Enhanced Electroactive Phases of Poly(vinylidene Fluoride) Fibers for Tissue Engineering Applications.

Nanofibrous materials generated through electrospinning have gained significant attention in tissue regeneration, particularly in the domain of bone reconstruction. There is high interest in designing a material resembling bone tissue, and many scientists are trying to create materials applicable to bone tissue engineering with piezoelectricity similar to bone. One of the prospective candidates is highly piezoelectric poly(vinylidene fluoride) (PVDF), which was used for fibrous scaffold formation by electrospinning. In this study, we focused on the effect of PVDF molecular weight (180,000 g/mol and 530,000 g/mol) and process parameters, such as the rotational speed of the collector, applied voltage, and solution flow rate on the properties of the final scaffold. Fourier Transform Infrared Spectroscopy allows for determining the effect of molecular weight and processing parameters on the content of the electroactive phases. It can be concluded that the higher molecular weight of the PVDF and higher collector rotational speed increase nanofibers' diameter, electroactive phase content, and piezoelectric coefficient. Various electrospinning parameters showed changes in electroactive phase content with the maximum at the applied voltage of 22 kV and flow rate of 0.8 mL/h. Moreover, the cytocompatibility of the scaffolds was confirmed in the culture of human adipose-derived stromal cells with known potential for osteogenic differentiation. Based on the results obtained, it can be concluded that PVDF scaffolds may be taken into account as a tool in bone tissue engineering and are worth further investigation.

Development of Poly(methyl methacrylate)/nano-hydroxyapatite (PMMA/nHA) Nanofibers for Tissue Engineering Regeneration Using an Electrospinning Technique.

The study explores the in vitro biocompatibility and osteoconductivity of poly(methyl methacrylate)/nano-hydroxyapatite (PMMA/nHA) composite nanofibrous scaffolds for bone tissue engineering (BTE). Electrospun scaffolds, exhibiting both low and high fiber orientation, were investigated. The inclusion of hydroxyapatite nanoparticles enhances the osteoconductivity of the scaffolds while maintaining the ease of fabrication through electrospinning. SEM analysis confirms the high-quality morphology of the scaffolds, with successful incorporation of nHA evidenced by SEM-EDS and FTIR methods. DSC analysis indicates that nHA addition increases the PMMA glass transition temperature (Tg) and reduces stress relaxation during electrospinning. Furthermore, higher fiber orientation affects PMMA Tg and stress relaxation differently. Biological studies demonstrate the composite material's non-toxicity, excellent osteoblast viability, attachment, spreading, and proliferation. Overall, PMMA/nHA composite scaffolds show promise for BTE applications.

Electrospun Antimicrobial Drug Delivery Systems and Hydrogels Used for Wound Dressings.

Wounds and chronic wounds can be caused by bacterial infections and lead to discomfort in patients. To solve this problem, scientists are working to create modern wound dressings with antibacterial additives, mainly because traditional materials cannot meet the general requirements for complex wounds and cannot promote wound healing. This demand is met by material engineering, through which we can create electrospun wound dressings. Electrospun wound dressings, as well as those based on hydrogels with incorporated antibacterial compounds, can meet these requirements. This manuscript reviews recent materials used as wound dressings, discussing their formation, application, and functionalization. The focus is on presenting dressings based on electrospun materials and hydrogels. In contrast, recent advancements in wound care have highlighted the potential of thermoresponsive hydrogels as dynamic and antibacterial wound dressings. These hydrogels contain adaptable polymers that offer targeted drug delivery and show promise in managing various wound types while addressing bacterial infections. In this way, the article is intended to serve as a compendium of knowledge for researchers, medical practitioners, and biomaterials engineers, providing up-to-date information on the state of the art, possibilities of innovative solutions, and potential challenges in the area of materials used in dressings.

On-Demand Sequential Release of Dual Drug from pH-Responsive Electrospun Janus Nanofiber Membranes toward Wound Healing and Infection Control.

