Η επίδραση του βιοϋλικού υδροξυαπατίτη/χιτοζάνης στην κατευθυνόμενη οστική ανάπλαση προκλητών κρανιακών ελλειμμάτων επίμυων

Abstract

Τhis study aims at determining the effect of 75/25 w/w nHAp/CS scaffolds on Guided Bone Regeneration (GBR) in rat calvarial critical-size defects (CSD) and the ability of Cone Beam Computed Tomography (CBCT) to visualize CSD created at rat calvaria and filled with 75/25 w/w nHAp/CS scaffolds. Materials and Methods: Thirty six Sprague Dawley adult rats, approximately 3 months old, 18 males and 18 females, weighing more than 250g, were used. These were housed in the Laboratory of Experimental Surgery of the Medical School of the University of Athens, where stable conditions of heating, ventilation and relative humidity were maintained. Also, daily animal health monitoring, as well as daily care and nutrition were performed by a specialized veterinarian and by a specialized animal dietician respectively. All animal handling and surgical procedures were conducted in accordance with the guidelines of animal care and the use of laboratory experimental animals. This experimental study was approved by the Directorate of Agricultural and Veterinary Policy with protocol number 1181/2-03-2017 and registration code EL 25 BIO 05, Athens, Greece. Also, this study protocol was in line with EU Directive 2010/63/EU, based on the concept of replacement, reduction and refinement of animal studies (the 3R principle). The sample size was minimized in accordance with the aims of “Animal Research: Reporting of In Vivo Experimental guidelines (ARRIVE)”. Also, the size of the sample was estimated with power analysis (Power 1-β err prob=0.6949) using one-way ANOVA Fixed effects (IBM SPSS 25.0, IBM Corp., Armonk, N.Y., USA). The introduction of gender as a variable was intended to limit the use of only male rats, in agreement with the National Institute of Health (NIH, NOT-OD-15-102) notice. Thus, three study groups were created for each time period up to euthanasia (2, 4 and 8 weeks, respectively). Each group comprised of twelve rats (6 males and 6 females). The number of animals used was comparable to that of previous GBR studies. Nanoparticles of hydroxyapatite (nHAp) were synthesized according to our previous work, in the presence of hyperbranched polyethylene imine (Lupasol G100, BASF, Greece), to regulate the size and morphology of hydroxyapatite crystals. Composite porous scaffolds were developed by preparing a 3% w/w chitosan (Aldrich, high molecular weight, deacetylation degree ≥75%) solution in aqueous acetic acid (1.5% w/w), and adding nHAp to a final HAp:CS weight ratio of 75:25. The resulting thick slurry was thoroughly mixed and molded in glass tubes (5mm inner diameter) that were subsequently frozen at -25oC and lyophilized. After lyophilization the resulting HAp:CS cylindrical porous scaffolds (5mm diameter) were cut into disks of 1mm thickness, ethanol sterilized and extensively washed with sterile phosphate buffer saline inside a laminar flow cabinet. By determining the volume of liquid infused in their pores, porosity and total pore volume were found to be 85±1% and 5.0±0.5mL/g respectively.Preoperatively, a 10-day preparation time was spent on standard rat health tests. In particular, prior to use each rat was subjected to a thorough clinical examination and complete hematological and biochemical tests prior to use. General anesthesia was given by intramuscular injection with xylazine 5mg/kg (Rompun, Bayer Animal Health GmbH D-51368, Leverkusen, Germany) and ketamine hydrochloride 100mg/kg (IMALGENE 