Development of Super-Paramagnetic Iron Oxide Nanoparticle-Coated Melt Electrowritten Scaffolds for Biomedical Applications.

Polycaprolactone (PCL) is usually the material chosen for melt electrowriting (MEW) due to its biocompatibility, mechanical strength, and melt processability. This work first investigates the effect of different processing parameters to obtain optimum PCL-MEW scaffolds. Secondly, to increase PCL`s hydrophilicity and cell affinity, and to enable coating with superparamagnetic iron oxide nanoparticles (SPIONs) and silica-coated-SPIONs (Si-SPIONs), the scaffolds are modified with alkaline surface treatment. Finally, SPIONs and Si-SPIONs are successfully coated on MEW scaffolds. Results show that reproducible scaffolds are fabricated. Additionally, the alkaline treatment does not change the three-dimensional morphology of scaffolds while reducing fiber diameter. Furthermore, SEM images and ATR-FTIR results confirmed that SPIONs and Si-SPIONs-were coated on scaffolds. A cytocompatibility assay showed a non-toxic effect on MG-63 osteoblast-like cells in all scaffolds. Additionally, higher MC3T3-E1 pre-osteoblastic cell adhesion efficiency and proliferation are achieved for the alkaline-treated scaffolds and SPIONs/Si-SPIONs-coated scaffolds. All samples demonstrated the ability to generate heat, useful for hyperthermia-treatment, when subjected to an alternating magnetic field. Overall, the findings suggest that the strategy of coating PCL-MEW scaffolds with SPIONs/Si-SPIONs has the potential to improve scaffold performance for biomedical applications, especially for producing magnetically responsive MEW scaffolds.

Multifunctional Chitosan Scaffold Platforms Loaded with Natural Polyphenolic Extracts for Wound Dressing Applications.

Chitosan (CS)-based scaffolds loaded with Pinus radiata extract bark (PE) and grape seed extract (GSE) were successfully developed for wound dressing applications. The effects of incorporating GSE and PE in CS scaffolds were investigated in relation to their physicochemical and biological properties. All scaffolds exhibited porous structures with the ability to absorb more than 70 times their weight when contacted with blood and phosphate buffer solution. The incorporation of GSE and PE into the CS scaffolds increased their blood absorption ability and degradation rates over time. All scaffolds showed a clotting ability above 95%, with their surfaces being favorable for red blood cell attachment. Both GSE and PE were released from the CS scaffolds in a sustained manner. Scaffolds loaded with GSE and PE inhibited the bacterial activity of S. aureus and E. coli by 40% and 44% after 24 h testing. In vitro cell viability studies demonstrated that all scaffolds were nontoxic to HaCaT cells. Importantly, the addition of GSE and PE further increased cell viability compared to that of the CS scaffold. This study provides a new synthesis method to immobilize GSE and PE on CS scaffolds, enabling the formation of novel material platforms with a high potential for wound dressing applications.

3D-Printed Multifunctional Hydrogels with Phytotherapeutic Properties: Development of Essential Oil-Incorporated ALG-XAN Hydrogels for Wound Healing Applications.

This study aimed to develop three-dimensional (3D)-printed hydrogels containing phytotherapeutic agents as multifunctional wound dressings. In this regard, 3D-printed sodium alginate (ALG)-xanthan gum (XAN) hydrogels incorporated with different clove essential oil (CLV) concentrations were produced by the extrusion-based 3D-printing technology. Rheology measurements, filament fusion, and filament collapse analyses indicated that XAN's blending overcame the challenges associated with ALG's printability and shape fidelity. Attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectra and total phenolic content assay confirmed the presence of CLV in the 3D-printed hydrogels. Additionally, the releasing profile showed that CLV exhibited long-term release for up to 28 days. Furthermore, the incorporation of CLV increased 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging while reducing the S. aureus and E. coli relative bacterial viability; thereby, the CLV incorporation enhanced the 3D-printed ALG-XAN hydrogel antioxidant and antibacterial activity. In addition, anti-inflammatory activity was assessed using Raw 264.7 macrophage-like cells, and the results demonstrated that CLV reduced nitric oxide (NO) concentration in medium, indicating a potential anti-inflammatory effect. Moreover, in vitro cytotoxicity results showed that the incorporation of CLV has no toxic effect on NHDF cells, whereas the proliferation of NHDF cells exhibited a dose-dependent response. In conclusion, the present study shows not only the development of a new ALG-XAN biomaterial ink but also the potential benefit of natural phytotherapeutics incorporated into 3D-printed hydrogels as a multifunctional wound dressing.

