Irradiation-induced changes in the mandibular bone were studied in both animals and humans. Histological analysis of the canine mandibular bone was performed with light microscopy (LM) and morphological analysis with micro-CT and SEM. Human mandibular cortical bone was investigated with Raman spectroscopy for the biochemical analysis and with LM and SEM for the histological assessment (Figure 4).
Figure 4. Overview of the thesis (Canine mandible - Study I, Human mandible – Studies II, III)
4.1 ETHICAL CONSIDERATIONS
The procedures to be carried out with the canine model were approved by the Ethics Board of the University of Kuopio (Nro 23 Zd: 04726 972 89 170;
11.7.1989), and the experiments to be carried out are as per the University’s Guidelines for Animal Experimentation.
Permission for the human material was approved by the Medical Ethical Committee of the VU University Medical Center in Amsterdam (registration number: 2011/220) and the Research Ethics Committee of the Northern
Savo Hospital District (754/2018; 21.4.2020). Human studies fulfilled the World Medical Association Declaration of Helsinki (Helsinki, Finland, 1964).
All patients signed a written informed consent to participate in the study.
4.2 CANINE MODELS AND BONE SAMPLES (STUDY I)
The study included mandibular bone samples from 5 beagle dogs of age 1-2 years (175). The timeline of the canine study is presented in Figure 5. In canines, all the premolars were extracted under intravenous anesthesia with sodium pentobarbital (Mebunat, Orion, Espoo, Finland), 20mg/kg.
After three months, radiation was given on the right-side mandible in two sessions, each lasting one week. The dose for each session (4 Gy/day) was divided into five successive days. To minimize mucosal damage, radiation sessions were given at an interval of approximately one month. The total dosage was 40 Gy (5 x 4 Gy = 20 Gy each in 2 sessions). The energy source used was a linear accelerator (Saturne III, CGR, France) with 6 MeV
electrons, and the size of the treatment area was limited to 4.5 x 3 cm.
Dental implants were inserted three months after the last radiation under IV anesthesia. A total of 20 (5 dogs x 4 implants) implants were inserted, two on the right irradiated side and two on the left control side, in each of the dogs. The size of the hollow titanium implant used was 3 mm x 6 mm.
Procaine penicillin with dihydrostreptomycin, 1 ml/10kg (Combiotic, Pfitzer, Switzerland) was administered postoperatively, daily for one week. Fixed prosthetic superstructures were placed three months after implantation.
After bridge fixation, the animals were allowed to chew rubber bones in order to stimulate the occlusal loading of the implants.
Figure 5. The timeline of the animal study.
The animals were sacrificed six months after prosthesis placement and the mandible bone blocks containing the implants were dissected using a hand saw in the buccolingual direction under physiological lubrication. Bone segments were processed by bulk staining en bloc in basic fuchsin (176).
The bone blocks were embedded in poly-methyl methacrylate resin (PMMA) without preliminary decalcification. All bone blocks were first subjected to micro-CT imaging, and later prepared for light microscopy (LM) and scanning electron microscopy (SEM).
4.3 HUMAN SUBJECTS AND BONE SAMPLES (STUDIES II, III) Both studies consisted of mandibular bone tissue samples from the control and the irradiated patients. Additionally, bone samples were included from the ORN patients in Study III. Table 14 presents a summary of the demographic data of the patients and the methods used in the studies.
Table 14. Demographic data of studies II, III
Study and Method
N Subjects Age range - years
Sex - Male/
Female
Total radiation dose (Gy)
Local radiation dose (Gy)
Radiation Biopsy Interval (months) Study II
LM Raman
33 Control-16 IR -17
33-74 51-79
6M/10F 13M/4F
-
54-70 Gy -
15-70 Gy - 11- 199 Study III
LM SEM
31 Control-11 IR -14 ORN - 6
33-74 51-74 51-84
7M/4F 11M/3F 5M/1F
-
54-70 Gy 56-70 Gy
-
18-62 Gy 56-66 Gy
- 11-199 13-90 LM = Light microscopy, SEM = Scanning electron microscopy,
ORN = Osteoradionecrosis, IR = Irradiated, N = Number of samples, Gy = Gray The study included patients without a history of cancer and RT as healthy controls and patients with a positive history of RT to treat HNC as an irradiated group. Patients who did not respond to conservative treatment (i.e. long-term systemic antibiotics and/or topical application of antiseptic agents) and visited for surgical management of the osteonecrotic lesion in
the mandible were included in the ORN group. All the irradiated patients who received an estimated dose of 50 Gy or higher on the anterior
mandible were treated with 20 sessions of HBO therapy preoperatively and 10 sessions postoperatively (Marx protocol) as a standard procedure.
