Development of bilayer tissue-engineered scaffolds: combination of 3D printing and electrospinning methodologies.

Although different fabrication methods and biomaterials are used in scaffold development, hydrogels and electrospun materials that provide the closest environment to the extracellular matrix have recently attracted considerable interest in tissue engineering applications. However, some of the limitations encountered in the application of these methods alone in scaffold fabrication have increased the tendency to use these methods together. In this study, a bilayer scaffold was developed using 3D-printed gelatin methacryloyl (GelMA) hydrogel containing ciprofloxacin (CIP) and electrospun polycaprolactone (PCL)-collagen (COL) patches. The bilayer scaffolds were characterized in terms of chemical, morphological, mechanical, swelling, and degradation properties; drug release, antibacterial properties, and cytocompatibility of the scaffolds were also studied. In conclusion, bilayer GelMA-CIP/PCL-COL scaffolds, which exhibit sufficient porosity, mechanical strength, and antibacterial properties and also support cell growth, are promising potential substitutes in tissue engineering applications.

Correction to "Design of Cinnamaldehyde- and Gentamicin-Loaded Double-Layer Corneal Nanofiber Patches with Antibiofilm and Antimicrobial Effects".

[This corrects the article DOI: 10.1021/acsomega.3c00914.].

Whey protein-loaded 3D-printed poly (lactic) acid scaffolds for wound dressing applications.

Chronic skin wounds pose a global clinical challenge, necessitating effective treatment strategies. This study explores the potential of 3D printed Poly Lactic Acid (PLA) scaffolds, enhanced with Whey Protein Concentrate (WPC) at varying concentrations (25, 35, and 50% wt), for wound healing applications. PLA's biocompatibility, biodegradability, and thermal stability make it an ideal material for medical applications. The addition of WPC aims to mimic the skin's extracellular matrix and enhance the bioactivity of the PLA scaffolds. Fourier Transform Infrared Spectroscopy (FTIR) results confirmed the successful loading of WPC into the 3D printed PLA-based scaffolds. Scanning Electron Microscopy (SEM) images revealed no significant differences in pore size between PLA/WPC scaffolds and pure PLA scaffolds. Mechanical strength tests showed similar tensile strength between pure PLA and PLA with 50% WPC scaffolds. However, scaffolds with lower WPC concentrations displayed reduced tensile strength. Notably, all PLA/WPC scaffolds exhibited increased strain at break compared to pure PLA. Swelling capacity was highest in PLA with 25% WPC, approximately 130% higher than pure PLA. Scaffolds with higher WPC concentrations also showed increased swelling and degradation rates. Drug release was found to be prolonged with increasing WPC concentration. After seven days of incubation, cell viability significantly increased in PLA with 50% WPC scaffolds compared to pure PLA scaffolds. This innovative approach could pave the way for personalized wound care strategies, offering tailored treatments and targeted drug delivery. However, further studies are needed to optimize the properties of these scaffolds and validate their effectiveness in clinical settings. .

A new strategy for the treatment of middle ear infection using ciprofloxacin/amoxicillin-loaded ethyl cellulose/polyhydroxybutyrate nanofibers.

