The role of melatonin in preventing amiodarone-induced rat liver damage.

Long-term exposure to amiodarone, an antiarrhythmic drug, can induce different organ damage, including liver. Cell damage included by amiodarone is a consequence of mitochondrial damage, reactive oxygen species production, and cell energy depletion leading to programmed cell death. In the present study, hepatoprotective potential of neurohormone melatonin (50 mg/kg/day) was evaluated in a chronic experimental model of liver damage induced by a 4-week application of amiodarone (70 mg/kg/day). The obtained results indicate that amiodarone induces an increase in xanthine oxidase activity, as well as the content of the lipid and protein oxidatively modified products and p53 levels. Microscopic analysis further corroborated the biochemical findings revealing hepatocyte degeneration, apoptosis, and occasional necrosis, with the activation of Kupffer cells. Coadministration of melatonin and amiodaron prevented an increase in certain damage associated parameters, due to its multiple targets. In conclusion, the application of melatonin together with amiodarone prevented an increase in tissue oxidative damage parameters and moderately prevented liver cell apoptosis, indicating that the damage of hepatocytes provoked by amiodarone supersedes the protective properties of melatonin in a given dose.

Optimization of solid-state fermentation for enhanced production of pectinolytic complex by Aspergillus tubingensis FAT43 and its application in fruit juice processing.

The main goal of this study was to examine the efficiency of a newly isolated fungus from quince, Aspergillus tubingensis FAT43, to produce the pectinolytic complex using agricultural and industrial waste as the substrate for solid state fermentation. Sugar beet pulp was the most effective substrate inducer of pectinolytic complex synthesis out of all the waste residues examined. For endo-pectinolytic and total pectinolytic activity, respectively, statistical optimization using Placked-Burman Design and Optimal (Custom) Design increased production by 2.22 and 2.15-fold, respectively. Liquification, clarification, and an increase in the amount of reducing sugar in fruit juices (apple, banana, apricot, orange, and quince) processed with pectinolytic complex were identified. Enzymatic pre-treatment considerably increases yield (14%-22%) and clarification (90%). After enzymatic treatment, the best liquefaction was observed in orange juice, whereas the best clarification was obtained in apricot juice. Additionally, the pectinolytic treatment of apricot juice resulted in the highest increase in reducing sugar concentration (11%) compared to all other enzymatically treated juices. Optimizing the production of a highly active pectinolytic complex and its efficient utilization in the processing of fruit juices, including the generation of an increasing amount of waste, are the significant outcomes of this research.

Novel D-modified heterocyclic androstane derivatives as potential anticancer agents: Synthesis, characterization, in vitro and in silico studies.

Cancer remains a major health concern worldwide. The most frequently diagnosed types of cancer are caused by abnormal production or action of steroid hormones. In the present study, the synthesis and structural characterization of new heterocyclic androstane derivatives with D-homo lactone, 17α-(pyridine-2''-ylmethyl) or 17(E)-(pyridine-2''-ylmethylidene) moiety are presented. All compounds were evaluated for their anti-proliferative activity against HeLa cervical cancer cell line and non-cancerous kidney MDCK cells, where A-homo lactam compound 9A showed the greatest selectivity. Based on in vitro binding assays, N-formyl lactam compound 18 appeared to be the strong and isoform-selective ligand for ERα, while compound 9A displayed binding affinity for the GR-LBD, but also inhibited aldo-keto reductase 1C4 enzyme. Out of four selected compounds, methylpyrazolo derivative 13 showed potential for aromatase binding, while in silico studies provided insight into experimentally confirmed protein-ligand interactions.

Analyses of the Cellular Interactions between the Ossification of Collagen-Based Barrier Membranes and the Underlying Bone Defects.