Patients with impaired bone metabolism (e.g., hyperparathyroidism, osteomalacia), bisphosphonate medication or systemic
immunosuppressive medication up to three months before sample harvesting were excluded from the study.
Before the dental implant surgical procedure, all the control patients were administered a single dose of antibiotic prophylaxis (amoxicillin 3 g orally). A single oral and maxillofacial surgeon (CB) performed the surgery under local anesthesia in the Alrijne Hospital in Leiderdorp, The
Netherlands. For the irradiated group, antibiotic prophylaxis
(amoxicillin/clavulanic acid 500/125mg three times daily) was given 24 hours before dental implant surgery and continued for 10 days post-surgery. The operation was performed (ES) under general anesthesia in the Department of Oral and Maxillofacial Surgery, Amsterdam University Medical Centers (Amsterdam UMC), location Vrije Universiteit Medical Center (VUmc), in Amsterdam, The Netherlands.
The control and irradiated group underwent similar dental implant procedures, beginning with the crestal incision in the interforaminal region of the mandible with a mid-line buccal release incision. Elevation of a full-thickness mucoperiosteal flap was made to expose the alveolar ridge. The alveolar ridge was leveled by vertical alveolotomy if required. A 3.5mm diameter trephine drill (Straumann® Dental Implant System; Straumann Holding AG, Basel, Switzerland) was used to a depth of 10mm for the implant (4.1mm diameter) preparation at the canine regions of the right and left lower mandible, under continuous irrigation with sterile saline. The bone specimen from the trephine drill was obtained carefully with an ejector pin, fixed in ethanol, and prepared for further analysis. ORN
patients with extensive necrotic lesions visited the department for surgical debridement. A sample of the excised tissue was sent to the Pathology department for routine examination to exclude the recurrence of
malignancy within the lesion. The remaining tissue was used for Study III.
4.4 METHODS
4.4.1Micro-computed tomography (micro-CT) (Study I)
The mandible bone blocks embedded in PMMA with the implants were scanned using Skyscan, 1172 micro-CT (Bruker microCT, Kontich, Belgium) for a series of images (N=352). Images were acquired with a tube voltage of 100 kV, at 1800rotation, Al-Cu filter, and 30 µm pixel size. Image
reconstruction and analyses were calculated with a Skyscan CT- analyzer.
3D visualization was accomplished with Mathematica® 8 (Wolfram Research Inc., Champaign, IL, USA), and Skyscan Analyser® and CT-Volume®.
4.4.2Light microscopy (LM) (Studies I, II, III)
From the canine bone blocks (Study I), thin sections of 40 µm were prepared using cutting and grinding systems (Exakt, Norderstedt, Germany). For the human samples (Studies II, III), the obtained bone specimens were fixed in 70% ethanol. After fixation, the specimens were dehydrated with increasing grades of ethanol before embedding in Poly-methyl methacrylate (PMMA: Merck KGaA). The fresh surface of bone revealed from the PMMA block was used for microscopic analysis. For light microscopy (LM), thin sections (10 µm) were prepared with a microtome and stained with Masson-Goldner trichrome to analyze the bone tissue histology.
4.4.3Raman spectroscopy (Study II)
The measurements were conducted with a dispersive Raman microscope (Thermo DXR2xi, Thermo Fisher Scientific) on the surfaces of the PMMA bone blocks. The wavelength of 785 nm at 20 mW laser power (source) was used for excitation. An objective with 10x magnification and a numerical aperture of 0.25 was chosen. The system was equipped with a grating of 400 lines/mm, resulting in 5 cm-1 spectral resolution and 3300-50 cm-1 spectral range. Three regions of interest (ROI) of 30 µm x 30 µm were
measured by two investigators (PSR, EU) from each sample with an
exposure time of 0.01 sec, 50 µm confocal pinhole aperture, 2.0 µm image pixel size, and 30 scans. All the data acquired were selected from the cortical bone areas under the supervision of a pathologist. A background spectrum of the embedding medium PMMA was also obtained under similar conditions.
4.4.4Scanning electron microscopy (SEM) (Studies I, III)
For SEM, the PMMA-embedded tissue blocks were acid-etched with 9%
phosphoric acid for 60 seconds (canine bone blocks) and 30 seconds (human bone blocks) (177). After etching, the blocks were immersed in 5%
sodium hypochlorite for 3 min and rinsed in distilled water. The tissue blocks were dried overnight in a desiccator. Thereafter they were coated with a thin layer of gold of 30–40 nm. The gold-coated specimens obtained were examined under SEM and imaged using a Zeiss Sigma HD VP (Carl Zeiss GmbH, Oberkochen, Germany) scanning electron microscope operated at 15 kV, for the analysis of the bone structural and cellular details.
