2.1

Surgical procedures

All experimental procedures have complied with the Italian D.L. no 116 of 1992 and associated guidelines in the European Communities Council Directive of the 24th of November 1986 (86/609/ECC). Titanium implants (Winsix Implant System, Biosafin, Ancona, Italy) modified with 3D scaffolds were used.

The proposed biomaterial was a hydrophilic hybrid composite made up of nanoinclusions of hydroxyapatite dipped in a ceramo-polymeric matrix composed by poly-hydroxylethylmethacrylate and fumed nanosilica.

The scaffolds were designed using a reverse engineering approach and were retained by modified implant prototypes. Standard titanium implants with a diameter of 5.2 mm, a length of 13 mm and a hexagonal internal connection were used. An implant was imported in a solid modeler and meshed to discretize its geometry. Then, a finite element modeling was used to modify the fixture and create the experimental prototype, whose shape was suitable to incorporate the scaffold ( Fig. 1 A). The coronal part of the screw thread was removed so as to incorporate the scaffold, that had a length of 5 mm and the shape of a truncated cone with the major base apically (major base 0.6 mm, minor base 0.4 mm). The titanium implant prototypes had a micro-rough surface that was acid etched ( Fig. 1 A).

A steel cylinder was used as tray for the fabrication of the modified implants. A double impression technique was performed using putty and light silicon impression materials. First, the shape of a standard unmodified implant was impressed in the silicon to obtain the external profile shape mold. Subsequently, the modified titanium implant prototypes were inserted in the silicon molds by means of an implant holder and the unpolymerized novel material liquid composite resin was injected in the empty space between the external shape mold and the modified implant. The material was let to self-polymerize at room temperature for 15 min. Then, the implant threads were thoroughly polished by means of silicon rubbers. Although, the new biomaterial was designed as self-setting at room temperature, an additional thermal treatement at 80 °C was added to the fabrication procedures in order to favor complete polymerization and stabilization of the scaffolds in their highly rigid glassy state (with an elastic modulus comparable to that of the bone) before the surgical insertion ( Fig. 1 B). The scaffolded implants were then sterilized by means of gamma-rays. The scaffolded implants were introduced unicortically into the left and right proximal tibial metaphysis of 4 male Yucatan minipigs (>18-month old, average body weight 65 kg) with an inter-implant distance of 5–9 mm. The implants were inserted flush with the cortical bone, so as the scaffolds could interface with both the cortical and the spongy bone ( Fig. 2 ). This was made to evaluate if the different elasticity of the bony tissues could affect the swelling of the scaffolds. The animals were sedated by an intramuscular injection (10 mg/kg) of ketamine (Inoketam 1000 ^® , Virbac S.r.l., Milan, Italy) and 0.5 mg/kg midazolam PHG (Hospira, Lake Forest, IL, USA). Anesthesia was induced by mask and, after endotracheal intubation, the minipigs were maintained in spontaneously breathing by inhalation of 3% isoflurane and oxigen. The surgical fields were shaved and disinfected with benzoxonium chloride (Citrosil ^® , Manetty & Roberts, Milan, Italy). The tibias were exposed by skin incisions and fascial-periosteal flaps. The implant sites were prepared using drills with increasing diameter and a torque-controlled handpiece implant unit at a rotation speed of 25–30 RPM, with continuous external sterile saline irrigation to minimize bone damage caused by overheating. Thereafter, 4 implants were placed in each tibia, according to local anatomy and bone quality. A total of 28 scaffolded fixtures was inserted in 7 tibias; 1 tibia was not used because of unfavorable local anatomy. The insertion torque values were recorded using an electronic torque-control implant handpiece. The implant position was checked by means of radiographs and intensification of brilliance, in order to verify that the scaffolds were in the transition area between the cortical and the spongy bone. The skin and the fascia-periosteum were closed in separate layers with synthetic monofilament non absorbable polypropylene suture (Prolene ^® 2-0, Ethicon, Somerville, NJ, USA) and a single absorbable suture (Vicryl ^® 3-0, Ethicon, Somerville, NJ, USA), respectively. Perioperatively, the animals received enrofloxacin 2.5 mg/kg/12 h (Baytril ^® , Bayer, Barmen, Germany) as antibiotic for 6 days and ketorolac 1 mg/kg/24 h as anti-inflammatory medication for 3 days. The animals were inspected after the first few postoperative days for signs of wound dehiscence or infection and, thereafter, weekly to assess general health. The animals did not show signs of physical impairments and were in stable condition throughout the study. After 8 weeks, animals were sacrificed and specimens containing the implants were fixed in 10% pH 7.0-buffered formalin.