Drugs against bacteria and abnormal cells, such as antibiotics and anticancer drugs, may save human lives. However, drug resistance is becoming more common in the clinical world. Nowadays, a synergistic action of multiple bioactive compounds and their combination with smart nanoplatforms has been considered an alternative therapeutic strategy to fight drug resistance in multidrug-resistant cancers and microorganisms. The present study reports a one-step fabrication of innovative pH-responsive Janus nanofibers loaded with two active compounds, each in separate polymer compartments for synergistic combination therapy. By dissolving one of the compartments from the nanofibers, we could clearly demonstrate a highly yielded anisotropic Janus structure with two faces by scanning electron microscopy (SEM) analysis. To better understand the distinctive attributes of Janus nanofibers, several analytical methods, such as X-ray diffraction (XRD), FTIR spectroscopy, and contact angle goniometry, were utilized to examine and compare them to those of monolithic nanofibers. Furthermore, a drug release test was conducted in pH 7.4 and 6.0 media since the properties of Janus nanofibers correlate significantly with different environmental pH levels. This resulted in the on-demand sequential codelivery of octenidine (OCT) and curcumin (CUR) to the corresponding pH stimulus. Accordingly, the antibacterial properties of Janus fibers against Escherichia coli and Staphylococcus aureus, tested in a suspension test, were pH-dependent, i.e., greater in pH 6 due to the synergistic action of two active compounds, and Eudragit E100 (EE), and highly satisfactory. The biocompatibility of the Janus fibers was confirmed in selected tests.

Nanofibrous Polycaprolactone Membrane with Bioactive Glass and Atorvastatin for Wound Healing: Preparation and Characterization.

Skin wound healing is one of the most challenging processes for skin reconstruction, especially after severe injuries. In our study, nanofiber membranes were prepared for wound healing using an electrospinning process, where the prepared nanofibers were made of different weight ratios of polycaprolactone and bioactive glass that can induce the growth of new tissue. The membranes showed smooth and uniform nanofibers with an average diameter of 118 nm. FTIR and XRD results indicated no chemical interactions of polycaprolactone and bioactive glass and an increase in polycaprolactone crystallinity by the incorporation of bioactive glass nanoparticles. Nanofibers containing 5% w/w of bioactive glass were selected to be loaded with atorvastatin, considering their best mechanical properties compared to the other prepared nanofibers (3, 10, and 20% w/w bioactive glass). Atorvastatin can speed up the tissue healing process, and it was loaded into the selected nanofibers using a dip-coating technique with ethyl cellulose as a coating polymer. The study of the in vitro drug release found that atorvastatin-loaded nanofibers with a 10% coating polymer revealed gradual drug release compared to the non-coated nanofibers and nanofibers coated with 5% ethyl cellulose. Integration of atorvastatin and bioactive glass with polycaprolactone nanofibers showed superior wound closure results in the human skin fibroblast cell line. The results from this study highlight the ability of polycaprolactone-bioactive glass-based fibers loaded with atorvastatin to stimulate skin wound healing.

A facile one-stone-two-birds strategy for fabricating multifunctional 3D nanofibrous scaffolds.

Local bacterial infections lead to delayed wound healing and in extreme cases, such as diabetic foot ulcers, to non-healing due to the impaired cellular function in such wounds. Thus, many scientists have focused on developing advanced therapeutic platforms to treat infections and promote cellular proliferation and angiogenesis. This study presents a facile approach for designing nanofibrous scaffolds in three dimensions (3D) with enhanced antibacterial activity to meet the need of treating chronic diabetic wounds. Being a cationic surfactant as well as an antimicrobial agent, octenidine (OCT) makes a 2D membrane hydrophilic, enabling it to be modified into a 3D scaffold in a "one stone, two birds" manner. Aqueous sodium borohydride (NaBH4) solution plays a dual role in the fabrication process, functioning as both a reducing agent for the in situ synthesis of silver nanoparticles (Ag NPs) anchored on the nanofiber surface and a hydrogen gas producer for expanding the 2D membranes into fully formed 3D nanofiber scaffolds, as demonstrated by morphological analyses. Various techniques were used to characterize the developed scaffold (e.g., SEM, XRD, DSC, FTIR, and surface wettability), demonstrating a multilayered porous structure and superhydrophilic properties besides showing sustained and prolonged release of OCT (61% ± 1.97 in 144 h). Thanks to the synergistic effect of OCT and Ag NPs, the antibacterial performance of the 3D scaffold was significantly higher than that of the 2D membrane. Moreover, cell viability was studied in vitro on mouse fibroblasts L929, and the noncytotoxic character of the 3D scaffold was confirmed. Overall, it is shown that the obtained multifunctional 3D scaffold is an excellent candidate for diabetic wound healing and skin repair.

Methylcellulose/agarose hydrogel loaded with short electrospun PLLA/laminin fibers as an injectable scaffold for tissue engineering/3D cell culture model for tumour therapies.