Barrier membranes are an essential tool in guided bone Regeneration (GBR), which have been widely presumed to have a bioactive effect that is beyond their occluding and space maintenance functionalities. A standardized calvaria implantation model was applied for 2, 8, and 16 weeks on Wistar rats to test the interactions between the barrier membrane and the underlying bone defects which were filled with bovine bone substitute materials (BSM). In an effort to understand the barrier membrane's bioactivity, deeper histochemical analyses, as well as the immunohistochemical detection of macrophage subtypes (M1/M2) and vascular endothelial cells, were conducted and combined with histomorphometric and statistical approaches. The native collagen-based membrane was found to have ossified due to its potentially osteoconductive and osteogenic properties, forming a "bony shield" overlying the bone defects. Histomorphometrical evaluation revealed the resorption of the membranes and their substitution with bone matrix. The numbers of both M1- and M2-macrophages were significantly higher within the membrane compartments compared to the underlying bone defects. Thereby, M2-macrophages significantly dominated the tissue reaction within the membrane compartments. Statistically, a correlation between M2-macropahges and bone regeneration was only found at 2 weeks post implantationem, while the pro-inflammatory limb of the immune response correlated with the two processes at 8 weeks. Altogether, this study elaborates on the increasingly described correlations between barrier membranes and the underlying bone regeneration, which sheds a light on the understanding of the immunomodulatory features of biomaterials.

The Use of Collagen-Based Materials in Bone Tissue Engineering.

Synthetic bone substitute materials (BSMs) are becoming the general trend, replacing autologous grafting for bone tissue engineering (BTE) in orthopedic research and clinical practice. As the main component of bone matrix, collagen type I has played a critical role in the construction of ideal synthetic BSMs for decades. Significant strides have been made in the field of collagen research, including the exploration of various collagen types, structures, and sources, the optimization of preparation techniques, modification technologies, and the manufacture of various collagen-based materials. However, the poor mechanical properties, fast degradation, and lack of osteoconductive activity of collagen-based materials caused inefficient bone replacement and limited their translation into clinical reality. In the area of BTE, so far, attempts have focused on the preparation of collagen-based biomimetic BSMs, along with other inorganic materials and bioactive substances. By reviewing the approved products on the market, this manuscript updates the latest applications of collagen-based materials in bone regeneration and highlights the potential for further development in the field of BTE over the next ten years.

Comparative In Vitro Biocompatibility Study of the Two Orthodontic Bonding Materials of Different Types.

In the present study, the in vitro biocompatibility and cell response to two commonly used orthodontic bonding materials of different types, one self-curing and one light-curing, were examined and compared in indirect and direct cell culture systems. The study was conducted on fibroblasts and macrophages as in vitro models to study the biocompatibility of dental materials. Differences were found between the light- and self-curing material in cytotoxicity and effects on fibroblasts' proliferation in indirect cell culture systems as well as in macrophages response in vitro in both direct and indirect cell culture systems. Based on the obtained results, we can conclude that the self-curing material is generally more cytotoxic for fibroblasts compared to the light-curing, while macrophages' response to these materials was dependent on the macrophages' state and differed between the examined materials. This indicates that more attention should be paid when choosing and applying these materials in practice due to their toxicity to cells. Prior to their use, all aspects should be considered regarding the patient's conditions, associated problems, microenvironment in the oral cavity, etc. Further studies on in vivo models should be conducted to fully understand the potential long-term effects of the use of mentioned materials in orthodontics.

Free-standing multilayer films as growth factor reservoirs for future wound dressing applications.

Chronic skin wounds place a high burden on patients and health care systems. The use of angiogenic and mitogenic growth factors can facilitate the healing but growth factors are quickly inactivated by the wound environment if added exogenously. Here, free-standing multilayer films (FSF) are fabricated from chitosan and alginate as opposing polyelectrolytes in an alternating manner using layer-by-layer technique. One hundred bilayers form an about 450 µm thick, detachable free-standing film that is subsequently crosslinked by either ethyl (dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide (E-FSF) or genipin (G-FSF). The characterization of swelling, oxygen permeability and crosslinking density shows reduced swelling and oxygen permeability for both crosslinked films compared to non-crosslinked films (N-FSF). Loading of fibroblast growth factor 2 (FGF2) into the films results in a sustained release from crosslinked FSF in comparison to non-crosslinked FSF. Biocompatibility studies in vitro with human dermal fibroblasts cultured underneath the films demonstrate increased cell growth and cell migration for all films with and without FGF2. Especially G-FSF loaded with FGF2 greatly increases cell proliferation and migration. In vivo biocompatibility studies by subcutaneous implantation in mice show that E-FSF causes an inflammatory tissue response that is absent in the case of G-FSF. N-FSF also represents a biocompatible film but shows early degradation. All FSF possess antibacterial properties against gram+ and gram- bacteria demonstrated by an agar diffusion disc assay. In summary, FSF made of alginate and chitosan crosslinked with genipin can act as a reservoir for the sustained release of FGF2, possessing high biocompatibility in vitro and in vivo. Moreover, G-FSF promotes growth and migration of human dermal fibroblasts and has antibacterial properties, which makes it an interesting candidate for bioactive wound.