Representative sagittal μCT slice showing scaffold-implants systems inserted into the tibia of minipigs after 8 weeks of healing time and the cylindrical volumetric region of interest (VOI), where measurements in peri-implant bone were made (A), at the level of the test zone and (B), at the level of reference zone.

2.2

Biomechanical analysis

Insertion torque values (N cm) were recorded during implant placement by means of a torque-controlled implant handpiece. Moreover, once implant placement was completed, a RFA was performed using a dedicated electronic device (Osstell ISQ, Osstell AB, Göteborg, Sweden). The values of resonance frequency were recorded as an implant stability quotient (ISQ) in order to allow a comparison of primary stability between different fixtures .

2.3

μCT and 3D reconstruction

The bone surrounding the implant scaffolds was examined using μCT (eXplore Locus, GE Healthcare, London, Canada). The detector of such μCT system was made up of 2 components, a scintillator and a CCD camera, which were connected with optical fibers. The scintillator is a thin screen of cesium iodide, that luminesces when exposed to X-rays. The camera behind it records the image that is projected onto the scintillator. The projection is saved in digital format. A total of 466 μCT slices, with a pixel size of 45 μm, were imaged at an X-ray energy level of 80 kVp and a current of 450 μA. Exposure time was 100 ms with a total scanning time of 11 min. Images were reconstructed using a modified Feldkamp algorithm and scaled into Hounsfield units (HU). A calibration standard, containing a compartment with 1073 mg/cm ³ SB3 (an epoxy based bone-mimicking material), was used for bone mineral density (BMD) calibration. All data were evaluated in MicroView, 2.1.2 (GE Healthcare, London, Canada). Datasets were resampled to reorient implants perpendicular to the cross-section plane. Before segmentation, threshold levels for bone, implant and scaffold were determined, based on visual inspection of the complete slices and on the gray-scale histogram. The upper and lower threshold levels for bone, implants and scaffold were determined in all 7 samples and did not overlap, allowing to make a clear distinction. An automatic segmentation technique, that utilizes the Otsu method, was used for calculate the value to be used as the threshold to extract the bone from the surrounding structures. The means were calculated and used for each sample. The inter- and intra-examiner variabilities within 1 standard deviation were calculated. Then, a cylindrical, volumetric region of interest (VOI) was placed in correspondence of the scaffold element of the implant circumferentially expanded 2.5 mm around the scaffold, including both cortical and cancellous bone ( Fig. 2 C), which was defined as the test zone ( Fig. 3 A ). Subsequently, bone, implant and scaffold in the ROI were differentiated based on their threshold levels. Outcome variables were: BMD (mg/cm ³ ) and bone mineral concentration (BMC, mg); bone volume fraction (BVF), being the % of the ROI which includes bone; bone volume (BV), being the mm ³ of bone that is present in the region of interest; the ratio of the volume of bone present (BV) to the total volume (TV) of interest (%); the ratio of the surface (BS) to the volume (BV) of bone (mm ⁻¹ ); the average trabecular thickness (Tb.Th, mm); the mean trabecular number (Tb.N, mm ⁻¹ ); the mean diameter to the marrow spaces (Tb.Sp, mm). These values were also measured in VOI at an equivalent distance between 2 median implants, as reference zone ( Fig. 3 B). 3-D volume rendering images were performed on 27 μm μCT dataset using Osirix Imaging Software 5.8.5 (Pixmeo, Bermex, Switzerland) ( Fig. 2 A, B, E, F).