1000, MERIAL, 29 Avenue Tony Garnier, 69007 Lyon, France). After shaving and painting with povidone-iodine (Betadine Solution, Lavipharm, Athens, Greece) a 2cm-sized longitudinal midsagittal cutaneous incision was made on the scalp. The musculature and the periosteum were exposed under the skin to allow for the periosteal dissection procedure. Subsequently, two symmetrical round bone CSD were created in the dorsal part of the right and the left parietal bones, using a diameter of 5mm dental trephine burr (MT-00500, MIS, Israel) operated at 10000rpm under sterile saline irrigation (Sodium Chloride 0.9% Intravenous Infusion, BIOSER, Greece). The diameter of the trephine burr was chosen in order to allow proper fitting of a 5mm in diameter and 1mm thick nHAp/CS scaffold. The procedure mentioned above was done with caution in order to avoid any damage to the dura mater or the superior sagittal sinus, also avoiding the engagement of the midsagittal suture. The CSD on the left parietal bone remained empty as no biomaterial was inserted (group A), while the CSD on the right parietal bone was loaded with a scaffold 75/25 w/w nHAp/CS (group B). Thus, two groups were created, with group A being used as control and group B as experimental. The wound was sutured in layers. The periosteal flap was reflected over the defects and sutured to the contralateral side using 4-0 polyglycolic acid suture (PGA 4-0, medipac, Greece). The skin was then closed using 3-0 polyglactin 910 suture (Coated VICRYL, Ethicon, Johnson&Johnson, USA). The postoperative stage included an antimicrobial treatment by intramuscular injection with enrofloxacin 2.5mg/kg (Baytril 5%, Bayer Animal Health GmbH D-51368 Leverkusen, Germany). Post-operatively, analgesic and anti-inflammatory treatment with caprofen (Rimadyl, Pfizer, USA) was administered also. Two female rats died post-operatively. The 34 rats, 18 males and 16 females, were euthanized with diethyl ether (Sigma Aldrich, USA) inhalation at 2, 4 and 8 weeks after surgery. More in detail, 12 rats (6 males, 6 females) were euthanized at 2 weeks, 11 rats (6 males, 5 females) at 4 weeks and 11 rats (6 males, 5 females) at 8 weeks after surgery accordingly. The rat calvaria were properly cut off with two horizontal and two vertical osteotomies and excised using a surgical sawmill. The 34 specimens included both the parietal bones and also the parts of occipital and frontal bones. The dimensions of each specimen were approximately 15mm wide, 2mm thick and 10 mm long (15x2x10mm). Subsequently, the specimens were immediately fixed in 10% neutral buffered formalin solution (Formaldehyde solution, Sigma Aldrich, USA) for 1 day. Ιnitially, a pilot study was conducted in 6 animals (3 males, 3 females) to investigate biocompatibility of the biomaterial. Τhen a CBCT analysis was conducted in 28 animals. The 6 animals of the pilot study were excluded from the CBCT analysis. The 28 specimens (15x2x10mm) housed in plastic cylindrical tubes (3.5cm in diameter, 7cm in height) filled with 10% neutral buffered formalin solution were scanned using a New Tom VGi CBCT imaging unit (QR s.r.l., Rev 2.6, 2014, Verona, Italy). The manufacturer’s software trace region profile tool (NNT v6.2, Verona, Italy) was used in selected axial slices. The greyscale value (in VGiHU) and the traced/selected region of interest (ROI, in mm2) of those areas were automatically calculated. Following CBCT, all the 34 specimens (including experimental animals of the pilot study) were submitted to decalcification by using EDTA-based solution (MicroDec, Diapath, Italy) for 