Aspergillus welwitschiae inulinase enzyme cocktails obtained on agro-material inducers for the purpose of fructooligosaccharides production.

Production of fructooligosaccharides (FOS) is a trending topic due to their prebiotic effect becoming increasingly important for the modern human diet. The most suitable process for FOS production is the one using fungal inulinases. Introduction of new fungal inulinase producers and their implementation in production of inulinase enzymes is therefore gaining interest. This study provides a new approach to FOS synthesis by fungal enzyme complex without prior separation of any specific enzyme. Inulinase enzyme complexes could be used for the synthesis of FOS in two possible ways - hydrolysis of inulin (FOSh) and transfructosylation process of sucrose (FOSs), as demonstrated here. Depending on the fungal growth inducing substrate, a variety of inulinase enzyme complexes was obtained - one of which was most successful in production of FOSh and another one of FOSs. Substrates derived from crops: triticale, wheat bran, Jerusalem artichoke and Aspergillus welwitschiae isolate, previously proven as safe for use in food, were utilized for production of inulinase enzyme cocktails. The highest FOSs production was obtained by enzyme complex rich in ß-fructofuranosidase, while the highest FOSh production was obtained by enzyme complex rich in endoinulinase. Both FOSh and FOSs showed antioxidant potential according to ABTS and ORAC, which classifies them as a suitable additive in functional food. Simultaneous zymographic detection of inulinase enzymes, which could contribute to expansion of the knowledge on fungal enzymes, was developed and applied here. It demonstrated the presence of different inulinase isoforms depending on fungal growth substrate. These findings, which rely on the innate ability of fungi to co-produce all inulinases from a cocktail, could be useful as a new, easy approach to FOS production by fungal enzymes without their separation and purification, contributing to cheaper and faster production processes.

In Vivo Analysis of the Immune Response to Strontium- and Copper-doped Bioglass.

BACKGROUND: Bioglass is a highly adoptable bone substitute material which can be combined with so-called therapeutic ions. However, knowledge is poor regarding the influence of therapeutic ions on immune reactions and associated bone healing. Thus, the aim of this work was to investigate the influence of strontium- and copper-doped bioglass on the induction of M1 and M2 macrophages, as well as vascularization. MATERIALS AND METHODS: Two types of alkali glass were produced based on ICIE16 bioglass via the melt-quench method with the addition of 5 wt% copper or strontium (ICIE16-Cu and ICIE16-Sr). Pure ICIE16 and 45S5 bioglass were used as control materials. The ion release and chemical composition of the bioglass were investigated, and an in vivo experiment was subcutaneously performed on Sprague-Dawley rats. RESULTS: Scanning electron microscopy revealed significant differences in the surface morphology of the bioglass materials. Energy dispersive X-ray spectroscopy confirmed the efficiency of the doping process by showing the ion-release kinetics. ICIE16-Cu exhibited a higher ion release than ICIE16-Sr. ICIE16-Cu induced low immune cell migration and triggered not only a low number of M1 and M2 macrophages but also of blood vessels. ICIE16-Sr induced higher numbers of M1 macrophages after 30 days. Both bioglass types induced numbers of M2 macrophages comparable with those found in the control groups. CONCLUSION: Bioglass doping with copper and strontium did not significantly influence the foreign body response nor vascularization of the implantation bed in vivo. However, all the studied bioglass materials seemed to be biocompatible.

Lyophilized Human Bone Allograft as an Antibiotic Carrier: An In Vitro and In Vivo Study.