A middle ear infection occurs due to the presence of several microorganisms behind the eardrum (tympanic membrane) and is very challenging to treat due to its unique location and requires a well-designed treatment. If not treated properly, the infection can result in severe symptoms and unavoidable side effects. In this study, excellent biocompatible ethyl cellulose (EC) and biodegradable polyhydroxybutyrate (PHB) biopolymer were used to fabricate drug-loaded nanofiber scaffolds using an electrospinning technique to overcome antibiotic overdose and insufficient efficacy of drug release during treatment. PHB polymer was produced from Halomonas sp., and the purity of PHB was found to around be 90 %. Additionally, ciprofloxacin (CIP) and amoxicillin (AMX) are highly preferable since both drugs are highly effective against gram-negative and gram-positive bacteria to treat several infections. Obtained smooth nanofibers were between 116.24 and 171.82 nm in diameter and the addition of PHB polymer and antibiotics improved the morphology of the nanofiber scaffolds. Thermal properties of the nanofiber scaffolds were tested and the highest Tg temperature resulted at 229 °C. The mechanical properties of the scaffolds were tested, and the highest tensile strength resulted in 4.65 ± 6.33 MPa. Also, drug-loaded scaffolds were treated against the most common microorganisms that cause the infection, such as S.aureus, E.coli, and P.aeruginosa, and resulted in inhibition zones between 10 and 21 mm. MTT assay was performed by culturing human adipose-derived mesenchymal stem cells (hAD MSCs) on the scaffolds. The morphology of the hAD MSCs' attachment was tested with SEM analysis and hAD MSCs were able to attach, spread, and live on each scaffold even on the day of 7. The cumulative drug release kinetics of CIP and AMX from drug-loaded scaffolds were analysed in phosphate-buffered saline (pH: 7.4) within different time intervals of up to 14 days using a UV spectrophotometer. Furthermore, the drug release showed that the First-Order and Korsmeyer-Peppas models were the most suitable kinetic models. Animal testing was performed on SD rats, matrix and collagen deposition occurred on days 5 and 10, which were observed using Hematoxylin-eosin and Masson's trichrome staining. At the highest drug concentration, a better repair effect was observed. Results were promising and showed potential for novel treatment.

Three-Dimensional-Printed GelMA-KerMA Composite Patches as an Innovative Platform for Potential Tissue Engineering of Tympanic Membrane Perforations.

Tympanic membrane (TM) perforations, primarily induced by middle ear infections, the introduction of foreign objects into the ear, and acoustic trauma, lead to hearing abnormalities and ear infections. We describe the design and fabrication of a novel composite patch containing photocrosslinkable gelatin methacryloyl (GelMA) and keratin methacryloyl (KerMA) hydrogels. GelMA-KerMA patches containing conical microneedles in their design were developed using the digital light processing (DLP) 3D printing approach. Following this, the patches were biofunctionalized by applying a coaxial coating with PVA nanoparticles loaded with gentamicin (GEN) and fibroblast growth factor (FGF-2) with the Electrohydrodynamic Atomization (EHDA) method. The developed nanoparticle-coated 3D-printed patches were evaluated in terms of their chemical, morphological, mechanical, swelling, and degradation behavior. In addition, the GEN and FGF-2 release profiles, antimicrobial properties, and biocompatibility of the patches were examined in vitro. The morphological assessment verified the successful fabrication and nanoparticle coating of the 3D-printed GelMA-KerMA patches. The outcomes of antibacterial tests demonstrated that GEN@PVA/GelMA-KerMA patches exhibited substantial antibacterial efficacy against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, cell culture studies revealed that GelMA-KerMA patches were biocompatible with human adipose-derived mesenchymal stem cells (hADMSC) and supported cell attachment and proliferation without any cytotoxicity. These findings indicated that biofunctional 3D-printed GelMA-KerMA patches have the potential to be a promising therapeutic approach for addressing TM perforations.

Osteoblastic Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth by Probiotic Hydroxyapatite.

OBJECTIVE: Multipotent cells derived from human exfoliated deciduous teeth (SHED) possess the ability to differentiate into various cell types, including osteoblasts. This study aims to simulate the growth induction and osteogenic differentiation of SHED cells using probiotics and their resultant biomaterials. MATERIALS AND METHODS: This experimental study proceeded in two stages. Initially, we evaluated the effect of autoclaved nutrient agar (NA) grown probiotic Bacillus coagulans (B. coagulans) on the SHED and MG-63 cell lines. Subsequently, probiotics grown on the Pikovskaya plus urea (PVKU) medium and their synthesised hydroxyapatite (HA) were identified using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and Fourier transform infrared spectroscopy (FTIR), and then used to stimulate growth and osteogenic differentiation of the SHED cell line. Osteoblast cell differentiation was assessed by morphological changes, the alkaline phosphatase (ALP) assay, and alizarin red staining. RESULTS: There was a substantial increase in SHED cell growth of about 14 and 33% due to probiotics grown on NA and PVKU medium, respectively. The PVKU grown probiotics enhanced growth and induced stem cell differentiation due to HA content. Evidence of this differentiation was seen in the morphological shift from spindle to osteocyte-shaped cells after five days of incubation, an increase in ALP level over 21 days, and detection of intracellular calcium deposits through alizarin red staining-all indicative of osteoblast cell development. CONCLUSION: The osteogenic differentiation process in stem cells, improved by the nano-HA-containing byproducts of probiotic bacteria in the PVKU medium, represents a promising pathway for leveraging beneficial bacteria and their synthesised biomaterials in tissue engineering.

