood clot (control sample).
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After 4 months from surgery, test and control sample follow-up have been performed to extract cylindrical bone sample by means of stainless-steel trephine bone cutter with 2 mm internal diameter.
Each extracted bone sample was fixed in 10% formalin and before micro- tomographic analysis has been carefully washed with physiological solution and fixed on suitable stub by plasticine.
After microtomographic analysis, bone samples have been again inserted in sterile physiological solution for about 10 min for rehydration, and then in 10%
formalin, to store them for histological investigations. Each bone sample was embedded in paraffin and 7mm sections were prepared and stained with hematoxy- lin and eosin.
Each histological obtained image, for test and control bone samples, will allows to display new bone formation and scaffolding residues.
For each test and control sample the morphometric parameters have been calculated and 2D – 3D dimensional images were processed by means of the same test protocol used for microtomographic analysis of biomaterials shaped as block, before the implant phase.
The biomaterial blocks have been acquired and processed with different resolution because of different size that are commercialized, according to the following values:
• Biomaterial 1 block at 25 X magnification, corresponding to 11.72mm pixel size;
• Biomaterial 2 block at 40 X magnification, corresponding to 7.32mm pixel size;
• Biomaterial 3 block at 25 X magnification, corresponding to 11.72mm pixel size.
For all control and test samples, the same acquisition parameters have been chosen (as showed in Table 1) to allow comparing qualitative and quantitative results.
To obtain a three-dimensional structure’s specimen, the range of values that corresponds to image grayscale should vary according to material density. In fact, in radiology, 255 grey’s levels correspond to different X-ray absorption coefficient of sample structures. It is known that absorption coefficient depends on the density of crossed material, especially the denser material that absorbs more rays and can be identified with white color, on the other hand X-rays can penetrate the less dense material, arriving on the CCD camera and be identified with black color. In this study images present inverted colours, which means that white color is used for less dense material and black for the denser.
Table 1 Microtomographic acquisition parameters used for bone sample analysis Acquisition parameters of microtomographic instrument
Magnification (cross-section pixel size) 95X (3.1mm)
Rotation angle 1801
Rotation step 0.45 0.01
Power source 100 KV/98 microA10%
Filter thickness (Al) (10.01) mm
The observation of images’ samples showed that in all the samples three main shades of gray can be identified which can correspond to three different bone densities. Therefore, three threshold ranges were chosen (0–195, 0–155, 0–115), as showed in Fig.1. For each range, a different color was selected to discriminate structures with different density in 3D image of the sample: yellow represents the less radiopaque component which corresponds to bone, orange represents areas that reached a first stage of calcification, and, at last, red represents areas that reached a higher stage of calcification (Fig.2).