Background: Antibiotics delivered from implanted bone substitute materials (BSM) can potentially be used to prevent acute infections and biofilm formation, providing high concentrations of antibiotics at the surgical site without systemic toxicity. In addition, BSM should allow osteoconductivity supporting bone healing without further surgery. Promising results have been achieved using lyophilized bone allografts mixed with antibiotics. Methods: In this study specially prepared human bone allografts were evaluated as an antibiotic carrier in vitro and in vivo. The efficacy of different antibiotic-impregnated bone allografts was measured by drug release tests in vitro and in vivo and bacterial susceptibility tests using four bacterial species usually responsible for implant-associated infections. Results: The loading procedures of allograft bone substitutes with antibiotics were successful. Some of the antibiotic concentrations exceeded the MIC90 for up to 7 days in vitro and for up to 72 h in vivo. The susceptibility tests showed that S. epidermidis ATCC 12228 was the most susceptible bacterial species in comparison to the other strains tested for all antibiotic substances. Vancomycin and rifampicin showed the best results against standard and patient-isolated strains in vitro. In vivo, new bone formation was comparable in all study groups including the control group without antibiotic loading. Conclusions: Human bone allografts showed the capacity to act as customized loaded antibiotic carriers to prevent acute infections and should be considered in the management of bone infections in combination with systemic antimicrobial therapy.

Mucinous cystadenoma of the liver with pathological-radiological correlation.

The paper presents a pathological-radiological correlation of the manifestation of mucosal cystadenoma with ovarian stroma of the liver with examination and correlation with the new stroma nomenclature and differential diagnostic dilemmas of radiologists and pathologists.

In Vivo Biocompatibility Analysis of a Novel Barrier Membrane Based on Bovine Dermis-Derived Collagen for Guided Bone Regeneration (GBR).

Collagen-based barrier membranes are nowadays the prevalent option for Guided Bone Regeneration (GBR) procedures. Xenogeneic collagen is highly biocompatible as it shares a similar structure to native human collagen, which prevents it from eliciting an exaggerated host immune response. Most commercially available collagen barrier membranes are porcine-derived, while bovine-derived alternatives are still rarely available. The aim of the present study was to investigate the tissue responses and the barrier functionality of a novel GBR membrane composed of bovine collagen type I (BM). Therefore, the subcutaneous implantation model in Wistar rats was performed to compare the novel medical device with two already clinically used native porcine-based barrier membranes, i.e., Jason® membrane (JM) and Bio-Gide® (BG), at 10-, 30-, 60-, and 90-days post implantationem. Histochemical and immunohistochemical stains were used for histopathological evaluation including a biocompatibility scoring according to the DIN EN ISO 10993-6 norm as well as histomorphometrical analyses of the occurrence of M1 and M2 macrophages and the transmembraneous vascularization. The bovine membrane exhibited a host tissue reaction that was comparable to both control materials, which was verified by the scoring results and the histomorphometrical macrophage measurements. Moreover, the novel membrane exhibited an integration pattern without material fragmentation up to day 60. At day 90, material fragmentation was observable that allowed for "secondary porosity" including transmembrane vascularization. The results of this study suggest that the novel bovine barrier membrane is fully biocompatible and suitable for indications that require GBR as a suitable alternative to porcine-sourced barrier membranes.

Electrospun Poly(lactide) Fibers as Carriers for Controlled Release of Biochanin A.

The aim of this study is to investigate the possibility of using electrospun polylactide (PLA) fibers as a carrier of the phytoestrogen biochanin A. Polylactide fibers were prepared with different contents of biochanin A by using an electrospinning method at specific process parameters. The obtained electrospun polylactide fibers, as carriers of biochanin A, were characterized by means of different methods. The presented results showed that the mechanical properties of PLA have not changed significantly in the presence of biochanin A. Scanning electron microscopy showed that the fine fiber structure is retained without visible deformations and biochanin A crystals on the surface of the fibres. The analysis by infrared spectroscopy showed that there are no strong interactions between polylactide and biochanin A molecules, which is a good prerequisite for the diffusion release of biochanin A from PLA fibers.The release of biochanin A from PLA fibers in buffer solution pH 7.4 at 37 °C was monitored by applying the HPLC method. The rate and time of the release of biochanin A from PLA fibers is in correlation with the amount of the active ingredient in the matrix of the carrier and follows zero-order kinetics. PLA fibers with biochanin A exhibit concentration-dependent activity on proliferation and migration of L929 fibroblasts in direct culture system in vitro, and proved to be suitable for a potential formulation for use in wound healing.

How to improve O-RADS MRI score for rating adnexal masses with cystic component?