(A) High-resolution μCT 3-D volume rendering of minipig tibias with implants. (B) Representative axial μCT image of minipig tibias with implant–bone interface visualization. (C) Representative photograph of minipig tibias with implant position visualization. (D and E) High-resolution μCT 3-D volume rendering of scaffold. (F) High-resolution μCT 3-D volume rendering peri-implant bone.

2.4

Histology and histomorphometry

The formalin-fixed specimens were transferred to 70% ethanol solution, dehydrated in ascending concentration of ethanol up to 100%, and then infiltrated and embedded in a hydrophilic acrylic resin (LR White, London Resin Company, Berkshire, England). After polymerization, the specimens were sectioned along the longitudinal axis with a high-precision diamond disk at about 100 μm, provided with a custom-built sawing and a grinding apparatus (TT System, TMA2, Grottammare, Italy). A total of 3 sections was obtained for each implant (1 longitudinal central section and 2 sections at a cutting distance of 600 μm). The sections were ground down to about 60 ± 10 μm and stained with acid fuchsine and toluidine blue. The investigation was conducted in a transmitted brightfield and circularly polarized Light Microscope Axiolab (Zeiss Oberchen, Germany) connected to a high-resolution digital camera (FinePix S2 Pro, Fuji Photo Film Co. Ltd, Minato-Ku, Japan). A histomorphometric software package with image capturing capabilities (Image-Pro Plus 6.0, Media Cybernetics Inc, Bethesda, MD, USA) was used. To ensure accuracy, the software was calibrated for each experimental image using a feature named “Calibration Wizard”, which creates a linear remapping of the pixel numbers. The unit of measurement was the pixel. The analyzed parameter was the bone-to-implant contact (BIC, %), defined as the ratio between the length of the scaffold material section in contact with bone and the perimeter of the scaffold area, at 25× magnification. At each measurement, the software was calibrated using the length of the implant as reference. Measurements were performed by a single experienced and calibrated operator. Intra-examiner variability was controlled by carrying out 2 measurements; if the difference between the 2 values was greater than 5%, the measuring was repeated.

2.5

Statistical analyses

The study variables were divided into 4 groups as follows: biomechanical (IT and RFA), histomorphometric (BIC), μCT of implant sites (BMD, BMC, BVF, BV, BV/TV, BS/BV, TbTh, TbN, TbSp) and μCT of control sites (cBMD, cBMC, cBVF, cBV, cBV/TV, cBS/BV, cTb/Th, cTbN, cTbSp). Mean, standard deviation, median, minimum and maximum values were calculated for all the variables. Normality was tested using Shapiro–Wilk test and variance homogeneity using Bartlett or Levene test. When data were normally distributed and variance homogeneity was met, the variables were compared using one-way analysis of variance (ANOVA). In case of violation of normality or variance homogeneity, Kruskall–Wallis or Mann–Whitney test were performed. The mean values of 21 variables for 7 tibial samples were used for the statistical analysis. The Pearson correlation was performed for all possible pairs of variables, for a total of 210 combinations. The linear regression coefficients were calculated for the 12 pairs of variables found to be correlated. Moreover, IT, RFA, BIC, BMD, BMC, TbTh, TbN and TbSp were analyzed for comparison among implant sites. Sample size required to reach a 5% significance with a 80% power was estimated for Mann–Whitney, Shapiro–Wilk, Anova and Pearson correlation tests. Statistical analysis was performed using R statistical software (version 3.1.2, R Foundation for Statistical Computing, Vienna, Austria) and the level of significance was set at p < 0.05 for all tests.

