Journal of Functional Biomaterials

The eggshell membrane (ESM) is a natural biomaterial with unique physical and mechanical properties that make it a promising candidate for wound-healing applications. However, the ESM’s inherent properties can be enhanced through incorporation of silver nanoparticles (AgNPs), which have been shown to have antimicrobial properties. In this study, commercially produced AgNPs and green-processed AgNPs were incorporated into ESM and evaluated for their physical, biological, and antimicrobial properties for potential dermal application. The ESM was extracted using various techniques, and then treated with either commercially produced AgNPs (Sigma-Aldrich, Poole, UK) or green-synthesized AgNPs (Metalchemy, London, UK) to produce AgNPs-ESM samples. The physical characteristics of the samples were evaluated using scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, and the biological properties were assessed through in vitro studies using human dermal fibroblasts (HDFs) and BJ cells. The SEM analysis of the AgNPs-ESM samples showed localization of AgNPs on the ESM surface, and that the ESM maintained its structural integrity following AgNP incorporation. The FTIR confirmed loading of AgNPs to ESM samples. The biological studies showed that the 5 μg/mL AgNPs-ESM samples were highly biocompatible with both HDFs and BJ cells, and had good viability and proliferation rates. Additionally, the AgNPs-ESM samples demonstrated pro-angiogenic properties in the CAM assay, indicating their potential for promoting new blood vessel growth. Assessment of the antimicrobial activity of the enhanced AgNPs/ESMs was validated using the International Standard ISO 16869:2008 methodology and exploited Cladosporium, which is one of the most commonly identified fungi in wounds, as the test microorganism (≥5 × 10⁶ cells/mL). The AgNPs-ESM samples displayed promising antimicrobial efficacy as evidenced by the measured zone of inhibition. Notably, the green-synthesized AgNPs demonstrated greater zones of inhibition (~17 times larger) compared to commercially available AgNPs (Sigma-Aldrich). Although both types of AgNP exhibited long-term stability, the Metalchemy-modified samples demonstrated a slightly stronger inhibitory effect. Overall, the AgNPs-ESM samples developed in this study exhibited desirable physical, biological, and antimicrobial properties for potential dermal wound-dressing applications. The use of green-processed AgNPs in the fabrication of the AgNPs-ESM samples highlights the potential for sustainable and environmentally friendly wound-healing therapies. Further research is required to assess the long-term biocompatibility and effectiveness of these biomaterials in vivo. Full article

Catecholamine metabolites are not only involved in primary metabolism, but also in secondary metabolism, serving a diverse array of physiologically and biochemically important functions. Melanin, which originates from dopa and dopamine, found in the hair, eye, and skin of all animals, is an important biopolymeric pigment. It provides protection against damaging solar radiation to animals. N-Acetyldopamine and N-β-alanyldopamine play a crucial role in the hardening of the exoskeletons of all insects. In addition, insects and other arthropods utilize the melanogenic process as a key component of their defense systems. Many marine organisms utilize dopyl peptides and proteins as bonding materials to adhere to various substrata. Moreover, the complex dopa derivatives that are precursors to the formation of the exoskeletons of numerous marine organisms also exhibit antibiotic properties. The biochemistry and mechanistic transformations of different catecholamine derivatives to produce various biomaterials with antioxidant, antibiotic, crosslinking, and gluing capabilities are highlighted. These reactivities are exhibited through the transient and highly reactive quinones, quinone methides, and quinone methide imine amide intermediates, as well as chelation to metal ions. A careful consideration of the reactivities summarized in this review will inspire numerous strategies for synthesizing novel biomaterials for future medical and industrial use. Full article

With their distinctive core–shell design, core–shell nanocrystals have drawn interest in catalysis, medicinal research, and nanotechnology. These nanocrystals have a variety of characteristics and possible uses. The application of core–shell nanocrystals offers significant potential in increasing diagnostic and therapeutic approaches for cancer research in apoptosis and in vitro cancer cell imaging. In the present study, we investigated the fluorescence behavior of hydrophilic CdSe (core-only) and CdSe@CdS (core–shell) nanocrystals (NCs) and their potential in cancer cell imaging. The addition of a CdS coating to CdSe NCs increased the fluorescence intensity tenfold. The successful fabrication of core–shell CdSe@CdS nanocrystals was proven by a larger particle size (evaluated via DLS and TEM) and their XRD pattern and surface morphology compared to CdSe (core-only) NCs. When these NCs were used for bioimaging in MCF-7 and HEK-293 cell lines, they demonstrated excellent cellular uptake due to higher fluorescence intensity within cancerous cells than normal cells. Comparative cytotoxicity studies revealed that CdSe NCs were more toxic to all three cell lines (HEK-293, MCF-7, and HeLa) than CdSe@CdS core–shell structures. Furthermore, a decrease in mitochondrial membrane potential and intracellular ROS production supported NCs inducing oxidative stress, which led to apoptosis via the mitochondria-mediated pathway. Increased cytochrome c levels, regulation of pro-apoptotic gene expression (e.g., p53, Bax), and down-regulation of Bcl-2 all suggested cellular apoptosis occurred via the intrinsic pathway. Significantly, at an equivalent dose of core–shell NCs, core-only NCs induced more oxidative stress, resulting in increased apoptosis. These findings shed light on the role of a CdS surface coating in reducing free radical release, decreasing cytotoxicity, and improving fluorescence, advancing the field of cell imaging. Full article