OBJECTIVES: To test the performance of the Ovarian-Adnexal Reporting Data System (O-RADS) MRI in characterizing adnexal masses with cystic components and to test new specific MRI features related to cystic components to improve the ability of the O-RADS MRI score to stratify lesions according to their risk of malignancy. METHODS: The EURopean ADnexal study (EURAD) database was retrospectively queried to identify adnexal masses with a cystic component. One junior and 13 radiologists independently reviewed cases blinded to the pathological diagnosis. For each lesion, the size of the whole lesion, morphological appearance, number of loculi, presence of a thickened wall, thickened septae, signal intensity of the cystic components on T1-weighted/T2-weighted/diffusion weighted, mean value of the apparent diffusion coefficient, and O-RADS MRI score were reported. Univariate and multivariate logistic regression analysis was performed to determine significant features to predict malignancy. RESULTS: The final cohort consisted of 585 patients with 779 pelvic masses who underwent pelvic MRI to characterize an adnexal mass(es). Histology served as the standard of reference. The diagnostic performance of the O-RADS MRI score was 0.944, 95%CI [0.922-0.961]. Significant criteria associated with malignancy included an O-RADS MRI score ≥ 4, ADCmean of cystic component > 1.69, number of loculi > 3, lesion size > 75 mm, the presence of a thick wall, and a low T1-weighted, a high T2-weighted, and a low diffusion-weighted signal intensity of the cystic component. Multivariate analysis demonstrated that an O-RADS MRI score ≥ combined with an ADC mean of the cystic component > 1.69, size > 75 mm, and low diffusion-weighted signal of the cystic component significantly improved the diagnostic performance up to 0.958, 95%CI [0.938-0.973]. CONCLUSION: Cystic component analysis may improve the diagnosis performance of the O-RADS MRI score in adnexal cystic masses. KEY POINTS: • O-RADS MRI score combined with specific cystic features (area under the receiving operating curve, AUROC = 0.958) improves the diagnostic performance of the O-RADS MRI score (AUROC = 0.944) for predicting malignancy in this cohort. • Cystic features that improve the prediction of malignancy are ADC mean > 1.69 (OR = 7); number of loculi ≥ 3 (OR = 5.16); lesion size > 75 mm (OR = 4.40); the presence of a thick wall (OR = 3.59); a high T2-weighted signal intensity score 4 or 5 (OR = 3.30); a low T1-weighted signal intensity score 1, 2, or 3 (OR = 3.45); and a low diffusion-weighted signal intensity (OR = 2.12). • An adnexal lesion with a cystic component rated O-RADS MRI score 4 and an ADC value of the cystic component < 1.69 associated with a low diffusion-weighted signal, has virtually a 0% risk of malignancy.

In Vivo Biocompatibility Investigation of an Injectable Calcium Carbonate (Vaterite) as a Bone Substitute including Compositional Analysis via SEM-EDX Technology.

Injectable bone substitutes (IBS) are increasingly being used in the fields of orthopedics and maxillofacial/oral surgery. The rheological properties of IBS allow for proper and less invasive filling of bony defects. Vaterite is the most unstable crystalline polymorph of calcium carbonate and is known to be able to transform into hydroxyapatite upon contact with an organic fluid (e.g., interstitial body fluid). Two different concentrations of hydrogels based on poly(ethylene glycol)-acetal-dimethacrylat (PEG-a-DMA), i.e., 8% (w/v) (VH-A) or 10% (w/v) (VH-B), were combined with vaterite nanoparticles and implanted in subcutaneous pockets of BALB/c mice for 15 and 30 days. Explants were prepared for histochemical staining and immunohistochemical detection methods to determine macrophage polarization, and energy-dispersive X-ray analysis (EDX) to analyze elemental composition was used for the analysis. The histopathological analysis revealed a comparable moderate tissue reaction to the hydrogels mainly involving macrophages. Moreover, the hydrogels underwent a slow cellular infiltration, revealing a different degradation behavior compared to other IBS. The immunohistochemical detection showed that M1 macrophages were mainly found at the material surfaces being involved in the cell-mediated degradation and tissue integration, while M2 macrophages were predominantly found within the reactive connective tissue. Furthermore, the histomorphometrical analysis revealed balanced numbers of pro- and anti-inflammatory macrophages, demonstrating that both hydrogels are favorable materials for bone tissue regeneration. Finally, the EDX analysis showed a stepwise transformation of the vaterite particle into hydroxyapatite. Overall, the results of the present study demonstrate that hydrogels including nano-vaterite particles are biocompatible and suitable for bone tissue regeneration applications.