2.1

Surgical procedures

All experimental procedures have complied with the Italian D.L. no 116 of 1992 and associated guidelines in the European Communities Council Directive of the 24th of November 1986 (86/609/ECC). Titanium implants (Winsix Implant System, Biosafin, Ancona, Italy) modified with 3D scaffolds were used.

The proposed biomaterial was a hydrophilic hybrid composite made up of nanoinclusions of hydroxyapatite dipped in a ceramo-polymeric matrix composed by poly-hydroxylethylmethacrylate and fumed nanosilica.

The scaffolds were designed using a reverse engineering approach and were retained by modified implant prototypes. Standard titanium implants with a diameter of 5.2 mm, a length of 13 mm and a hexagonal internal connection were used. An implant was imported in a solid modeler and meshed to discretize its geometry. Then, a finite element modeling was used to modify the fixture and create the experimental prototype, whose shape was suitable to incorporate the scaffold ( Fig. 1 A). The coronal part of the screw thread was removed so as to incorporate the scaffold, that had a length of 5 mm and the shape of a truncated cone with the major base apically (major base 0.6 mm, minor base 0.4 mm). The titanium implant prototypes had a micro-rough surface that was acid etched ( Fig. 1 A).

A steel cylinder was used as tray for the fabrication of the modified implants. A double impression technique was performed using putty and light silicon impression materials. First, the shape of a standard unmodified implant was impressed in the silicon to obtain the external profile shape mold. Subsequently, the modified titanium implant prototypes were inserted in the silicon molds by means of an implant holder and the unpolymerized novel material liquid composite resin was injected in the empty space between the external shape mold and the modified implant. The material was let to self-polymerize at room temperature for 15 min. Then, the implant threads were thoroughly polished by means of silicon rubbers. Although, the new biomaterial was designed as self-setting at room temperature, an additional thermal treatement at 80 °C was added to the fabrication procedures in order to favor complete polymerization and stabilization of the scaffolds in their highly rigid glassy state (with an elastic modulus comparable to that of the bone) before the surgical insertion ( Fig. 1 B). The scaffolded implants were then sterilized by means of gamma-rays. The scaffolded implants were introduced unicortically into the left and right proximal tibial metaphysis of 4 male Yucatan minipigs (>18-month old, average body weight 65 kg) with an inter-implant distance of 5–9 mm. The implants were inserted flush with the cortical bone, so as the scaffolds could interface with both the cortical and the spongy bone ( Fig. 2 ). This was made to evaluate if the different elasticity of the bony tissues could affect the swelling of the scaffolds. The animals were sedated by an intramuscular injection (10 mg/kg) of ketamine (Inoketam 1000 ^® , Virbac S.r.l., Milan, Italy) and 0.5 mg/kg midazolam PHG (Hospira, Lake Forest, IL, USA). Anesthesia was induced by mask and, after endotracheal intubation, the minipigs were maintained in spontaneously breathing by inhalation of 3% isoflurane and oxigen. The surgical fields were shaved and disinfected with benzoxonium chloride (Citrosil ^® , Manetty & Roberts, Milan, Italy). The tibias were exposed by skin incisions and fascial-periosteal flaps. The implant sites were prepared using drills with increasing diameter and a torque-controlled handpiece implant unit at a rotation speed of 25–30 RPM, with continuous external sterile saline irrigation to minimize bone damage caused by overheating. Thereafter, 4 implants were placed in each tibia, according to local anatomy and bone quality. A total of 28 scaffolded fixtures was inserted in 7 tibias; 1 tibia was not used because of unfavorable local anatomy. The insertion torque values were recorded using an electronic torque-control implant handpiece. The implant position was checked by means of radiographs and intensification of brilliance, in order to verify that the scaffolds were in the transition area between the cortical and the spongy bone. The skin and the fascia-periosteum were closed in separate layers with synthetic monofilament non absorbable polypropylene suture (Prolene ^® 2-0, Ethicon, Somerville, NJ, USA) and a single absorbable suture (Vicryl ^® 3-0, Ethicon, Somerville, NJ, USA), respectively. Perioperatively, the animals received enrofloxacin 2.5 mg/kg/12 h (Baytril ^® , Bayer, Barmen, Germany) as antibiotic for 6 days and ketorolac 1 mg/kg/24 h as anti-inflammatory medication for 3 days. The animals were inspected after the first few postoperative days for signs of wound dehiscence or infection and, thereafter, weekly to assess general health. The animals did not show signs of physical impairments and were in stable condition throughout the study. After 8 weeks, animals were sacrificed and specimens containing the implants were fixed in 10% pH 7.0-buffered formalin.