Fabrication of ethosuximide loaded alginate/polyethylene oxide scaffolds for epilepsy research using 3D-printing method.

Epilepsy is a medical condition that causes seizures and impairs the mental and physical activities of patients. Unfortunately, over one-third of patients do not receive adequate relief from oral Antiepileptic Drugs (AEDs) and continue to experience seizures. In addition to that, long term usage of Antiepileptic Drugs can cause a range of side effects. To overcome this problem, the precision of 3D printing technology is combined with the controlled release capabilities of biodegradable polymers, allowing for tailored and localized AED delivery to specific seizure sites. As a result of this novel technique, therapeutic outcomes can be enhanced, side effects of AEDs are minimized, and patient-specific dosage forms can be created. This study focused on the use of ethosuximide, an antiepileptic drug, at different concentrations (10, 13, and 15 mg) loaded into 3D-printed sodium alginate and polyethylene oxide scaffolds. The scaffolds contained varying concentrations (0.25%, 0.50%, and 0.75% w/v) and had varying pores created by 3D patterning sizes from 159.86 ± 19.9 µm to 240.29 ± 10.7 µm to optimize the releasing system for an intracranial administration. The addition of PEO changed the Tg and Tm temperatures from 65°C to 69°C and from 262°C to 267°C, respectively. Cytotoxicity assays using the human neuroblastoma cell line (SH-SY5Y) showed that cell metabolic activity reached 130% after 168 h, allowing the cells to develop into mature neural cells. In vitro testing demonstrated sustained ethosuximide release lasting 2 hours despite crosslinking with 3% CaCl2. The workpaves the way for the use of ethosuximide -loaded scaffolds for treating epilepsy.

Advanced Applications of Silk-Based Hydrogels for Tissue Engineering: A Short Review.

Silk has been consistently popular throughout human history due to its enigmatic properties. Today, it continues to be widely utilized as a polymer, having first been introduced to the textile industry. Furthermore, the health sector has also integrated silk. The Bombyx mori silk fibroin (SF) holds the record for being the most sustainable, functional, biocompatible, and easily produced type among all available SF sources. SF is a biopolymer approved by the FDA due to its high biocompatibility. It is versatile and can be used in various fields, as it is non-toxic and has no allergenic effects. Additionally, it enhances cell adhesion, adaptation, and proliferation. The use of SF has increased due to the rapid advancement in tissue engineering. This review comprises an introduction to SF and an assessment of the relevant literature using various methods and techniques to enhance the tissue engineering of SF-based hydrogels. Consequently, the function of SF in skin tissue engineering, wound repair, bone tissue engineering, cartilage tissue engineering, and drug delivery systems is therefore analysed. The potential future applications of this functional biopolymer for biomedical engineering are also explored.

Fucoidan-loaded electrospun Polyvinyl-alcohol/Chitosan nanofibers with enhanced antibacterial activity for skin tissue engineering.

The polymeric nanofiber may interact and control certain regeneration processes at the molecular level to repair damaged tissues. This research focuses on the development of characterization and antibacterial capabilities of polyvinyl alcohol (PVA)/chitosan (CS) nanofibres containing fucoidan (FUC) for tissue engineering as a skin tissue substitute. A control group consisting of 13% PVA/(0.1)% CS nanofiber was prepared. To confer antibacterial properties to the nanofiber, 10, 20, and 30 mg of FUC were incorporated into this control group. The scanning electron microscope (SEM) proved the homogeneous and beadless structures of the nanofibers. The antibacterial activity of the 13% PVA/(0.1)% CS/(10, 20, 30) FUC was tested against the S.aureus and E.coli and the results showed that with FUC addition, the antibacterial activities of the nanofibers increased. The biocompatibility test was performed with a fibroblast cell line for 1, 3, and 7 days of incubation and the results demonstrated that FUC addition enhanced the bioactivity of the 13% PVA/(0.1)% CS nanofibers. In addition, the biocompatibility results showed that 13% PVA/(0.1)% CS/10 FUC had the highest viability value for all incubation periods compared to the others. In addition, the tensile test results showed that; the maximum tensile strength value was observed for 13% PVA/(0.1)% CS/10 FUC nanofibers.