7 days. The histological samples were serially dehydrated in ethanol in a tissue processor (Citadel Shandon 1000, Thermo Scientific, Langenselbold, Germany) and paraffin embedded in another tissue processor (EMBED 503, Kaltek, Padova, Italy). Histological sections of 5μm in thickness were prepared from the mid-point of the critical sized defects in the coronal plane using a Microtome (RM 2145, Leica, Buffalo Grove, USA). The slides were deparaffinized with xylene and rehydrated with serial concentrations of ethanol. The slides were stained with hematoxylin-eosin (Eosin G Acqeuous Solution 1%, Diapath, Martinengo, Italy-Papanicolaou’s Solution 1a Harris hematoxylin solution, Merk, Darmstadt, Germany) for the use of optical microscope (Olympus Microscope, Shinjuku, Tokyo, Japan). Histomorphometric assessment was performed by a certified in oral pathology, who was blind to the intervention and to euthanasia period. Digital image analysis required a semi-automated system with Intel CORE i5 7th Gen (Intel Corporation, California, USA), SAMSUNG Color Display Unit LS24F356FHUXEN (Samsung Electronics Ltd, United Kingdom), Creative Pen Tablet WACOM (Wacom Europe GmbH, Germany), Digital Camera OLYMPUS U-CMAD3 T7, U-TV1X-2 T7 (Olympus Corporation, Tokyo, Japan), Optical Microscope OLYMPUS CX 23LEDRFS2 (Olympus Corporation, Tokyo, Japan) hardware features, and the following soft¬ware: Windows 10 (Microsoft Corporation, NY, USA), Adobe Photoshop CC 2015 (San Jose, USA), Image Pro-Plus v6.0.0.260 (Media Cybernetics, Rockville, MD, USA)Parameters assessed were new bone formation in CSDs expressed as μm2 and the total number of osteocytes in newly formed bone surface. This number may be considered as a surrogate maker of bone formation speed, as the more rapid the bone formation the more osteocytes are present. For those purposes, division of the corresponding histological sections was implemented by splitting the whole image in continuous areas. A digital drawing and crop tool separated the area of interest from the near environment and at the final stage digital analysis was performed. As bone regeneration process from the edge towards the center of the defect, bone formation was evaluated separately on the central and two lateral areas of the defect, defined as Regions of Interest (ROI), as follows: (a) lateral area inward of the middle sagittal seam (lateral 1, l1); (b) lateral area outward of the middle sagittal seam (lateral 2, l2), (c) central area (c, central), (d) area of the original defect (defi). These measurements were used in the following formula to determine the fraction of bone regeneration (FBR): FBR= (lateral 1 + lateral 2 + central) / (original defect) x 100%. Βiodegradability of the nHAp/CS scaffolds was also investigated. Τhe absolute number of osteocytes (Ost) was measured, as well as cell density of the osteocytes (CD, osteocytes/mm2). These parameters were calculated for each ROI of both the experimental (group B) and the control group (group A).All the data were analyzed using IBM SPSS 25.0 (Chicago, IL). The greyscale value (in VGiHU) and the ROI (in mm2) of 28 specimens were assessed as mean±Standard Deviation (SD). Multivariate ANOVA and Post Hoc Tests were used for statistical analysis. The data, i.e. new bone surface as μm2 (lateral 1, lateral 2, cental, original defect area, biomaterial’s area) and total number of osteocytes (Ost), fraction of bone regeneration (FBR), cell density (CD) of al the 34 specimens were estimated as mean±Standard Deviation (SD). One way Analysis of Variance (one way ANOVA), T-test