Standardized wild apple fruit extract as a bioactive agent in dermocosmetic products for efficacy skin hydration-In vitro and in vivo evaluation.

BACKGROUND: Wild apple fruit is rich source of bioactive substances, which can be potentially used for a production of dermocosmetic products. AIM: The aim of study was in vitro and in vivo investigation of standardized wild apple fruit extracts (obtained by purified water and maceration-EM, percolation-EP, Soxhlet extraction-ES and ultrasonic extraction-EU) and dermocosmetic creams of o/w type containing 6%, 12%, and 15% of extract EU, as extract with the best content of polyphenolic compounds (CE6, CE12, and CE15, respectively), in order to determine the optimal concentration of the extract, which manifests the best effect on the biophysical characteristics of the skin after application. METHODS: Content of polyphenolic substances-PP was investigated using HPLC analysis, while in vitro biological activity was examined on L929 fibroblasts using MTT viability test. In vivo efficacy included screening and comparison of skin hydration potential, transepidermal water loss-TEWL, and skin pH after 28 days of creams application on healthy volunteers' skin. RESULTS: Content of identified PP was the highest in the extract EU (2001.57 ± 0.87 mg PP/100 g EU) and satisfactory in cream CE6 (53.83 ± 0.23 mg PP/100 g CE6). Procyanidin B2 was the most common PP. Biological activity analysis showed that extract EU could be considered as non-cytotoxic for fibroblasts in examined concentrations. In vivo investigation revealed an increase of skin hydration (after 28 days ΔEC was 21.19 ± 7.59, 26.71 ± 7.94, and 29.60 ± 10.95 for CE6, CE12, and CE15, respectively), with unchanged TEWL and skin pH values. CONCLUSION: Obtained results indicate that wild apple fruit extract represents a valuable source of bioactive substances, with good hydration effects of creams on human skin (effect of creams increased proportionally to the increase of concentration of incorporated extract), so it might be considered as a great potential for application in dermocosmetic industry.

Selection of Non-Mycotoxigenic Inulinase Producers in the Group of Black Aspergilli for Use in Food Processing.

Research background: Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach: Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions: The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution: The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally.

The Granule Size Mediates the In Vivo Foreign Body Response and the Integration Behavior of Bone Substitutes.

The physicochemical properties of synthetically produced bone substitute materials (BSM) have a major impact on biocompatibility. This affects bony tissue integration, osteoconduction, as well as the degradation pattern and the correlated inflammatory tissue responses including macrophages and multinucleated giant cells (MNGCs). Thus, influencing factors such as size, special surface morphologies, porosity, and interconnectivity have been the subject of extensive research. In the present publication, the influence of the granule size of three identically manufactured bone substitute granules based on the technology of hydroxyapatite (HA)-forming calcium phosphate cements were investigated, which includes the inflammatory response in the surrounding tissue and especially the induction of MNGCs (as a parameter of the material degradation). For the in vivo study, granules of three different size ranges (small = 0.355-0.5 mm; medium = 0.5-1 mm; big = 1-2 mm) were implanted in the subcutaneous connective tissue of 45 male BALB/c mice. At 10, 30, and 60 days post implantationem, the materials were explanted and histologically processed. The defect areas were initially examined histopathologically. Furthermore, pro- and anti-inflammatory macrophages were quantified histomorphometrically after their immunohistochemical detection. The number of MNGCs was quantified as well using a histomorphometrical approach. The results showed a granule size-dependent integration behavior. The surrounding granulation tissue has passivated in the groups of the two bigger granules at 60 days post implantationem including a fibrotic encapsulation, while a granulation tissue was still present in the group of the small granules indicating an ongoing cell-based degradation process. The histomorphometrical analysis showed that the number of proinflammatory macrophages was significantly increased in the small granules at 60 days post implantationem. Similarly, a significant increase of MNGCs was detected in this group at 30 and 60 days post implantationem. Based on these data, it can be concluded that the integration and/or degradation behavior of synthetic bone substitutes can be influenced by granule size.

Novel Histomorphometrical Approach to Evaluate the Integration Pattern and Functionality of Barrier Membranes.