This research aimed at designing and fabricating a smart thermosensitive injectable methylcellulose/agarose hydrogel system loaded with short electrospun bioactive PLLA/laminin fibers as a scaffold for tissue engineering applications or 3D cell culture models. Considering ECM-mimicking morphology and chemical composition, such a scaffold is capable of ensuring a hospitable environment for cell adhesion, proliferation, and differentiation. Its viscoelastic properties are beneficial from the practical perspective of minimally invasive materials that are introduced to the body via injection. Viscosity studies showed the shear-thinning character of MC/AGR hydrogels enabling the potential injection ability of highly viscous materials. Injectability tests showed that by tuning the injection rate, even a high amount of short fibers loaded inside of hydrogel could be efficiently injected into the tissue. Biological studies showed the non-toxic character of composite material with excellent viability, attachment, spreading, and proliferation of fibroblasts and glioma cells. These findings indicate that MC/AGR hydrogel loaded with short PLLA/laminin fibers is a promising biomaterial for both tissue engineering applications and 3D tumor culture models.

A Comprehensive Review of Electrospun Fibers, 3D-Printed Scaffolds, and Hydrogels for Cancer Therapies.

Anticancer therapies and regenerative medicine are being developed to destroy tumor cells, as well as remodel, replace, and support injured organs and tissues. Nowadays, a suitable three-dimensional structure of the scaffold and the type of cells used are crucial for creating bio-inspired organs and tissues. The materials used in medicine are made of non-degradable and degradable biomaterials and can serve as drug carriers. Developing flexible and properly targeted drug carrier systems is crucial for tissue engineering, regenerative medicine, and novel cancer treatment strategies. This review is focused on presenting innovative biomaterials, i.e., electrospun nanofibers, 3D-printed scaffolds, and hydrogels as a novel approach for anticancer treatments which are still under development and awaiting thorough optimization.

A Comparative Study of Three Approaches to Fibre's Surface Functionalization.

Polyester-based scaffolds are of research interest for the regeneration of a wide spectrum of tissues. However, there is a need to improve scaffold wettability and introduce bioactivity. Surface modification is a widely studied approach for improving scaffold performance and maintaining appropriate bulk properties. In this study, three methods to functionalize the surface of the poly(lactide-co-ε-caprolactone) PLCL fibres using gelatin immobilisation were compared. Hydrolysis, oxygen plasma treatment, and aminolysis were chosen as activation methods to introduce carboxyl (-COOH) and amino (-NH2) functional groups on the surface before gelatin immobilisation. To covalently attach the gelatin, carbodiimide coupling was chosen for hydrolysed and plasma-treated materials, and glutaraldehyde crosslinking was used in the case of the aminolysed samples. Materials after physical entrapment of gelatin and immobilisation using carbodiimide coupling without previous activation were prepared as controls. The difference in gelatin amount on the surface, impact on the fibres morphology, molecular weight, and mechanical properties were observed depending on the type of modification and applied parameters of activation. It was shown that hydrolysis influences the surface of the material the most, whereas plasma treatment and aminolysis have an effect on the whole volume of the material. Despite this difference, bulk mechanical properties were affected for all the approaches. All materials were completely hydrophilic after functionalization. Cytotoxicity was not recognized for any of the samples. Gelatin immobilisation resulted in improved L929 cell morphology with the best effect for samples activated with hydrolysis and plasma treatment. Our study indicates that the use of any surface activation method should be limited to the lowest concentration/reaction time that enables subsequent satisfactory functionalization and the decision should be based on a specific function that the final scaffold material has to perform.

A methylcellulose/agarose hydrogel as an innovative scaffold for tissue engineering.