Alternative techniques have been investigated for effectiveness in caries removal because conventional metallic dental burs can lead to an excessive loss of sound tissue. The aim of the present study is to realize a preliminary approach in obtaining effective polymer mixtures for polymeric bur development, capable of removing primary dental caries using combinations of polymers to ensure the requirements for such instruments, but also a greater compatibility with the teeth structure. This study assessed the main mechanical properties, water sorption, solubility and microscopic structure of four new polymer mixture recipes to provide essential features in obtaining experimental self-limited dental burs. Two mixtures have in their composition polymer mixtures of Bis-phenol A diglycidyl ether dimethacrylate/Triethylene glycol dimethacrylate/Urethane dimethacrylates (R1, R2), and two other mixtures have Bis-phenol A diglycidyl ether dimethacrylate/Polymethyl methacrylate/Methyl methacrylates (R3, R4). The incorporation of nanoparticles into the polymer matrix has become essential due to the need of polymer biocompatibility increasing along with teeth surface remineralization, so that the powder charge was added to four recipes, such as 5% glass with BaF₂ and 0.5% graphene with silver particles. All data sets were analyzed using the One-Way ANOVA test. R3, R4 showed higher compressive strength and diametrical compression values; these values increased when glass and graphene were added. Moreover, the addition of glass particles lead to an increase in flexural strength. Regarding the sorption, sample R3 had the most significant differences between day 69 and the rest of the investigation days, while the solubility varied at different intervals. From the mechanical evaluation, we could conclude that the Bis-GMA/PMMA/MMA mixtures fit the mechanical characteristics supported by polymer burs, following future studies regarding their use on the affected dentin. Full article

Background: Regenerative dentistry is the operation of restoring dental, oral and maxillofacial tissues. Currently, there are no guidelines for the ideal cement/material in regenerative endodontic treatments (RET). Hydraulic calcium silicate-based cements (hCSCs) are currently the material of choice for RET. Objectives: This systematic review was conducted to gather all of the different direct and indirect approaches of using hCSCs in RET in vitro and in vivo, and to ascertain if there are any superiorities to indirect approaches. Methods and Materials: This systematic review was conducted according to the 2020 PRISMA guidelines. The study question according to the PICO format was as follows: Comparison of the biological behavior (O) of stem cells (P) exposed to hCSCs through direct and indirect methods (I) with untreated stem cells (C). An electronic search was executed in Scopus, Google Scholar, and PubMed. Results: A total of 78 studies were included. Studies were published between 2010 and 2022. Twenty-eight commercially available and eighteen modified hCSCs were used. Seven exposure methods (four direct and three indirect contacts) were assessed. ProRoot MTA and Biodentine were the most used hCSCs and had the most desirable results. hCSCs were either freshly mixed or set before application. Most studies allowed hCSCs to set in incubation for 24 h before application, which resulted in the most desirable biological outcomes. Freshly mixed hCSCs had the worst outcomes. Indirect methods had significantly better viability/proliferation and odonto-/osteogenesis outcomes. Conclusion: Biodentine and ProRoot MTA used in indirect exposure methods result in desirable biological outcomes. Full article

The design of the implant prosthesis–abutment complex appears crucial for shaping healthy and stable peri-implant soft tissues. The aim of the present animal study was to compare two implants with different healing abutment geometries: a concave design (TEST) and a straight one (CTRL). Transmission electron microscopy (TEM) was used to quantify the three-dimensional topography and morphological properties of collagen at nanoscale resolution. Two swine were included in the experiment and six implants per animal were randomly placed in the left or right hemimandible in either the physiologically mature bone present between the lower canine and first premolar or in the mandibular premolar area. Each CTRL implant was positioned across from its respective TEST implant on the other side of the jaw. After 12 weeks of healing, eight specimens (four CTRL and four TEST) were retrieved and prepared for histological and TEM analysis. The results showed a significantly higher percentage of area covered by collagen bundles and average bundle size in TEST implants, as well as a significant decrease in the number of longitudinally oriented bundles with respect to CTRL implants, which is potentially due to the larger size of TEST bundles. These data suggest that a concave transmucosal abutment design serves as a scaffold, favoring the deposition and growth of a well-organized peri-implant collagen structure over the implant platform in the early healing phase, also promoting the convergence of collagen fibers toward the abutment collar. Full article