In vitro cytocompatibility and antibacterial studies on biodegradable Zn alloys supplemented by a critical assessment of direct contact cytotoxicity assay.

In vitro cytotoxicity assessment is indispensable in developing new biodegradable implant materials. Zn, which demonstrates an ideal corrosion rate between Mg- and Fe-based alloys, has been reported to have excellent in vivo biocompatibility. Therefore, modifications aimed at improving Zn's mechanical properties should not degrade its biological response. As sufficient strength, ductility and corrosion behavior required of load-bearing implants has been obtained in plastically deformed Zn-3Ag-0.5Mg, the effect of simultaneous Ag and Mg additions on in vitro cytocompatibility and antibacterial properties was studied, in relation to Zn and Zn-3Ag. Direct cell culture on samples and indirect extract-based tests showed almost no significant differences between the tested Zn-based materials. The diluted extracts of Zn, Zn-3Ag, and Zn-3Ag-0.5Mg showed no cytotoxicity toward MG-63 cells at a concentration of ≤12.5%. The cytotoxic effect was observed only at high Zn2+ ion concentrations and when in direct contact with metallic samples. The highest LD50 (lethal dose killing 50% of cells) of 13.4 mg/L of Zn2+ ions were determined for the Zn-3Ag-0.5Mg. Similar antibacterial activity against Escherichia coli and Staphylococcus aureus was observed for Zn and Zn alloys, so the effect is attributed mainly to the released Zn2+ ions exhibiting bactericidal properties. Most importantly, our experiments indicated the limitations of water-soluble tetrazolium salt-based cytotoxicity assays for direct tests on Zn-based materials. The discrepancies between the WST-8 assay and SEM observations are attributed to the interference of Zn2+ ions with tetrazolium salt, therefore favoring its transformation into formazan, giving false cell viability quantitative results.

Fabrication of Biocompatible Electrospun Poly(ε-caprolactone)/Gelatin Nanofibers Loaded with Pinus radiata Bark Extracts for Wound Healing Applications.

In this study, poly(ε-caprolactone) (PCL)/gelatin (GEL) electrospun nanofibers loaded with two different concentrations of Pinus radiata bark extracts (PEs) were fabricated via electrospinning for wound healing applications. The effects of incorporating PE into PCL/GEL electrospun nanofibers were investigated regarding their physicochemical properties and in vitro biocompatibility. All electrospun nanofibers showed smooth, uniform, and bead-free surfaces. Their functional groups were detected by ATR-FTIR spectroscopy, and their total phenol content was measured by a Folin-Ciocalteu assay. With PE addition, the electrospun nanofibers exhibited an increase in their wettability and degradation rates over time and a decrease in their tensile stress values from 20 ± 4 to 8 ± 2 MPa for PCL/GEL and PCL/GEL/0.36%PE samples, respectively. PE was also released from the fibrous mats in a rather controlled fashion. The PCL/GEL/0.18%PE and PCL/GEL/0.36%PE electrospun nanofibers inhibited bacterial activity at around 6 ± 0.1% and 23 ± 0.3% against E. coli and 14 ± 0.1% and 18 ± 0.2% against S. aureus after 24 h incubation, respectively. In vitro cell studies showed that PE-loaded electrospun nanofibers enhanced HaCaT cell growth, attachment, and proliferation, favoring cell migration towards the scratch area in the wound healing assay and allowing a complete wound closure after 72 h treatment. These findings suggested that PE-loaded electrospun nanofibers are promising materials for antibiotic-free dressings for wound healing applications.

Melaleuca armillaris Essential Oil as an Antibacterial Agent: The Use of Mesoporous Bioactive Glass Nanoparticles as Drug Carrier.