GBR (guided bone regeneration) is a standard procedure for building up bony defects in the jaw. In this procedure, resorbable membranes made of bovine and porcine collagen are increasingly being used, which, in addition to many possible advantages, could have the potential disadvantage of a shorter barrier functionality, especially when augmenting large-volume defects. Thus, it is of importance to evaluate the integration behavior and especially the standing time of barrier membranes using specialized methods to predict its respective biocompatibility. This study is intended to establish a new histomorphometrical analysis method to quantify the integration rate of collagen-based barrier membranes. Three commercially available barrier membranes, i.e., non-crosslinked membranes (BioGide® and Jason® membrane), a ribose-crosslinked membrane (Ossix® Plus), and a newly developed collagen-hyaluronic acid-based (Coll-HA) barrier membrane were implanted in the subcutaneous tissue of 48 6-8-week-old Wistar rats. The explants, after three timepoints (10, 30, and 60 days), were processed and prepared into histological sections for histopathological (host tissue response) and histomorphometrical (cellular invasion) analyses. 10 days after implantation, fragmentation was not evident in any of the study groups. The sections of the Coll-HA, Jason® and BioGide® membranes showed a similar mild inflammatory reaction within the surrounding tissue and an initial superficial cell immigration. Only in the Ossix® Plus group very little inflammation and no cell invasion was detected. While the results of the three commercially available membranes remained intact in the further course of the study, only fragments of the Coll-HA membrane were found 30 and 60 days after implantation. Histomorphometrically, it can be described that although initially (at 10 days post-implantation) similar results were found in all study groups, after 30 days post-implantation the cellular penetration depth of the hyaluronic acid-collagen membrane was significantly increased with time (**** p < 0.0001). Similarly, the percentage of cellular invasion per membrane thickness was also significantly higher in the Coll-HA group at all timepoints, compared to the other membranes (**** p < 0.0001). Altogether, these results show that the histomorphometrical analysis of the cellular migration can act as an indicator of integration and duration of barrier functionality. Via this approach, it was possible to semi-quantify the different levels of cellular penetration of GBR membranes that were only qualitatively analyzed through histopathological approaches before. Additionally, the results of the histopathological and histomorphometrical analyses revealed that hyaluronic acid addition to collagen does not lead to a prolonged standing time, but an increased integration of a collagen-based biomaterial. Therefore, it can only partially be used in the dental field for indications that require fast resorbed membranes and a fast cell or tissue influx such as periodontal regeneration processes.

Biocompatibility Analyses of HF-Passivated Magnesium Screws for Guided Bone Regeneration (GBR).

Background: Magnesium (Mg) is one of the most promising materials for human use in surgery due to material characteristics such as its elastic modulus as well as its resorbable and regenerative properties. In this study, HF-coated and uncoated novel bioresorbable magnesium fixation screws for maxillofacial and dental surgical applications were investigated in vitro and in vivo to evaluate the biocompatibility of the HF coating. Methods: Mg alloy screws that had either undergone a surface treatment with hydrofluoric-acid (HF) or left untreated were investigated. In vitro investigation included XTT, BrdU and LDH in accordance with the DIN ISO 10993-5/-12. In vivo, the screws were implanted into the tibia of rabbits. After 3 and 6 weeks, degradation, local tissue reactions and bony integration were analyzed histopathologically and histomorphometrically. Additionally, SEM/EDX analysis and synchrotron phase-contrast microtomography (µCT) measurements were conducted. The in vitro analyses revealed that the Mg screws are cytocompatible, with improved results when the surface had been passivated with HF. In vivo, the HF-treated Mg screws implanted showed a reduction in gas formation, slower biodegradation and a better bony integration in comparison to the untreated Mg screws. Histopathologically, the HF-passivated screws induced a layer of macrophages as part of its biodegradation process, whereas the untreated screws caused a slight fibrous tissue reaction. SEM/EDX analysis showed that both screws formed a similar layer of calcium phosphates on their surfaces and were surrounded by bone. Furthermore, the µCT revealed the presence of a metallic core of the screws, a faster absorbing corrosion front and a slow absorbing region of corroded magnesium. Conclusions: Overall, the HF-passivated Mg fixation screws showed significantly better biocompatibility in vitro and in vivo compared to the untreated screws.