Endocrowns are designed to restore endodontically treated teeth with root canal treatment (Rct). Recently, endocrowns were proposed for teeth treated with full pulpotomy (FP). No data exist on in vitro evaluations for this combination. This study aimed to evaluate the mechanical behavior of pulpotomy-treated teeth with endocrowns according to different protocols for preparation design and materials and to assess whether 3D-printed resin simulators could be a reliable alternative for human teeth during in vitro strength tests. One hundred and ten extracted natural molars were randomized into 11 groups according to the type of endodontic treatment, the material used, and the design of peripheric preparation. One hundred and ten resin simulators were separated similarly. The samples were embedded in epoxy resin blocks before being subjected to oblique compressive load until failure. For natural teeth, the variance analysis separated two homogeneous groups, one regrouping the endodontically treated or pulpotomy-treated teeth without coronal restoration and the other one regrouping all the other samples, i.e., the untreated teeth (positive controls) and the treated and restored teeth. The strength resistance was lower for the resin simulators than for natural teeth in all groups. Within the limit of this study, strength resistance is not the most important criterion for choosing the type of material, preparation, or endodontic treatment for endocrowns. Resin simulators are not efficient for in vitro strength studies. Full article

The main purpose of these studies was to obtain carbon–carbon composites with a core built of carbon fibers and a matrix in the form of pyrolytic carbon (PyC), obtained by using the chemical vapor deposition (CVD) method with direct electrical heating of a bundle of carbon fibers as a potential electrode material for nerve tissue stimulation. The methods used for the synthesis of PyC proposed in this paper allow us, with the appropriate selection of parameters, to obtain reproducible composites in the form of rods with diameters of about 300 µm in 120 s (CF_PyC_120). To evaluate the materials, various methods such as scanning electron microscopy (SEM), scanning transmission electron microscope (STEM), high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and tensiometer techniques were used to study their microstructural, structural, chemical composition, surface morphology, and surface wettability. Assessing their applicability for contact with nervous tissue cells, the evaluation of cytotoxicity and biocompatibility using the SH-SY5Y human neuroblastoma cell line was performed. Viability and cytotoxicity tests (WST-1 and LDH release) along with cell morphology examination demonstrated that the CF_PyC_120 composites showed high biocompatibility compared to the reference sample (Pt wire), and the best adhesion of cells to the surface among all tested materials. Full article

Severe peripheral nerve injuries, such as deficits over long distances or proximal nerve trunk injuries, pose complex reconstruction challenges that often result in unfavorable outcomes. Innovative techniques, such as nerve transposition repair with conduit suturing, can be employed to successfully treat severe peripheral nerve damage. However, cylindrical nerve guides are typically unsuitable for nerve transposition repair. Furthermore, angiogenic and neurotrophic factors are necessary to stimulate the emergence of axonal lateral sprouts, proximal growth, and the rehabilitation of neuron structures and functions. In the current study, we used chitosan to make chitin conduits with different inner diameters at both ends, combined with gelatin methacrylate hydrogels that can continuously release dual growth factors, namely, the vascular endothelial growth factor (VEGF) and the nerve growth factor (NGF), and evaluated its impact on nerve transposition repair in rats. At 16 weeks after the operation, our findings showed that the conduit combined with the dual growth factor hydrogel significantly improved the restoration of both motor and conduction functions of the nerve. In addition, histological analysis showed significant recovery of nerve fibers, target muscles, and neurons. In conclusion, the combination of chitin conduits with different inner diameters and dual growth factor hydrogels can significantly improve the effect of nerve transposition repair, which has important potential clinical value. Full article