Development of gallic acid-loaded ethylcellulose fibers as a potential wound dressing material.

In this study, novel fibers were designed based on ethylcellulose (EC), loaded with different concentrations of gallic acid (GA) using the electrospinning technique, in order to investigate the potential of these materials as wound dressings. The chemical structure and morphology, along with the antimicrobial and biocompatibility tests of the EC_GA fibers were investigated. To observe the chemical interactions between the components, fourier transform infrared spectroscopy (FTIR) was used. The morphological analyzes were performed using scanning electron microscope (SEM). The uniaxial tensile test machine was used to obtain mechanical performance of the fibers. MTT assay was applied to get the biocompatibility properties of the fibers and antimicrobial test was applied to obtain the antimicrobial activity of the fibers. Based on the obtained results, the highest viability value of 67.4 % was obtained for 10%EC_100GA on the third day of incubation, demonstrating that with the addition of a higher concentration of GA, the cell viability increases. The antimicrobial tests, evaluated against Staphylococcus (S.) aureus, Escherichia (E.) coli, Pseudomonas (Ps.) aeruginosa and Candida (C.) albicans, showed a >90 % microbial reduction capacity correlated with a logarithmic reduction ranging from 0.63 to 1, for 10%EC_100 GA. In vitro release tests of GA from the fibers showed that GA was totally released from 10%EC_100 GA fibers after 2880 min, demonstrating a controlled release profile. These findings demonstrated that EC_GA fibers may be suitable for application in biomedical fields such as wound dressing materials. However, further studies should be performed to increase the biocompatibility properties of the fibers.

Synthesis, physicochemical characteristics, cytocompatibility, and antibacterial properties of iron-doped biphasic calcium phosphate nanoparticles with incorporation of silver.

The application of biphasic calcium phosphate (BCP) in tissue engineering and regenerative medicine has been widely explored due to its extensively documented multi-functionality. The present study attempts to synthesize a new type of BCP nanoparticles, characterised with favourable cytocompatibility and antibacterial properties via modifications in their structure, functionality and assemblage, using dopants. In this regard, this study initially synthesized iron-doped BCP (FB) nanoparticles with silver subsequently incorporated into FB nanoparticles to create a nanostructured composite (FBAg). The FB and FBAgnanoparticles were then characterized using Fourier transform infrared spectroscopy, x-ray diffraction, ultraviolet-visible spectroscopy, and x-ray photoelectron spectroscopy. The results showed that silver was present in the FBAgnanoparticles, with a positive correlation observed between increasing AgNO3concentrations and increasing shape irregularity and reduced particle size distribution. Additionally, cell culture tests revealed that both FB and FBAgnanoparticles were compatible with bone marrow-derived mesenchymal stem cells (hBMSCs). The antibacterial activity of the FBAgnanoparticles was also tested using Gram-negativeE. coliand Gram-positiveS. aureus, and was found to be effective against both bacteria. The inhibition rates of FBAgnanoparticles againstE. coliandS. aureuswere 33.78 ± 1.69-59.03 ± 2.95%, and 68.48 ± 4.11-89.09 ± 5.35%, respectively. These findings suggest that the FBAgnanoparticles have potential use in future biomedical applications.

Biocompatible polyvinyl alcohol nanofibers loaded with amoxicillin and salicylic acid to prevent wound infections.

Diabetic wounds are one of the most challenging clinical conditions in diabetes, necessitating the development of new treatments to foster healing and prevent microbial contamination. In this study, polyvinyl alcohol was used as a matrix polymer, and amoxicillin (AMX) and salicylic acid (SA) were selected as bioactive compounds with antimicrobial (with AMX) and anti-inflammatory action (with SA) to obtain innovative drug-loaded electrospun nanofiber patches for the management of diabetic wounds. Scanning electron microscope images revealed the uniform and beadless structure of the nanofiber patches. Mechanical tests indicated that AMX minimally increased the tensile strength, while SA significantly reduced it. The patches demonstrated effective antibacterial activity against both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) strains. The potential of these patches in the development of novel wound dressings is highlighted by the excellent biocompatibility with fibroblast cells maintained for up to 7 d.