4.5 ANALYSIS
4.5.1Canine bone sample analysis (Study I)
Micro-CT analyses were carried out by two investigators (JM, TS) from each control and irradiated specimen in order to evaluate the following
parameters: Bone-to-implant contact (BIC), BS/BV, pore volume, porosity, degree of anisotropy, buccal and lingual bone thickness. BIC is an
important parameter for the evaluation of osseointegration, which is calculated as the length of bone contact with the implant divided by the total length of implant surface at the cortical bone X 100. The parameters such as the thickness of the new bone layer attached to implants, the number and diameter of Haversian canals, the number of veins within the lamellar bone and in contact with the periosteum were assessed by two investigators (TR, AMK) using SEM.
4.5.2Raman biochemical analysis (Study II)
The obtained Raman spectroscopy data were preprocessed and analyzed using MATLAB (MathWorks, v.R2017b) by two investigators (DS, SPS). Bone spectra were interpolated to the 1800-820 cm-1. The cosmic background was removed by filtering and the background fluorescence was removed by fitting a fourth-order polynomial function. Subsequently, the PMMA spectrum was mathematically subtracted from the bone spectra to minimize the influence of the background. The mean spectrum for the study group was calculated by averaging the variations at X-axis in the Raman spectra. Normalized spectra were used for multivariate Principal Component Analysis (PCA). Curve fitting was performed using Origin2021b software to accurately identify changes in Raman bands corresponding to inorganic and organic contents. First, the location of sub-peaks was
identified by intensity minima in a second derivative spectrum. The sum of the squared differences between observed and computed spectra was minimized to acquire the best fit (169). Peaks fitting was modeled using the Gaussian function and spectral band areas were measured. The
biochemical parameters of the control and irradiated samples were obtained by the curve-fitting analysis and were compared. Mineral
components were assessed by computing the intensity of phosphate, and carbonate bands. Mineral crystallinity and carbonate to phosphate ratio (CPR) were also evaluated. Matrix components were computed through the intensity of the phenylalanine and CH2 stretch bands, and then the mineral-to-matrix ratios (MMR) were assessed.
4.5.3Human bone SEM image analysis (Study III)
SEM images were acquired at 400x and 2000x magnification. Three
Regions of Interest (ROIs) at 400x with higher osteon density were selected from each bone sample. Further, in each ROI, three small areas (two osteonal areas and one interstitial bone area) were selected for
examination at 2000x. The images were measured by three investigators (PSR, BK, KV) for the parameters presented in Table 15. The diameters were measured using Carl Zeiss SmartTiff V3 imaging software and were
measured as the shortest perpendicular distance to the longest axis of the osteon and Haversian canal (HC). The osteons with well-defined or almost complete cement-lined boundaries were taken intoaccount. The osteonal wall thickness was calculated for each osteon as half of the difference between the osteonal diameter and HC diameter (178).
Table 15. The SEM parameters of human bone samples analyzed at magnifications 400x and 2000x
400x magnification 2000x magnification Number of osteons
Number of HC Diameter of osteon
Diameter of HC Osteonal wall thickness
Number of osteocytes/100 x 100 µm2
Near to HC (osteocytes/100 x 50 µm2) Far to HC osteocytes/100 x 50 µm2) Number of osteocytic dendrites SEM = Scanning electron microscopy, HC = Haversian canal
4.6 STATISTICAL METHODS (STUDIES I, II, III)
Study I: The statistical analysis was performed using IBM SPSS statistics software version 24 for Windows (SPSS, Chicago, IL). The microarchitecture of different parts of the bone was compared using a one-way analysis of variance (ANOVA). The difference between the study groups was compared using the paired t-test. A p-value of <0.05 was considered statistically significant.
Study II: The statistical analysis of the data used GraphPad Prism 6.1 software to express the mean and standard deviation. The comparison among groups was made using an unpaired Student’s t-test coupled with welch correction. The p-value for statistical significance was considered as p<0.05 statistically significant; p<0.01 very significant; and p<0.001 or less as extremely significant.
Study III: The Statistical Package for Social Sciences (SPSS v 27.0, IBM) was used for statistics. The normality of the data was checked using the Shapiro-Wilk test. The comparison among groups was obtained using a non-parametric Kruskal-Wallis H test and p-values of <0.05 were
considered statistically significant.