and Post Hoc Tests (Bonferroni) were used for statistical analysis. The significance level was set to PResultsNo statistically significant difference between male and female subjects (P=0.188) was observed with respect to VGiHU. A new unified dependent variable was created that included gender, time in weeks, groups (A and B) and time’s relationship (in weeks). The aforementioned variable had a statistically significant effect (P Fifty eight CSDs were available for microscopic evaluation. Newly formed bone appeared as fibrous bone with numerous osteocytic lacunae. Consequently it could be easily distinguished by the pre-existing calvarial bone at the edges of the CSDs that was lamellar and sparsely cellular. The biomaterial of nHAp/CS showed good integration at 2, 4 and 8 weeks, as shown by lack of foreign body granuloma formation, pus formation, or necrosis in any of the CSD of the experimental or control group. At 2 weeks following surgery, a few newly formed bone was observed on the lateral region of both the CSDs (group A and group B). Mild inflammatory infiltration and few osteoclast-like, multinucleated giant cells were observed around the biomaterial. In the central portion no newly formed bone was observed. At 4 and 8 weeks after surgery, more newly formed bone was observed on the lateral region of group B. Newly bone formation in the central area of CSD with the biomaterial was observed in 13 rats. There was no inflammatory infiltration or multinucleated giant cells in relation to the biomaterial. No statistically significant difference between male and female subjects (P=0.06) was observed with respect to FBR. The mean value of FBR of experimental group (group B) was 33.8394% while the FBR of control group (group A) was 15.9155%. A statistically significant increase in FBR of group B was observed compared to FBR of group A (P=0.000). Conclusions: The present study shows that nHAp/CS scaffolds exhibits a positive effect on Guided Bone Regeneration (GBR) in rat calvarial critical-size defects (CSD) and they can be visualized using a particular high-resolution CBCT imaging unit. The higher the GVs (in VGiHU), the more radiopaque the scaffolds of nHAp/CS were. It is worth noting that only high-resolution CBCT systems may allow these measurements to be performed. Both CBCT measurements and histological results showed that the nHAp/CS scaffold presence contributes to new bone formation in rat calvarial CSD.The nHAp/CS 75/25 w/w scaffold should be considered as a suitable, biocompatible bone graft material for GBR studies. The biodegradability of the nHAp/CS 75/25 w/w scaffold is considered adequate. The surface of biomaterial decreases with respect to time, as demonstrated by histomorphometric analysis. Based on histological and histomorphometric analysis, the scaffold of nHAp/CS 75/25 w/w appears to promote newly bone formation in rat calvarial critical-size defects (CSD), as demonstrated by elevated the fraction of bone regeneration (FBR), the total number of osteocytes (Ost), as well as cell density of the osteocytes (CD) in the experimental group relative to the control group. In conclusion, scaffolds of nHAp/CS integrate bone tissue and provide an effective space for new bone formation. However, those results are not necessarily applicable to alveolar bone. Thus, further investigation of nHAp/CS scaffolds for GBR in oral and periodontal reconstructive procedures is required.