Bioactive glasses have been proposed for bone tissue engineering due to their excellent biocompatibility and osteo-inductive behaviour. The generation of mesoporous bioactive glass (nano) particles adds a high surface area for the dissolution and release of bioactive ions, and the possibility to load them with different drugs for antibacterial purposes. Essential oils (EO) are an interesting resource for alternative medical therapy, providing antimicrobial compounds that come from organic/natural resources like aromatic plants. Also, a biological polymer, such as chitosan, could be used to control the release of active agents from mesoporous bioactive glass (MBG) loaded particles. This work presents MBG particles with nominal composition (in mol) 60% SiO2, 30% CaO and 10% P2O5, loaded with essential oil of Melaleuca armillaris, which contains 1,8-cineol as the main active component, with an inhibitory in vitro activity against several bacterial species. Also, co-loading with a broad-spectrum antibiotic, namely gentamicin, was investigated. The MBG particles were found to be of around 300nm in diameter and to exhibit highly porous open structure. The release of EO from the particles reached 72% of the initial content after the first 24 h, and 80% at 48 h of immersion in phosphate buffered solution. Also, the MBG particles with EO and EO-gentamicin loading presented in vitro apatite formation after 7 days of immersion in simulated body fluid. The antibacterial tests indicated that the main effect, after 24 h of contact with the bacteria, was reached either for the MBG EO or MBG EO-gentamicin particles against E. coli, while the effect against S. aureus was less marked. The results indicate that MBG particles are highly bioactive with the tested composition and loaded with EO of Melaleuca armillaris. The EO, also combined with gentamicin, acts as an antibacterial agent but with different efficacy depending on the bacteria type.

Antibacterial and antioxidant activity of cinnamon essential oil-laden 45S5 bioactive glass/soy protein composite scaffolds for the treatment of bone infections and oxidative stress.

This study aimed to fabricate cinnamon essential oil (CO)-laden 45S5 bioactive glass (BG)/soy protein (SP) scaffolds exhibiting antioxidant and antibacterial activity. In this regard, 45S5 BG-based scaffolds were produced by the foam replica method, and subsequently the scaffolds were coated with various concentrations of CO (2.5, 5 and 7 (v/v) %) incorporated SP solution. Scanning electron microscopy images revealed that the CO-laden SP effectively attached to the 45S5 BG scaffold struts. The presence of 45S5 BG, SP and CO was confirmed using Fourier transform infrared spectroscopy. Compressive strength results indicated that SP based coatings improved the scaffolds' mechanical properties compared to uncoated BG scaffolds. The loading efficiency and releasing behaviour of the different CO concentrations were tested by gas chromatography-mass spectroscopy and UV-Vis spectroscopy. The results showed that CO incorporated scaffolds have controlled releasing behaviour over seven days. Furthermore, the coating on the scaffold surfaces slightly retarded, but it did not inhibit, the in vitro bioactivity of the scaffolds. Moreover, the antioxidant and antibacterial activity of CO was studied. The free radical scavenging activity measured by DPPH was 5 ± 1, 41 ± 3, 44 ± 1 and 43 ± 1 % for BGSP, CO2.5, CO5 and CO7, respectively. The antioxidant activity was thus enhanced by incorporating CO. Agar diffusion and colony counting results indicated that the incorporation of CO increased the antibacterial activity of scaffolds against S. aureus and E. coli. In addition, cytotoxicity of the scaffolds was investigated using MG-63 osteoblast-like cells. The results showed that the BG-SP scaffold was non-toxic under the investigated conditions, whereas dose-dependent toxicity was observed in CO-laden scaffolds. Considered together, the developed phytotherapeutic agent laden 45S5 BG-based scaffolds are promising for bone tissue engineering exhibiting capability to combat bone infections and to protect against oxidative stress damage.

Fabrication and Characterization of Cinnamaldehyde-Loaded Mesoporous Bioactive Glass Nanoparticles/PHBV-Based Microspheres for Preventing Bacterial Infection and Promoting Bone Tissue Regeneration.