MicroRNAs (miRNAs) are short non-coding RNAs that are found in various cellular compartments and play an important role in regulating gene expression. Extracellular miRNAs, such as those found within extracellular vesicles such as exosomes are involved in cell-to-cell communication. The intercellular transfer of miRNAs has been implicated in various diseases’ pathogenesis including cancer and has been studied extensively as potential cancer biomarkers. However, the extraction of miRNA from exosomes is still a challenging task. The current nucleic acid extraction assays are expensive and labor-intensive. In this study, we demonstrated a microfluidic device for aptamer-based magnetic separation of the exosomes and subsequent detection of the miRNA using a fluorescence switching assay, which was enabled by carbon nanomaterials coated on magnetic beads. In the OFF state, the fluorophore-labelled cDNA is quenched using carbon nanomaterials. However, when the target miRNA210 is introduced, the cDNA detaches from the bead’s surface, which leads to an increase in the fluorescence intensity (ON state). This increment was found to be proportional to miRNA concentration within the dynamic range of 0–100 nM with a detection limit of 5 pM. The assay was validated with spiked miRNA using the standard RT-PCR method. No notable cross-reactivity with other closely related miRNAs was observed. The developed method can be utilized for the minimally invasive detection of cancer biomarkers. Full article

Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (≤168 nm) and polydispersity indexes (≤0.16) and ζ-potentials (≤−35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56–78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs. Full article

This comparative study investigated the tissue regeneration and inflammatory response induced by xenografts comprised of hydroxyapatite (HA) and demineralized bone matrix (DBM) extracted from porcine (P) and bovine (B) sources. First, extraction of HA and DBM was independently conducted, followed by chemical and morphological characterization. Second, mixtures of HA/DBM were prepared in 50/50 and 60/40 concentrations, and the chemical, morphological, and mechanical properties were evaluated. A rat calvarial defect model was used to evaluate the tissue regeneration and inflammatory responses at 3 and 6 months. The commercial allograft DBM Puros^® was used as a clinical reference. Different variables related to tissue regeneration were evaluated, including tissue thickness regeneration (%), amount of regenerated bone area (%), and amount of regenerated collagen area (%). The inflammatory response was evaluated by quantifying the blood vessel area. Overall, tissue regeneration from porcine grafts was superior to bovine. After 3 months of implantation, the tissue thickness regeneration in the 50/50P compound and the commercial DBM was significantly higher (~99%) than in the bovine materials (~23%). The 50/50P and DBM produced higher tissue regeneration than the naturally healed controls. Similar trends were observed for the regenerated bone and collagen areas. The blood vessel area was correlated with tissue regeneration in the first 3 months of evaluation. After 6 months of implantation, HA/DBM compounds showed less regenerated collagen than the DBM-only xenografts. In addition, all animal-derived xenografts improved tissue regeneration compared with the naturally healed defects. No clinical complications associated with any implanted compound were noted. Full article

The use of bioactive materials, such as Ximenia americana L., to stimulate the bone repair process has already been studied; however, the synergistic effects of its association with light emitting diode (LED) have not been reported. The present work aims to evaluate the effect of its stem bark extract incorporated into methacrylate gelatin hydrogel (GelMA) on the bone repair process using pure hydrogel and hydrogel associated with LED therapy. For this purpose, the GelMA hydrogel loaded with Ximenia americana L. extract (steam bark) was produced, characterized and applied in animal experiments. The tests were performed using 50 male Wistar rats (divided into 5 groups) submitted to an induced tibia diaphyseal fracture. The therapy effects were verified for a period of 15 and 30 days of treatment using histological analysis and Raman spectroscopy. After 15 days of induced lesion/treatment, the new bone formation was significantly higher in the GXG (GelMA + X. americana L.) group compared to the control group (p < 0.0001). After 30 days, a statistically significant difference was observed when comparing the GXLEDG (GelMA + X. americana L. + LED) and the control group (p < 0.0001), the GXG and the control group (p < 0.001), and when comparing the GG, GXG (p < 0.005) and GXLEDG (p < 0.001) groups. The results shows that the Ximenia americana L. stem extract incorporated into GelMA hydrogel associated with LED therapy is a potentiator for animal bone repair. Full article

Lipid nanoparticles (LNPs) are spherical vesicles composed of ionizable lipids that are neutral at physiological pH. Despite their benefits, unmodified LNP drug delivery systems have substantial drawbacks, including a lack of targeted selectivity, a short blood circulation period, and in vivo instability. lipid–polymer hybrid nanoparticles (LPHNPs) are the next generation of nanoparticles, having the combined benefits of polymeric nanoparticles and liposomes. LPHNPs are being prepared from both natural and synthetic polymers with various techniques, including one- or two-step methods, emulsification solvent evaporation (ESE) method, and the nanoprecipitation method. Varieties of LPHNPs, including monolithic hybrid nanoparticles, core–shell nanoparticles, hollow core–shell nanoparticles, biomimetic lipid–polymer hybrid nanoparticles, and polymer-caged liposomes, have been investigated for various drug delivery applications. However, core–shell nanoparticles having a polymeric core surrounded by a highly biocompatible lipid shell are the most commonly explored LPHNPs for the treatment of various diseases. In this review, we will shed light on the composition, methods of preparation, classification, surface functionalization, release mechanism, advantages and disadvantages, patents, and clinical trials of LPHNPs, with an emphasis on core–shell-structured LPHNPs. Full article

