Macrophages, the main marker in biocompatibility evaluation of new hydrogels after subcutaneous implantation in rats.

Biocompatibility of materials is one of the most important conditions for their successful application in tissue regeneration and repair. Cell-surface interactions stimulate adhesion and activation of macrophages whose acquaintance can assist in designing novel biomaterials that promote favorable macrophage-biomaterial surface interactions for clinical application. This study is designed to determine the distribution and number of macrophages as a means of biocompatibility evaluation of two newly synthesized materials [silver/poly(vinyl alcohol) (Ag/PVA) and silver/poly(vinyl alcohol)/graphene (Ag/PVA/Gr) nanocomposite hydrogels] in vivo, with approval of the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade. Macrophages and giant cells were analyzed in tissue sections stained by routine H&E and immunohistochemical methods (CD68+). Statistical relevance was determined in the statistical software package SPSS 20 (IBM corp). The results of the study in terms of the number of giant cells localized around the implant showed that their number was highest on the seventh postoperative day (p.o.d.) in the group implanted with Ag/PVA hydrogels, and on the 30th p.o.d. in the group implanted with Ag/PVA/Gr. Interestingly, the number of macrophages measured in the capsular and pericapsular space was highest in the group implanted with the commercial Suprasorb© material. The increased macrophage number, registered around the Ag/PVA/Gr implant on 60th p.o.d. indicates that the addition of graphene can, in a specific way, modulate different biological responses of tissues in the process of wound healing, regeneration, and integration.

In Vivo/Ex Vivo EPR Investigation of the Brain Redox Status and Blood-Brain Barrier Integrity in the 5xFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive decline and total brain atrophy. Despite the substantial scientific effort, the pathological mechanisms underlying neurodegeneration in AD are currently unknown. In most studies, amyloid ß peptide has been considered the key pathological change in AD. However, numerous Aß-targeting treatments have failed in clinical trials. This implies the need to shift the research focus from Aß to other pathological features of the disease. OBJECTIVE: The aim of this study was to examine the interplay between mitochondrial dysfunction, oxidative stress and blood-brain barrier (BBB) disruption in AD pathology, using a novel approach that involves the application of electron paramagnetic resonance (EPR) spectroscopy. METHODS: In vivo and ex vivo EPR spectroscopy using two spin probes (aminoxyl radicals) exhibiting different cell-membrane and BBB permeability were employed to assess BBB integrity and brain tissue redox status in the 5xFAD mouse model of AD. In vivo spin probe reduction decay was analyzed using a two-compartment pharmacokinetic model. Furthermore, 15 K EPR spectroscopy was employed to investigate the brain metal content. RESULTS: This study has revealed an altered brain redox state, BBB breakdown, as well as ROS-mediated damage to mitochondrial iron-sulfur clusters, and up-regulation of MnSOD in the 5xFAD model. CONCLUSION: The EPR spin probes were shown to be excellent in vivo reporters of the 5xFAD neuronal tissue redox state, as well as the BBB integrity, indicating the importance of in vivo EPR spectroscopy application in preclinical studies of neurodegenerative diseases.

Toxicological Profile of Nanostructured Bone Substitute Based on Hydroxyapatite and Poly(lactide-co-glycolide) after Subchronic Oral Exposure of Rats.

Novel three-dimensional (3D) nanohydroxyapatite-PLGA scaffolds with high porosity was developed to better mimic mineral component and microstructure of natural bone. To perform a final assessment of this nanomaterial as a potential bone substitute, its toxicological profile was particularly investigated. Therefore, we performed a comet assay on human monocytes for in vitro genotoxicity investigation, and the systemic subchronic toxicity investigation on rats being per oral feed with exactly administrated extract quantities of the nano calcium hydroxyapatite covered with tiny layers of PLGA (ALBO-OS) for 120 days. Histological and stereological parameters of the liver, kidney, and spleen tissue were analyzed. Comet assay revealed low genotoxic potential, while histological analysis and stereological investigation revealed no significant changes in exposed animals when compared to controls, although the volume density of blood sinusoids and connective tissue, as well as numerical density and number of mitosis were slightly increased. Additionally, despite the significantly increased average number of the Ki67 and slightly increased number of CD68 positive cells in the presence of ALBO-OS, immunoreactive cells proliferation was almost neglected. Blood analyses showed that all of the blood parameters in rats fed with extract nanomaterial are comparable with corresponding parameters of no feed rats, taken as blind probe. This study contributes to the toxicological profiling of ALBO-OS scaffold for potential future application in bone tissue engineering.

Developing a novel resorptive hydroxyapatite-based bone substitute for over-critical size defect reconstruction: physicochemical and biological characterization and proof of concept in segmental rabbit's ulna reconstruction.