Representative sagittal μCT slice showing scaffold-implants systems inserted into the tibia of minipigs after 8 weeks of healing time and the cylindrical volumetric region of interest (VOI), where measurements in peri-implant bone were made (A), at the level of the test zone and (B), at the level of reference zone.

2.2

Biomechanical analysis

Insertion torque values (N cm) were recorded during implant placement by means of a torque-controlled implant handpiece. Moreover, once implant placement was completed, a RFA was performed using a dedicated electronic device (Osstell ISQ, Osstell AB, Göteborg, Sweden). The values of resonance frequency were recorded as an implant stability quotient (ISQ) in order to allow a comparison of primary stability between different fixtures .

2.3

μCT and 3D reconstruction

The bone surrounding the implant scaffolds was examined using μCT (eXplore Locus, GE Healthcare, London, Canada). The detector of such μCT system was made up of 2 components, a scintillator and a CCD camera, which were connected with optical fibers. The scintillator is a thin screen of cesium iodide, that luminesces when exposed to X-rays. The camera behind it records the image that is projected onto the scintillator. The projection is saved in digital format. A total of 466 μCT slices, with a pixel size of 45 μm, were imaged at an X-ray energy level of 80 kVp and a current of 450 μA. Exposure time was 100 ms with a total scanning time of 11 min. Images were reconstructed using a modified Feldkamp algorithm and scaled into Hounsfield units (HU). A calibration standard, containing a compartment with 1073 mg/cm ³ SB3 (an epoxy based bone-mimicking material), was used for bone mineral density (BMD) calibration. All data were evaluated in MicroView, 2.1.2 (GE Healthcare, London, Canada). Datasets were resampled to reorient implants perpendicular to the cross-section plane. Before segmentation, threshold levels for bone, implant and scaffold were determined, based on visual inspection of the complete slices and on the gray-scale histogram. The upper and lower threshold levels for bone, implants and scaffold were determined in all 7 samples and did not overlap, allowing to make a clear distinction. An automatic segmentation technique, that utilizes the Otsu method, was used for calculate the value to be used as the threshold to extract the bone from the surrounding structures. The means were calculated and used for each sample. The inter- and intra-examiner variabilities within 1 standard deviation were calculated. Then, a cylindrical, volumetric region of interest (VOI) was placed in correspondence of the scaffold element of the implant circumferentially expanded 2.5 mm around the scaffold, including both cortical and cancellous bone ( Fig. 2 C), which was defined as the test zone ( Fig. 3 A ). Subsequently, bone, implant and scaffold in the ROI were differentiated based on their threshold levels. Outcome variables were: BMD (mg/cm ³ ) and bone mineral concentration (BMC, mg); bone volume fraction (BVF), being the % of the ROI which includes bone; bone volume (BV), being the mm ³ of bone that is present in the region of interest; the ratio of the volume of bone present (BV) to the total volume (TV) of interest (%); the ratio of the surface (BS) to the volume (BV) of bone (mm ⁻¹ ); the average trabecular thickness (Tb.Th, mm); the mean trabecular number (Tb.N, mm ⁻¹ ); the mean diameter to the marrow spaces (Tb.Sp, mm). These values were also measured in VOI at an equivalent distance between 2 median implants, as reference zone ( Fig. 3 B). 3-D volume rendering images were performed on 27 μm μCT dataset using Osirix Imaging Software 5.8.5 (Pixmeo, Bermex, Switzerland) ( Fig. 2 A, B, E, F).