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is considered a suitable polymer for drug delivery systems and bone tissue engineering due to its biocompatibility and biodegradability. However, the lack of bioactivity and antibacterial activity hinders its biomedical applications. In this study, mesoporous bioactive glass nanoparticles (MBGN) were incorporated into PHBV to enhance its bioactivity, while cinnamaldehyde (CIN) was loaded in MBGN to introduce antimicrobial activity. The blank (PHBV/MBGN) and the CIN-loaded microspheres (PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20) were fabricated by emulsion solvent extraction/evaporation method. The average particle size and zeta potential of all samples were investigated, as well as the morphology of all samples evaluated by scanning electron microscopy. PHBV/MBGN/CIN5, PHBV/MBGN/CIN10, and PHBV/MBGN/CIN20 significantly exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli in the first 3 h, while CIN releasing behavior was observed up to 7 d. Human osteosarcoma cell (MG-63) proliferation and attachment were noticed after 24 h cell culture, demonstrating no adverse effects due to the presence of microspheres. Additionally, the rapid formation of hydroxyapatite on the composite microspheres after immersion in simulated body fluid (SBF) during 7 d revealed the bioactivity of the composite microspheres. Our findings indicate that this system represents an alternative model for an antibacterial biomaterial for potential applications in bone tissue engineering.

Cellular Response to Sol-Gel Hybrid Materials Releasing Boron and Calcium Ions.

Poly(dimethylsiloxane) (PDMS)-SiO2-CaO-based hybrid materials prepared by sol-gel have proved to be very promising materials for tissue engineering applications and drug-delivery systems. These hybrids are biocompatible and present osteogenic and bioactive properties supporting osteoblast attachment and bone growth. The incorporation of therapeutic elements in these materials, such as boron (B) and calcium (Ca), was considered in this study as an approach to develop biomaterials capable of stimulating bone regeneration. The main purpose of this work was thus to produce, by sol-gel, bioactive and biocompatible hybrid materials of the PDMS-SiO2-B2O3-CaO system, capable of a controlled Ca and B release. Different compositions with different boron amounts were prepared using the same precursors resulting in different monolithic materials, with distinct structures and microstructures. Structural features were assessed by Fourier transform infrared (FT-IR) spectrometry and solid-state nuclear magnetic resonance (NMR) techniques, which confirmed the presence of hybrid bonds (Si-O-Si) between organic (PDMS) and inorganic phase (tetraethyl orthosilicate (TEOS)), as well as borosiloxane bonds (B-O-Si). From the 11B NMR results, it was found that Ca changes the boron coordination, from trigonal (BO3) to tetrahedral (BO4). Scanning electron microscopy (SEM) micrographs and N2 isotherms showed that the incorporation of boron modifies the material's microstructure by increasing the macroporosity and decreasing the specific surface area (SSA). In vitro tests in simulated body fluid (SBF) showed the precipitation of a calcium phosphate layer on the material surface and the controlled release of therapeutic ions. The cytocompatibility of the prepared hybrids was studied with bone marrow stromal cells (ST-2 cell line) by analyzing the cell viability and cell density. The results demonstrated that increasing the dilution rate of extraction medium from the hybrids leads to improved cell behavior. The relationship between the in vitro response and the structural and microstructural features of the materials was explored. It was shown that the release of calcium and boron ions, determined by the hybrid structure was crucial for the observed cells behavior. Although not completely understood, the encouraging results obtained constitute an incentive for further studies on this topic.

Evaluation of Electrospun Poly(ε-Caprolactone)/Gelatin Nanofiber Mats Containing Clove Essential Oil for Antibacterial Wound Dressing.

The objective of this study was to produce antibacterial poly(ε-caprolactone) (PCL)-gelatin (GEL) electrospun nanofiber mats containing clove essential oil (CLV) using glacial acetic acid (GAA) as a "benign" (non-toxic) solvent. The addition of CLV increased the fiber diameter from 241 ± 96 to 305 ± 82 nm. Aside from this, the wettability of PCL-GEL nanofiber mats was increased by the addition of CLV. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the presence of CLV, and the actual content of CLV was determined by gas chromatography-mass spectrometry (GC-MS). Our investigations showed that CLV-loaded PCL-GEL nanofiber mats did not have cytotoxic effects on normal human dermal fibroblast (NHDF) cells. On the other hand, the fibers exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. Consequently, PCL-GEL/CLV nanofiber mats are potential candidates for antibiotic-free wound healing applications.

Bioactive glass (45S5)-based 3D scaffolds coated with magnesium and zinc-loaded hydroxyapatite nanoparticles for tissue engineering applications.