The aim of this study was to develop novel hydroxyapatite (HAP)-based bioactive bone replacement materials for segmental osteotomy reconstruction. Customized three-dimensional (3D) bone construct was manufactured from nanohydroxyapatite (nHAP) with poly(lactide-co-glycolide) (PLGA) coating using 3D models derived from the computed tomography (CT) scanning of the rabbit's ulna and gradient 3D printing of the bone substitute mimicking the anatomical shape of the natural bone defect. Engineered construct revealed adequate micro-architectural design for successful bone regeneration having a total porosity of 64% and an average pore size of 256 µm. Radiography and micro-CT analysis depicted new bone apposition through the whole length of the reconstructed ulna with a small area of non-resorbed construct in the central area of defect. Histological analysis revealed new bone formation with both endochondral and endesmal type of ossification. Immunohistochemistry analysis depicted the presence of bone formation indicators - bone morphogenetic protein (BMP), osteocalcin (OCN) and osteopontin (OPN) within newly formed bone. Manufactured personalized construct acts as a "smart" responsive biomaterial capable of modulating the functionality and potential for the personalized bone reconstruction on a clinically relevant length scale.

Mandibular lateral deviation induces alteration in vascular endothelial growth factor expression and oxidative stress/nitric oxide generation in rat condyle, synovial membrane and masseter muscle.

OBJECTIVE: We aimed to investigate alteration in cellular signaling mediated by vascular endothelial growth factor (VEGF) and parameters of oxidative stress/nitric oxide generation, superoxide dismutase (SOD) and neuronal nitric oxide synthase (nNOS), underlying altered functional mechanical loading of TMJ (temporomandibular joint) during lateral mandibular deviation. DESIGN: Thirty-eight 5-week-old male Wistar rats were divided into experimental group, which received acrylic resin appliance that shifted mandible to the left during closure, and control group. Computed tomography and histomorphometry were used for condyle analyses, while samples of condyle, synovial membrane and m. masseter were analyzed with enzyme-linked immunosorbent assay and spectrophotometry to determine VEGF and nNOS protein concentrations, and SOD activity. RESULTS: Experimental group of rats developed smaller and asymmetrical mandibles. Less of new bone and cartilage formation and larger bone marrow cavities area were found in the experimental group. Higher VEGF expression in condyle and m. masseter as well as higher nNOS expression in m. masseter and synovial membrane were found in the experimental compared to the control group. Alteration of SOD activity was found in m. masseter and synovial membrane in the experimental group. CONCLUSIONS: Lateral mandibular deviation induces mandibular and condylar morphological changes as well as significant cellular signaling alterations in condyle, synovial membrane and masticatory muscle. Cellular VEGF protein overexpression and oxidative stress/nitric oxide disbalance could be the mechanisms underlying unbalanced functional TMJ loading due to mandibular deviation.

Nanostructured endodontic materials mixed with different radiocontrast agents-biocompatibility study.

The aim of this study was to investigate the biocompatibility of nanostructured materials based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA+ using in vitro and in vivo model. Morphology of materials' samples was analyzed using SEM while the phase compositions were identified by XRD. pH values of materials' suspensions were conducted by pH-meter. The cytotoxicity of materials' solutions was tested by MTT test (100, 50, 25 and 12.5 mg/ml). LDH and 3H-thymidine assay were utilized for biocompatibility investigations of materials' eluates (24 h, 7 day and 21 day). Eighteen Guinea pigs were used for intramuscular implantation, as teflon tubes with freshly prepared materials were placed into intramuscular pockets. All samples were composed of round and needle-like particles equally distributed with Ca/Si ratio ~2.7 at%, with the presence of hydrated calcium silicate phases. The pH values of ALBO-MPCA1 and ALBO-MPCA2 were high alkaline, while in case of MTA+ they were lower and continuously declined (p < 0.05). Investigated materials didn't exhibit dose-dependent effect on metabolic activity of L929 cells (p > 0.05). Significant differences in the percentage of cytotoxicity between diluted and undiluted extracts between all tested materials after 24 h and 7 day were noticed (p < 0.05). Increase in L929 cells proliferation was noticed in case of undiluted eluates of ALBO-MPCA1 and ALBO-MPCA2 after 7 day (p < 0.05). There were no statistically significant differences in the intensity of inflammatory response between investigated materials and control group after 60 day (p > 0.05). Evaluation of biocompatibility of both ALBO-MPCA1 and ALBO-MPCA2 indicate their potential clinical use.

Biocompatibility Investigation of New Endodontic Materials Based on Nanosynthesized Calcium Silicates Combined with Different Radiopacifiers.