In situ crosslinked materials are the main interests of both scientific and industrial research. Methylcellulose (MC) aqueous solution is one of the representatives that belongs to this family of thermosensitive materials. At room temperature, MC is a liquid whereupon during temperature increase up to 37 °C, it crosslinks physically and turns into a hydrogel. This feature makes it unique, especially for tissue engineering applications. However, the crosslinking rate of MC alone is relatively slow considering tissue engineering expectations. According to these expectations, the crosslinking should take place slowly enough to allow for complete injection and fill the injury avoiding clogging in the needle, and simultanously, it should be sufficiently fast to prevent it from relocation from the lesion. One of the methods to overcome this problem is MC blending with another substance that increases the crosslinking rate of MC. In these studies, we used agarose (AGR). These studies aim to investigate the effect of different AGR amounts on MC crosslinking kinetics, and thermal, viscoelastic, and biological properties. Differential Scanning Calorimetry (DSC) and dynamic mechanical analysis (DMA) measurements proved that AGR addition accelerates the beginning of MC crosslinking. This phenomenon resulted from AGR's greater affinity to water, which is crucial in this particular crosslinking part. In vitro tests, carried out using the L929 fibroblast line and mesenchymal stem cells (MSCs), confirmed that most of the hydrogel samples were non-cytotoxic in contact with extracts and directly with cells. Not only does this type of thermosensitive hydrogel system provide excellent mechanical and biological cues but also its stimuli-responsive character provides more novel functionalities for designing innovative scaffold/cell delivery systems for tissue engineering applications.

Multifluid electrospinning for multi-drug delivery systems: pros and cons, challenges, and future directions.

The electrospinning method has been widely used to produce nano/micro fibers for various applications. As a drug delivery system, electrospun fibers display many advantages such as controlled drug delivery kinetics and the ability to deliver drugs locally. A drug delivery system improves delivery efficiency and reduces possible toxic effects. In particular, multiaxial fibers consisting of two or more fluid components have drawn attention for the simultaneous administration of multiple therapeutic agents for sustained delivery and effective treatment. This review discusses recently studied multi-compartment electrospun fibers, including side-by-side (Janus) and axially symmetric fibers - coaxial and triaxial - from the perspective of multi-drug incorporation. It begins with an overview of conventional uniaxial single-fluid electrospinning methods for drug delivery applications, then highlights the advantages of multi-compartment fibers for multi-substance loading/delivery and the advances in triaxial fibers that seem to be promising from the perspective of challenges for dressings and tissue regeneration. Furthermore, drug release mechanisms and kinetics are discussed in the controlled delivery of multiple therapeutics in fibers. In the conclusion, current biomedical applications of multi-drug delivery systems in selected applications and future perspectives are presented.

Immobilization of Gelatin on Fibers for Tissue Engineering Applications: A Comparative Study of Three Aliphatic Polyesters.

Immobilization of cell adhesive proteins on the scaffold surface has become a widely reported method that can improve the interaction between scaffold and cells. In this study, three nanofibrous scaffolds obtained by electrospinning of poly(caprolactone) (PCL), poly(L-lactide-co-caprolactone) (PLCL) 70:30, or poly(L-lactide) (PLLA) were subjected to chemical immobilization of gelatin based on aminolysis and glutaraldehyde cross-linking, as well as physisorption of gelatin. Two sets of aminolysis conditions were applied to evaluate the impact of amine group content. Based on the results of the colorimetric bicinchoninic acid (BCA) assay, it was shown that the concentration of gelatin on the surface is higher for the chemical modification and increases with the concentration of free NH2 groups. XPS (X-ray photoelectron spectroscopy) analysis confirmed this outcome. On the basis of XPS results, the thickness of the gelatin layer was estimated to be less than 10 nm. Initially, hydrophobic scaffolds are completely wettable after coating with gelatin, and the time of waterdrop absorption was correlated with the surface concentration of gelatin. In the case of all physically and mildly chemically modified samples, the decrease in stress and strain at break was relatively low, contrary to strongly aminolyzed PLCL and PLLA samples. Incubation testing performed on the PCL samples showed that a chemically immobilized gelatin layer is more stable than a physisorbed one; however, even after 90 days, more than 60% of the initial gelatin concentration was still present on the surface of physically modified samples. Mouse fibroblast L929 cell culture on modified samples indicates a positive effect of both physical and chemical modification on cell morphology. In the case of PCL and PLCL, the best morphology, characterized by stretched filopodia, was observed after stronger chemical modification, while for PLLA, there was no significant difference between modified samples. Results of metabolic activity indicate the better effect of chemical immobilization than of physisorption of gelatin.

Mucoadhesive brinzolamide-loaded nanofibers for alternative glaucoma treatment.