Design of Cinnamaldehyde- and Gentamicin-Loaded Double-Layer Corneal Nanofiber Patches with Antibiofilm and Antimicrobial Effects.

In this study, two-layer poly(vinyl alcohol)/gelatin (PVA/GEL) nanofiber patches containing cinnamaldehyde (CA) in the first layer and gentamicin (GEN) in the second layer were produced by the electrospinning method. The morphology, chemical structures, and thermal temperatures of the produced pure (PVA/GEL), CA-loaded (PVA/GEL/CA), GEN-loaded (PVA/GEL/GEN), and combined drug-loaded (PVA/GEL/CA/GEN) nanofiber patches were determined by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and differential scanning calorimetry, respectively. Their mechanical properties, swelling and degradation behavior, and drug release kinetics were investigated. SEM images showed that both drug-free and drug-loaded nanofiber patches possess smooth and monodisperse structures, and nanofiber size increase occurred as the amount of drug increased. The tensile test results showed that the mechanical strength decreased as the drug was loaded. According to the drug release results, CA release ended at the 96th hour, while GEN release continued until the 264th hour. The antibacterial and antibiofilm activities of PVA/GEL, PVA/GEL/CA, PVA/GEL/GEN, and PVA/GEL/CA/GEN nanofiber patches against Pseudomonas aeruginosa and Staphylococcus aureus were evaluated. Results showed that PVA/GEL/GEN and PVA/GEL/CA/GEN nanofiber patches have excellent antibacterial and antibiofilm activities. Moreover, all materials were biocompatible, with no cytotoxic effects in the mammalian cell model for 8 days. PVA/GEL/GEN nanofiber patches were the most promising material for a high cell survival ratio, which was confirmed by SEM images. This research aims to develop an alternative method to stop and treat the rapid progression of bacterial keratitis.

3D printed Styrax Liquidus (Liquidambar orientalis Miller)-loaded poly (L-lactic acid)/chitosan based wound dressing material: Fabrication, characterization, and biocompatibility results.

The medicinal plant of Styrax liquidus (ST) (sweet gum balsam) which extracted from Liquidambar orientalis Mill tree, was loaded into the 3D printed polylactic acid (PLA)/chitosan (CS) based 3D printed scaffolds to investigate its wound healing and closure effect, in this study. The morphological and chemical properties of the ST loaded 3D printed scaffolds with different concentrations (1 %, 2 %, and 3 % wt) were investigated by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR), respectively. In addition, the mechanical and thermal properties of the materials were investigated by Tensile test and Differential Scanning Calorimetry (DSC), respectively. The antimicrobial activities of the ST loaded 3D printed scaffolds and their incubation media in the PBS (pH 7.4, at 37 °C for 24 h) were investigated on two Gram-positive and two Gram-negative standard pathogenic bacteria with the agar disc diffusion method. The colorimetric MTT assay was used to determine the cell viability of human fibroblast cells (CCD-1072Sk) incubated with free ST, ST loaded, and unloaded 3D printed scaffolds. The 1 % and 2 % (wt) ST loaded PLA/CS/ST 3D printed scaffolds showed an increase in the cell number. Annexin V/PI double stain assay was performed to test whether early or late apoptosis was induced in the PLA/CS/1 % ST and PLA/CS/2 % ST loaded groups and the results were consistent with the MTT assay. Furthermore, a wound healing assay was carried out to investigate the effect of ST loaded 3D printed scaffolds on wound healing in CCD-1072Sk cells. The highest wound closure compared to the control group was observed on cells treated with PLA/CS/1 % ST for 72 h. According to the results, novel biocompatible ST loaded 3D printed scaffolds with antimicrobial effect can be used as wound healing material for potential tissue engineering applications.

An Eco-Friendly Process to Extract Hydroxyapatite from Sheep Bones for Regenerative Medicine: Structural, Morphologic and Electrical Studies.