INTRODUCTION: The aim of this article was to analyze biocompatibility and bioactivity of new endodontic materials on the basis of nanosynthesized calcium silicates (ALBO-MPCA1 and ALBO-MPCA2) combined with different radiopacifiers in comparison with MTA+. METHODS: Morphology of the samples was studied by scanning electron microscopy, and the pH and ion release analysis were also assessed. Biocompatibility of materials' eluates (24-hour, 7-day, and 21-day) was conducted by using MTT test. Twelve New Zealand white rabbits were used for intraosseous implantation. Four calvarial defects per animal were created and filled with freshly prepared investigated materials. RESULTS: Samples mostly consisted of agglomerates built up from nanoparticles, preferably spherical and rod-like. There was no significant difference among pH values of materials' eluates after 24 hours (P > .05). The amount of calcium and aluminum ion release decreased, whereas the amount of magnesium and bismuth (ALBO-MPCA1, MTA+) and barium (ALBO-MPCA2) increased during 21-day period. The metabolic activity of cells increased after the extraction time, except in case of undiluted elutes of ALBO-MPCA2 and ALBO-MPCA1 (21-day). Histologic analysis of the samples revealed newly formed bone tissue with moderate inflammation for all investigated materials, which subsided during 90-day period to mild. Both MTA+ and ALBO-MPCA1 were in direct contact with the newly formed bone tissue. After 90 days, statistically significant difference in hard tissue formation was observed in comparison of MTA+ and ALBO-MPCA1 with control group (P < .05). CONCLUSIONS: Experimental materials ALBO-MPCA1 and ALBO-MPCA2 possess both biocompatibility and bioactivity. Because ALBO-MPCA1 provokes favorable biological response, it is especially good candidate for further clinical investigations.

Histomorphometric evaluation of bone regeneration using autogenous bone and beta-tricalcium phosphate in diabetic rabbits.

Background/Aim: The mechanism of impaired bone healing in diabetes mellitus includes different tissue and cellular level activities due to micro- and macrovascular changes. As a chronic metabolic disease with vascular complications, diabetes affects a process of bone regeneration as well. The therapeutic approach in bone regeneration is based on the use of osteoinductive autogenous grafts as well as osteoconductive synthetic material, like a ß-tricalcium phosphate. The aim of the study was to determine the quality and quantity of new bone formation after the use of autogenous bone and ß-tricalcium phosphate in the model of calvarial critical-sized defect in rabbits with induced diabetes mellitus type I. Methods: The study included eight 4-month-old Chincilla rabbits with alloxan-induced diabetes mellitus type I. In all animals, there were surgically created two calvarial bilateral defects (diameter 12 mm), which were grafted with autogenous bone and ß-tricalcium phosphate (n = 4) or served as unfilled controls (n = 4). After 4 weeks of healing, animals were sacrificed and calvarial bone blocks were taken for histologic and histomorphometric analysis. Beside descriptive histologic evaluation, the percentage of new bone formation, connective tissue and residual graft were calculated. All parameters were statistically evaluated by Friedman Test and post hock Wilcoxon Singed Ranks Test with a significance of p < 0.05. Results: Histology revealed active new bone formation peripherally with centrally located connective tissue, newly formed woven bone and well incorporated residual grafts in all treated defects. Control samples showed no bone bridging of defects. There was a significantly more new bone in autogeonous graft (53%) compared with ß-tricalcium phosphate (30%), (p < 0.030) and control (7%), (p < 0.000) groups. A significant difference was also recorded between ß-tricalcium phosphate and control groups (p < 0.008). Conclusion: In the present study on the rabbit grafting model with induced diabetes mellitus type I, the effective bone regeneration of critical bone defects was obtained using autogenous bone graft. [Projekat Ministarstva nauke Republike Srbije, br. 175021].

Histological Effects of Enamel Matrix Derivative on Exposed Dental Pulp.

INTRODUCTION: Direct pulp capping procedure is a therapeutic application of a drug on exposed tooth pulp in order to ensure the closure of the pulp chamber and to allow the healing process to take place. OBJECTIVE: The aim of this study was to examine the histological effects of Emdogain® on exposed tooth pulp of a Vietnamese pig (Sus scrofa verus). METHODS: The study comprised 20 teeth of a Vietnamese pig. After class V preparation on the buccal surfaces of incisors, canines and first premolars, pulp was exposed. In the experimental group, the perforations were capped with Emdogain® (Straumann, Basel, Switzerland), while in the control group pulp capping was performed with MTA® (Dentsply Tulsa Dental, Johnson City, TN, USA). All cavities were restored with glass-ionomer cement (GC Fuji VIII, GC Corporation, Tokyo, Japan). The observational period was 28 days, after which the animal was sacrificed and histological preparations were made. A light microscope was used to analyze dentin bridge formation, tissue reorganization and inflammation, and the presence of bacteria in the pulp. RESULTS: The formation of dentin bridge was observed in the experimental and control groups. Inflammation of the pulp was mild to moderate in both groups. Angiogenesis and many odontoblast-like cells, responsible for dentin bridge formation, were observed. Necrosis was not observed in any case, nor were bacteria present in the pulp. CONCLUSION: Histological analysis indicated a favorable therapeutic effect of Emdogain® Gel in direct pulp capping of Vietnamese pigs. Pulp reaction was similar to that of MTA®.