Despite the advances in the field of pharmaceutical materials and technology, topical administration remains a method of choice for the treatment of eye diseases such as glaucoma, with eye drops being a leading dosage form. Their main disadvantage is a very short drug residence time and thus poor drug bioavailability, leading to the necessity of continuous repeated dosing. Mucoadhesive electrospun nanofibers are promising candidates for overcoming these challenges, while still benefiting from topical ocular administration. As an alternative for eye drops, a nanofibrous drug delivery system (DDS) for the delivery of brinzolamide (BRZ), based on ß-cyclodextrin (ß-CD), hydroxypropyl cellulose (HPC) and polycaprolactone (PCL), was designed. The results showed ß-CD/BRZ guest-host interactions, successful drug incorporation into the nanofibers, and the possibility of more accurate dosing in comparison with the control eye drops. Drug permeation through sheep corneas was almost linear in time, achieving therapeutic concentrations in the receptor medium, and mucoadhesion to sheep eye mucosa was relatively high in case of formulations with high HPC content. All formulations were biocompatible, their mechanical properties were sufficient to handle them without caution and UV irradiation was suitable to reduce bioburden of the fibers matrix, yet no antibacterial properties of BRZ were observed.

Control of drug release from cotton fabric by nanofibrous mat.

A drug delivery system (DDSs) was developed in the present study based on textile substrates as drug carriers and electrospun nanofibers as a controller of release rate. Three types of drugs consisting of ciprofloxacin (CIP), clotrimazole (CLO), and benzalkonium chloride (BEN) were loaded into the cover glass (CG) and cotton fabrics (CF1 and CF2) separately. Then, the drug-loaded substrates were coated with polycaprolactone (PCL) and polycaprolactone/gelatin (PCL/Gel) nanofibers with various thicknesses. The morphology and hydrophilicity of the electrospun nanofibers and the release profile of drug-loaded samples were investigated. FTIR, XRD, and in vitro biodegradability analysis were analyzed to characterize the drug delivery system. A morphological study of electrospun fibers showed the mean diameter of the PCL and PCL/Gel nanofibers 127 ± 25 and 178 ± 38 nm, respectively. The drug delivery assay revealed that various factors affect the rate of drug releases, such as the type of drug, the type of drug carrier, and the thickness of the covered nanofibers. The study highlights the ability of drugs to load substrates with coated nanofibers as controlled drug delivery systems. In conclusion, it is shown that the obtained samples are excellent candidates for future wound dressing applications.

Toward a Better Understanding of the Gelation Mechanism of Methylcellulose via Systematic DSC Studies.

A methylcellulose (MC) is one of the materials representatives performing unique thermal-responsive properties. While reaching a critical temperature upon heating MC undergoes a physical sol-gel transition and consequently becomes a gel. The MC has been studied for many years and researchers agree that the MC gelation is related to the lower critical solution temperature (LCST). Nevertheless, a precise description of the MC gelation mechanism remains under discussion. In this study, we explained the MC gelation mechanism through examination of a wide range of MC concentrations via differential scanning calorimetry (DSC). The results evidenced that MC gelation is a multistep thermoreversible process, manifested by three and two endotherms depending on MC concentration. The occurrence of the three endotherms for low MC concentrations during heating has not been reported in the literature before. We justify this phenomenon by manifestation of three various transitions. The first one manifests water-water interactions, i.e., spanning water network breakdown into small water clusters. It is clearly evidenced by additional normalization to the water content. The second effect corresponds to polymer-water interactions, i.e., breakdown of water cages surrounded methoxy groups of MC. The last one is related to the polymer-polymer interactions, i.e., fibril hydrophobic domain formation. Not only did these results clarify the MC crosslinking mechanism, but also in the future will help to assess MC relevance for various potential application fields.

Aminolysis as a surface functionalization method of aliphatic polyester nonwovens: impact on material properties and biological response.

It is reported in the literature that introducing amino groups on the surface improves cellular behaviour due to enhanced wettability and the presence of the positive charge. In this work, electrospun fibers were subjected to aminolysis under various conditions to investigate the impact of reaction parameters on the concentration of free NH2 groups, change of fiber properties, and the response of L929 cells. Three types of electrospun nonwovens obtained from poly(caprolactone) (PCL), poly(l-lactide-co-caprolactone) (PLCL) 70 : 30 and poly(l-lactide) (PLLA) were investigated. For all polymers, the concentration of NH2 groups increased with the diamine concentration and time of reaction. However, it was observed that PCL fibers require much stronger conditions than PLCL and PLLA fibers to reach the same level of introduced amine groups. X-ray photoelectron spectroscopy results clearly demonstrate that an aminolysis reaction is not limited to the surface of the material. Gel permeation chromatography results support this conclusion indicating global molecular weight reduction. However, it is possible to reach a compromise between the concentration of introduced amine groups and the change of mechanical properties. For most of the investigated conditions, aminolysis did not significantly change the water contact angle. Despite this, the change of L929 and MG63 cell shape to being more spread confirmed the positive effect of the presence of the amine groups.