(A) High-resolution μCT 3-D volume rendering of minipig tibias with implants. (B) Representative axial μCT image of minipig tibias with implant–bone interface visualization. (C) Representative photograph of minipig tibias with implant position visualization. (D and E) High-resolution μCT 3-D volume rendering of scaffold. (F) High-resolution μCT 3-D volume rendering peri-implant bone.

2.4

Histology and histomorphometry

The formalin-fixed specimens were transferred to 70% ethanol solution, dehydrated in ascending concentration of ethanol up to 100%, and then infiltrated and embedded in a hydrophilic acrylic resin (LR White, London Resin Company, Berkshire, England). After polymerization, the specimens were sectioned along the longitudinal axis with a high-precision diamond disk at about 100 μm, provided with a custom-built sawing and a grinding apparatus (TT System, TMA2, Grottammare, Italy). A total of 3 sections was obtained for each implant (1 longitudinal central section and 2 sections at a cutting distance of 600 μm). The sections were ground down to about 60 ± 10 μm and stained with acid fuchsine and toluidine blue. The investigation was conducted in a transmitted brightfield and circularly polarized Light Microscope Axiolab (Zeiss Oberchen, Germany) connected to a high-resolution digital camera (FinePix S2 Pro, Fuji Photo Film Co. Ltd, Minato-Ku, Japan). A histomorphometric software package with image capturing capabilities (Image-Pro Plus 6.0, Media Cybernetics Inc, Bethesda, MD, USA) was used. To ensure accuracy, the software was calibrated for each experimental image using a feature named “Calibration Wizard”, which creates a linear remapping of the pixel numbers. The unit of measurement was the pixel. The analyzed parameter was the bone-to-implant contact (BIC, %), defined as the ratio between the length of the scaffold material section in contact with bone and the perimeter of the scaffold area, at 25× magnification. At each measurement, the software was calibrated using the length of the implant as reference. Measurements were performed by a single experienced and calibrated operator. Intra-examiner variability was controlled by carrying out 2 measurements; if the difference between the 2 values was greater than 5%, the measuring was repeated.

2.5

Statistical analyses

The study variables were divided into 4 groups as follows: biomechanical (IT and RFA), histomorphometric (BIC), μCT of implant sites (BMD, BMC, BVF, BV, BV/TV, BS/BV, TbTh, TbN, TbSp) and μCT of control sites (cBMD, cBMC, cBVF, cBV, cBV/TV, cBS/BV, cTb/Th, cTbN, cTbSp). Mean, standard deviation, median, minimum and maximum values were calculated for all the variables. Normality was tested using Shapiro–Wilk test and variance homogeneity using Bartlett or Levene test. When data were normally distributed and variance homogeneity was met, the variables were compared using one-way analysis of variance (ANOVA). In case of violation of normality or variance homogeneity, Kruskall–Wallis or Mann–Whitney test were performed. The mean values of 21 variables for 7 tibial samples were used for the statistical analysis. The Pearson correlation was performed for all possible pairs of variables, for a total of 210 combinations. The linear regression coefficients were calculated for the 12 pairs of variables found to be correlated. Moreover, IT, RFA, BIC, BMD, BMC, TbTh, TbN and TbSp were analyzed for comparison among implant sites. Sample size required to reach a 5% significance with a 80% power was estimated for Mann–Whitney, Shapiro–Wilk, Anova and Pearson correlation tests. Statistical analysis was performed using R statistical software (version 3.1.2, R Foundation for Statistical Computing, Vienna, Austria) and the level of significance was set at p < 0.05 for all tests.