Hydroxyapatite (HA) promotes excellent bone regeneration in bone-tissue engineering, due to its similarity to bone mineral and its ability to connect to living tissues. These factors promote the osteointegration process. This process can be enhanced by the presence of electrical charges, stored in the HA. Furthermore, several ions can be added to the HA structure to promote specific biological responses, such as magnesium ions. The main objective of this work was to extract hydroxyapatite from sheep femur bones and to study their structural and electrical properties by adding different amounts of magnesium oxide. The thermal and structural characterizations were performed using DTA, XRD, density, Raman spectroscopy and FTIR analysis. The morphology was studied using SEM, and the electrical measurements were registered as a function of frequency and temperature. Results show that: (i) an increase of MgO amount indicates that the solubility of MgO is below 5%wt for heat treatments at 600 °C; (ii) the rise of MgO content increases the capacity for electrical charge storage; (iii) sheep hydroxyapatite presents itself as a natural source of hydroxyapatite, environmentally sustainable and low cost, and promising for applications in regenerative medicine.

Fabrication and optimization of 3D printed gelatin methacryloyl microneedle arrays based on vat photopolymerization.

Microneedles (MNs) are micrometer-sized arrays that can penetrate the skin in a minimally invasive manner; these devices offer tremendous potential for the transdermal delivery of therapeutic molecules. Although there are many conventional techniques for manufacturing MNs, most of them are complicated and can only fabricate MNs with specific geometries, which restricts the ability to adjust the performance of the MNs. Herein, we present the fabrication of gelatin methacryloyl (GelMA) MN arrays using the vat photopolymerization 3D printing technique. This technique allows for the fabrication of high-resolution and smooth surface MNs with desired geometries. The existence of methacryloyl groups bonded to the GelMA was verified by 1H NMR and FTIR analysis. To examine the effects of varying needle heights (1000, 750, and 500 µm) and exposure times (30, 50, and 70 s) on GelMA MNs, the height, tip radius, and angle of the needles were measured; their morphological and mechanical properties were also characterized. It was observed that as the exposure time increased, the height of the MNs increased; moreover, sharper tips were obtained and tip angles decreased. In addition, GelMA MNs exhibited good mechanical performance with no breakage up to 0.3 mm displacement. These results indicate that 3D printed GelMA MNs have great potential for transdermal delivery of various therapeutics.

A Novel Approach for the Fabrication of 3D-Printed Dental Membrane Scaffolds including Antimicrobial Pomegranate Extract.

In this study, a dental membrane scaffold was fabricated using a 3D printing technique, and the antimicrobial effect of pomegranate seed and peel extract were investigated. For the production of the dental membrane scaffold, a combination of polyvinyl alcohol, starch, and pomegranate seed and peel extracts was used. The aim of the scaffold was to cover the damaged area and aid in the healing process. This can be achieved due to the high antimicrobial and antioxidant content of pomegranate seed and peel extracts (PPE: PSE). Moreover, the addition of starch and PPE: PSE improved the biocompatibility of the scaffold, and their biocompatibility was tested using human gingival fibroblast (HGF) cells. The addition of PPE: PSE into the scaffolds resulted in a significant antimicrobial effect on S. aureus and E. faecalis bacteria. Moreover, different concentrations of starch (1%, 2%, 3% w/v) and pomegranate peel and seed extract (3%, 5%, 7%, 9%, and 11% PE v/v) were analyzed to obtain the ideal dental membrane structure. The optimum starch concentration was chosen as 2% w/v due to it giving the scaffold the highest mechanical tensile strength (23.8607 ± 4.0796 MPa). The pore sizes of each scaffold were studied by SEM analysis, and pore sizes were arranged between 155.86 and 280.96 µm without any plugging problems. Pomegranate seed and peel extracts were obtained by applying the standard extraction method. High-performance liquid chromatography was performed using the diode-array detection (HPLC-DAD) technique to analyze the phenolic content of the pomegranate seed and peel extracts. Two phenolic components of the pomegranate seed and peel extracts were investigated in the following amounts: fumaric acid (17.56 µg analyte/mg extract) and quinic acid (18.79 µg analyte/mg extract) in pomegranate seed extract and fumaric acid (26.95 µg analyte/mg extract) and quinic acid (33.79 µg analyte/mg extract) in pomegranate peel extract.