Effect of crystallinity and related surface properties on gene expression of primary fibroblasts.

The biomaterial-cells interface is one of the most fundamental issues in tissue regeneration. Despite many years of scientific work, there is no clear answer to what determines the desired adhesion of cells and the synthesis of ECM proteins. Crystallinity is a characteristic of the structure that influences the surface and bulk properties of semicrystalline polymers used in medicine. The crystallinity of polycaprolactone (PCL) was varied by changing the molecular weight of the polymer and the annealing procedure. Measurements of surface free energy showed differences related to substrate crystallinity. Additionally, the water contact angle was determined to characterise surface wettability which was crucial in the analysis of protein absorption. X-ray photoelectron spectroscopy was used to indicate oxygen bonds amount on the surface. Finally, the impact of the crystallinity, and related properties were demonstrated on dermal fibroblasts' response. Cellular proliferation and expression of selected genes: α-SMA, collagen I, TIMP, integrin were analysed.

Hydrogel, Electrospun and Composite Materials for Bone/Cartilage and Neural Tissue Engineering.

Injuries of the bone/cartilage and central nervous system are still a serious socio-economic problem. They are an effect of diversified, difficult-to-access tissue structures as well as complex regeneration mechanisms. Currently, commercially available materials partially solve this problem, but they do not fulfill all of the bone/cartilage and neural tissue engineering requirements such as mechanical properties, biochemical cues or adequate biodegradation. There are still many things to do to provide complete restoration of injured tissues. Recent reports in bone/cartilage and neural tissue engineering give high hopes in designing scaffolds for complete tissue regeneration. This review thoroughly discusses the advantages and disadvantages of currently available commercial scaffolds and sheds new light on the designing of novel polymeric scaffolds composed of hydrogels, electrospun nanofibers, or hydrogels loaded with nano-additives.

Polyurethane-Nanolignin Composite Foam Coated with Propolis as a Platform for Wound Dressing: Synthesis and Characterization.

This piece of research explores porous nanocomposite polyurethane (PU) foam synthesis, containing nanolignin (NL), coated with natural antimicrobial propolis for wound dressing. PU foam was synthesized using polyethylene glycol, glycerol, NL, and 1, 6-diisocyanato-hexane (NCO/OH ratio: 1.2) and water as blowing agent. The resultant foam was immersed in ethanolic extract of propolis (EEP). PU, NL-PU, and PU-NL/EEP foams were characterized from mechanical, morphological, and chemical perspectives. NL Incorporation into PU increased mechanical strength, while EEP coating showed lower strength than PU-NL/EEP. Morphological investigations confirmed an open-celled structure with a pore diameter of 150-200 µm, a density of nearly 0.2 g/cm3,, and porosity greater than 85%, which led to significantly high water absorption (267% for PU-NL/EEP). The hydrophilic nature of foams, measured by the contact angle, proved to be increased by NL addition and EEP coating. PU and PU-NL did not show important antibacterial features, while EEP coating resulted in a significant antibacterial efficiency. All foams revealed high biocompatibility toward L929 fibroblasts, with the highest cell viability and cell attachment for PU-NL/EEP. In vivo wound healing using Wistar rats' full-thickness skin wound model confirmed that PU-NL/EEP exhibited an essentially higher wound healing efficacy compared with other foams. Hence, PU-NL/EEP foam could be a promising wound dressing candidate.

Crosslinking of Gelatin in Bicomponent Electrospun Fibers.

Four chemical crosslinking methods were used in order to prevent gelatin leaching in an aqueous environment, from bicomponent polycaprolactone/gelatin (PCL/Gt) nanofibers electrospun from an alternative solvent system. A range of different concentrations and reaction times were employed to compare genipin, 1-(3-dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), 1,4-butanediol diglycidyl ether (BDDGE), and transglutaminase. The objective was to optimize and find the most effective method in terms of reaction time and solution concentration, that at the same time provides satisfactory gelatin crosslinking degree and ensures good morphology of the fibers, even after 24 h in aqueous medium in 37 °C. The series of experiments demonstrated that, out of the four compared crosslinking methods, EDC/NHS was able to yield satisfactory results with the lowest concentrations and the shortest reaction times.