Η πειραματική μελέτη αυτή έχει σκοπό να προσδιοριστεί η επίδραση του βιοϋλικού νάνο-Υδροξυαπατίτη/Χιτοζάνης 75/25 w/w στην κατευθυνόμενη οστική ανάπλαση κρανιακών οστικών ελλειμμάτων επίμυων. Ο προσδιορισμός της επίδρασης του βιοϋλικού στην κατευθυνόμενη οστική ανάπλαση θα γίνει με ανάλυση Κωνικής Δέσμης Υπολογιστικής Τομογραφίας (Cone Beam Computed Tomography, CBCT) και με ιστομορφομετρική ανάλυση στις περιοχές των κρανιακών οστικών ελλειμμάτων σε τρεις διαφορετικούς χρόνους ελέγχου (2, 4 και 8 εβδομάδες). Υλικά και Μέθοδοι: Χρησιμοποιήθηκαν 36 ενήλικοι επίμυες Sprague Dawley (18 αρσενικοί και 18 θηλυκοί). Με τη χρήση χειρουργικής φρέζας δημιουργήθηκαν 2 αμφοτερόπλευρα, τυποποιημένα οστικά ελλείμματα, διαμέτρου 5mm, στο δεξιό και αριστερό βρεγματικό οστούν της κεφαλής κάθε επίμυ. Στο δεξιό έλλειμμα τοποθετήθηκε το ικρίωμα του νάνο-Υδροξυαπατίτη/Χιτοζάνης και αποτέλεσε την πειραματική ομάδα, ενώ το αριστερό έλλειμμα παρέμεινε άδειο, αποτελώντας την ομάδα ελέγχου. Δύο θηλυκοί επίμυες πέθαναν μετεγχειρητικά. Οι 34 επίμυες ευθανατώθηκαν στις 2, 4 και 8 εβδομάδες μετά τη χειρουργική διαδικασία. Διεξήχθη ακτινολογική ανάλυση των 28 ιστοτεμαχίων με τη χρήση CBCT (ανάλυση του επιπέδου διαβάθμισης του γκρι, grayscale value, VGiHU) και ιστολογική και ιστομορφομετρική ανάλυση των 34 ιστοτεμαχίων σε επιλεγμένες περιοχές ενδιαφέροντος: περιφερική περιοχή προς τα έσω της μέσης οβελιαίας ραφής (lateral 1, l1), περιφερική περιοχή προς τα έξω της μέσης οβελιαίας ραφής (lateral 2, l2), κεντρική περιοχή (central). Στις περιοχές ενδιαφέροντος (ROI) μετρήθηκαν το εμβαδόν τους (μm2) και ο απόλυτος αριθμός των οστεοκυττάρων (Osteocytes, Ost). Στις περιοχές των αρχικών τρυπανισμών της πειραματικής ομάδας (Defects-Deficits, Defi) μετρήθηκε η επιφάνεια κάλυψης και η βιοαποικοδομησιμότητα του βιοϋλικού (Material, Mat) σε μm2 . Με βάση τα ανωτέρω μελετήθηκαν οι εξής παράμετροι: Κλάσμα Οστικής Ανάπλασης (Fraction of Bone Regeneration, FBR) [%], η βιοαποικοδομησιμότητα του βιοϋλικού (Material, Mat) [μm2], η Κυτταρική πυκνότητα Οστεοκυττάρων (Cell Density, CD) [οστεοκύτταρα/ μm2] και [οστεοκύτταρα/ mm2] και ο Απόλυτος Αριθμός Οστεοκυττάρων (Οsteocytes, Ost). Αποτελέσματα: Η πειραματική ομάδα παρουσίαζε σε στατιστικά σημαντικό βαθμό υψηλότερες μέσες τιμές VGiHU (856.35VGiHU) σε σχέση με την ομάδα ελέγχου (541.19 VGiHU) σε όλες τις εβδομάδες. Συνεπώς, το τυποποιημένο οστικό έλλειμμα της πειραματικής ομάδας απεικονίζεται περισσότερο ακτινοσκιερό σε σχέση με την ομάδα ελέγχου (Ρ=0,000Η μέση τιμή του κλάσματος οστικής ανάπλασης της πειραματικής ομάδας ήταν 33,8394% ενώ του κλάσματος οστικής ανάπλασης της ομάδας ελέγχου ήταν 15,9155% (P=0.000). Συμπεράσματα: Το βιοϋλικό νάνο-Υδροξυαπατίτης/Χιτοζάνη (nHAp/CS) 75/25 w/w επιδρά θετικά στην κατευθυνόμενη οστική ανάπλαση τυποποιημένων προκλητών κρανιακών ελλειμμάτων επίμυων, όπως καταδεικνύεται από τις αυξημένες τιμές του κλάσματος οστικής ανάπλασης, της κυτταρικής πυκνότητας των οστεοκυττάρων και του απόλυτου αριθμού των οστεοκυττάρων στην πειραματική ομάδα σε σχέση με την ομάδα ελέγχου. Το ικρίωμα του nHAp/CS απεικονίζεται ως ακτινοσκιερό υλικό με τη χρήση CBCT. Η βιοαποικοδομησιμότητα του ικριώματος nHAp/CS 75/25 w/w κρίνεται επαρκής. Συμπερασματικά, τα ικριώματα του nHAp/CS ενσωματώνονται άριστα εντός το οστίτη ιστού και παρέχουν ένα αποτελεσματικό χώρο για τον σχηματισμό νεόπλαστου οστού σε τυποποιημένα κρανιακά οστικά ελλείμματα επίμυων. Ωστόσο, αυτά τα αποτελέσματα δεν είναι κατά ανάγκη εφαρμόσιμα στο οστούν της ανθρώπινης φατνιακής ακρολοφίας. Συνεπώς, απαιτείται περαιτέρω διερεύνηση των ικριωμάτων nHAp/CS σε μελέτες κατευθυνόμενης οστικής ανάπλασης σε χειρουργικές διαδικασίες αποκατάστασης οστικών ελλειμμάτων της ανθρώπινης φατνιακής ακρολοφίας.