Bioactive glass (BG)-based scaffolds of 45S5 composition covered with hydroxyapatite nanoparticles loaded with Mg2+, Zn2+ and, both Mg2+ and Zn2+ ions, were developed and tested as materials for tissue engineering applications. The scaffolds were prepared by the foam replica technique and mono- and bi-metal loaded and unloaded hydroxyapatite nanoparticles (HA, Zn-HA, Mg-HA and Mg-Zn-HA) were obtained by an adaptation of the wet chemical deposition method. Coating of BG with these nanoparticles was performed by dip-coating to obtain HA-BG, Zn-HA-BG, Mg-HA-BG and Mg-Zn-HA-BG scaffolds. As predictor of the bone bonding ability of the produced scaffolds, in this study we investigated the formation of an apatite layer on the scaffold surfaces in the presence of simulated body fluid. The cytotoxicity and osteogenic properties of the materials in vitro was evaluated using human osteoblast-like MG-63 cell cultures. The mineralization assay following Kokubo's protocol indicated that bi-metal loaded Mg-Zn-HA-BG scaffolds exhibited higher/faster bioactivity than mono-metal loaded scaffolds while mineralization of HA-BG, Zn-HA-BG and Mg-HA-BG was similar to that of uncoated scaffolds. Moreover, an increase of proliferation of MG-63 cells after 48 h and 7 days was measured by BrdU assays for Mg-Zn-HA-BG scaffolds. In agreement with these results, SEM images confirmed increased interaction between these scaffolds and cells, in comparison to that observed for mono-metal-loaded HA-coated scaffolds. Altogether, the obtained results suggest that nanocrystalline Mg-Zn-HA coatings enhance the biological performance of standard scaffolds of 45S5 BG composition. Thus these novel ion doped HA coated scaffolds are attractive systems for bone tissue engineering.

Physical and Antibacterial Properties of Peppermint Essential Oil Loaded Poly (ε-caprolactone) (PCL) Electrospun Fiber Mats for Wound Healing.

The aim of this study was to fabricate and characterize various concentrations of peppermint essential oil (PEP) loaded on poly(ε-caprolactone) (PCL) electrospun fiber mats for healing applications, where PEP was intended to impart antibacterial activity to the fibers. SEM images illustrated that the morphology of all electrospun fiber mats was smooth, uniform, and bead-free. The average fiber diameter was reduced by the addition of PEP from 1.6 ± 0.1 to 1.0 ± 0.2 µm. Functional groups of the fibers were determined by Raman spectroscopy. Gas chromatography-mass spectroscopy (GC-MS) analysis demonstrated the actual PEP content in the samples. In vitro degradation was determined by measuring weight loss and their morphology change, showing that the electrospun fibers slightly degraded by the addition of PEP. The wettability of PCL and PEP loaded electrospun fiber mats was measured by determining contact angle and it was shown that wettability increased with the incorporation of PEP. The antimicrobial activity results revealed that PEP loaded PCL electrospun fiber mats exhibited inhibition against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. In addition, an in-vitro cell viability assay using normal human dermal fibroblast (NHDF) cells revealed improved cell viability on PCL, PCLPEP1.5, PCLPEP3, and PCLGEL6 electrospun fiber mats compared to the control (CNT) after 48 h cell culture. Our findings showed for the first time PEP loaded PCL electrospun fiber mats with antibiotic-free antibacterial activity as promising candidates for wound healing applications.

Biocompatibility of plasma-treated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofiber mats modified by silk fibroin for bone tissue regeneration.

The objective of this study was to produce biocompatible plasma-treated and silk-fibroin (SF) modified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofiber mats. The mats were plasma-treated using O2 or N2 gas to increase their hydrophilicity followed by SF immobilization for the improvement of biocompatibility. Contact angle measurements and SEM showed increased hydrophilicity and no disturbed morphology, respectively. Cell proliferation assay revealed that SF modification together with N2 plasma (PS/N2) promoted higher osteoblastic (SaOs-2) cell viability. Although, O2 plasma triggered more mineral formation on the mats, it showed poor cell viability. Consequently, the PS/N2 nanofiber mats would be a potential candidate for bone tissue engineering applications.