Chemotherapy (CT) plays an important role in the antitumor process, but the unsatisfactory therapeutic efficacy and the obvious toxic side effects of CT seriously restrict its application. To overcome the limitations of CT, the strategy of chemotherapy enhanced by chemodynamic therapy (CDT) and photothermal therapy (PTT) has been considered a promising approach to improve the anticancer effect. Herein, a novel GSH-activatable Cu²⁺-Quercetin network (QC) was synthesized via a convenient strategy to load Au nanoparticles (NPs) and DOX, named QCDA, for the synergistic therapy of CT/CDT/PTT. The results showed that QCDA exhibited GSH-sensitive degradation and “cargos” release in cancer cells, and then PTT and CDT caused by Au NPs and Cu⁺ significantly enhanced the CT effect of DOX and Quercetin on anticancer. More importantly, the PTT and depleted GSH accelerated the Fenton-like ionization process resulting in facilitating the CDT efficiency. Collectively, the multi-mode synergistic strategy of CT/CDT/PTT, which showed an excellent therapeutic effect, maybe a potential therapeutic pathway for anticancer. Full article

The objective of this study is to clarify the effect of restoring the lowered masticatory muscle functional pressure and correcting bilateral differences in masticatory muscle functional pressure on jawbone growth during growth and development with a quantitative evaluation of the changes in the micro/nanostructural characteristics of entheses. Male Wistar rats aged 4 weeks were divided into an experimental group injected with a botulinum toxin serotype A (BoNT/A) formulation to reduce muscle function (BTX group) and a control group (CTRL group). They were euthanised after 6, 8, 10, 12, and 16 weeks after measuring the difference between the midline of the upper and lower incisors. The mandibles were harvested for histological examination, second harmonic generation imaging, and the quantitative evaluation of biological apatite (BAp) crystal alignment. The midline difference decreased with age in weeks. In rats from 6 weeks after BoNT/A administration to 12 weeks after administration, the collagen fibre bundle diameter was significantly smaller in the BTX group; the difference between the two groups decreased with increasing age. BAp crystal alignment was significantly different on the x-axis and the y-axis on the BTX group from 6 weeks after BoNT/A administration to 10 weeks after administration. Asymmetry of mandibular bone formation caused by load imbalance during growth could be corrected by the adjustment of the function of the masseter muscle on either side. Full article

Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines. Full article

A nanosuspension of Artemisia absinthium extract was formulated and characterized for the enhancement of bioavailability and better hepatoprotective efficacy. The nanosuspension of A. absinthium extract was formulated using an antisolvent precipitation technique, and various formulation parameters were optimized using response surface methodology (RSM). The optimized nanosuspension was characterized using AFM and FT–IR spectroscopy. The drug-release profile and oral bioavailability of the optimized nanosuspension were assessed with reference to coarse suspension. The DPPH radical scavenging method was used to measure the nanosuspension’s antioxidant activity, and its in vivo hepatoprotective potential was assessed against CCl4-induced hepatic injury in rats. The developed optimized nanosuspension had suitable zeta potential of −11.9 mV, PDI of 0.285, and mean particle size of 253.8 nm. AFM study demonstrated a homogeneous population of nanoparticles with average size of 25 nm. The formulated nanosuspension of A. absinthium showed faster dissolution rate and 1.13-fold enhanced bioavailability as compared to the coarse suspension (plant extract). Furthermore, the nanoformulation had stronger antioxidant and hepatoprotective potential as compared to the unprocessed coarse extract. These results demonstrated that nanosuspension is a promising strategy for improving the oral bioavailability and bioactivities of A. absinthium extract. Full article

Despite great success with artificial implants for the human body, modern implants cannot solve major health problems. The reason is an immune reaction of organisms to artificial implants, known as the foreign body reaction. We have found a way to avoid or decrease the foreign body reaction. The surface of an artificial implant is modified with condensed aromatic structures containing free radicals, which provide a covalent attachment of host proteins in a native conformation. The total protein coverage prevents the direct contact of immune cells with the implant surface, and the immune cells are not activated. As a result, the immune response of the organism is not generated, and the artificial implant is not isolated from the tissue; there is no collagen capsule, low activity of macrophages, low cell proliferation, and low inflammatory activity. Full article