Three-dimensional (3D) human skin constructs are gaining increasing relevance important in regenerative research, drug testing and the cosmetics industry. They offer great potential to overcome the anatomical and physiological limitations of animal models, yet current models are often limited by high costs, batch-to-batch variability, and mechanical properties that poorly replicate native skin. In particular, discrepancies in elasticity and tensile strength compared to the natural extracellular matrix (ECM) of skin remain a major challenge. To address this, we developed a novel hybrid hydrogel composed of 20% gelatin-methacrylamide (GelMA) and 5% methacrylated elastin (ElMA) derived from porcine aorta, forming the basis of a bi-layered 3D skin construct that mimics both epidermal and dermal ECM structures. Using this bioink, we created two distinct model variants: a GelMA-ElMA (GE) formulation and a GelMA-ElMA composition reinforced with a GelMA electrospun nanofleece (GEN) to support enhanced epidermal maturation. The resulting constructs support fibroblast proliferation within the dermal layer and epidermal stratification marked by expression of keratin 14, Keratin 10 and involucrin, reflecting the architecture of native skin. The presence of elastin contributed to high biocompatibility and cytocompatibility, enhancing both cell proliferation and metabolic activity. Furthermore, the model proved suitable for studying IL-1α-induced inflammation and wound healing processes. This work demonstrates the potential of GelMA-ElMA-based bioinks to advance skin tissue engineering by integrating mechanical stability with biological relevance, offering a promising platform for both fundamental research and applied testing.

Elastin is a key protein responsible for elasticity, resilience, and deformability of tissues. Elastin is subject to an understudied posttranslational modification, prolyl hydroxylation, where a hydroxyl group replaces a hydrogen atom at C-γ in proline residues during assembly. Recent experimental studies suggest elastin-like peptides with hydroxyproline modifications are more resistant to enzymatic digestion and subject to abnormal assembly. We hypothesize that hydroxylation modulates protein-solvent interactions, thereby altering elastin behavior. To test our hypothesis, we build representative models with and without prolyl hydroxylation and perform extensive molecular dynamics simulations. Our findings suggest that hydroxyproline increases hydrogen bonding with water by an average of 135% compared to proline, which reduces the local configurational space, thereby negatively impacting elastin's global dynamics, essential for its biological functions. This modification may potentially protect the molecule from targeted degradation and modulate canonical hierarchical assembly. In addition, our study provides design insights for engineered elastin-based materials through fine-tuning of hydroxyproline content.

The modification of biomaterial surfaces with polyelectrolyte multilayers (PEM) represents a promising strategy for regulation of cell proliferation, and differentiation. This study examines how uploading cobalt (Co²⁺), copper (Cu²⁺), or iron (Fe³⁺) ions into chitosan-based PEM assembled with either hyaluronic acid (HA) or alginate (Alg) as polyanions affects the adhesion and osteogenic differentiation of a murine stem-cell line. Metal ion content in PEM is quantified by inductively coupled plasma mass spectrometry (ICP-MS) showing the highest concentration of Fe³⁺, followed by Cu²⁺ and Co²⁺. Interestingly, copper- and iron-doped [Chi/Alg] and [Chi/HA] PEM promote cell adhesion, spreading, and proliferation most, whereas cobalt-doped PEM exhibit reduced cellular interactions. In both PEM systems, metal-ion doping enhances osteogenic differentiation relative to undoped PEM, as shown by RT-PCR with up-regulation of alkaline phosphatase (ALP), Noggin, and Osterix particularly with Fe3+ and Cu2+. Microscopy confirms greater calcium-phosphate deposition and stronger collagen I and osteocalcin staining, after doping with Fe³⁺ and Cu2+ producing the most pronounced effects. Furthermore, [Chi/HA] PEM doped with metal ions demonstrate higher osteogenic marker expression than [Chi/Alg] PEM. These findings suggest that PEM composed of alginate or hyaluronic acid combined with chitosan doped with Cu²⁺, and Fe³⁺ yield promising bioactive coatings on implants and scaffolds, which may promote regeneration of bone tissue.

Poly(lactic-co-glycolic acid) (PLGA) is a prominent biodegradable polymer used in biomedical applications, including drug delivery systems (DDS) and tissue engineering. PLGA's ability to control drug release is often hindered by nonlinear release profiles and slow initial drug release for hydrophobic drugs. This study investigates the incorporation of dexamethasone phosphate (DEXP) into polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA) implants to enhance the initial release rate of dexamethasone (DEX). Implants were fabricated via hot-melt extrusion with varying DEX to DEXP ratios. X-ray diffraction (XRD) analysis confirmed that DEX remained crystalline in all formulations, whereas DEXP's crystallinity was detectable only in higher concentrations. Energy-dispersive X-ray spectroscopy (EDX) provided insights into the distribution of DEX and DEXP within the polymer matrix. Drug release studies revealed that PEG-PLGA implants accelerated initial drug release with increasing quantity of DEXP, though it also led to a shorter overall release duration. Despite these improvements, all implants exhibited a biphasic release profile. DEXP also influenced the characteristics of the polymer matrix, evidenced by increased swelling, water absorption, and mass loss. 1H NMR analysis revealed a faster decrease in glycolic acid monomers in DEXP-containing implants. These findings demonstrate that DEXP enhances early drug release of DEX-loaded PEG-PLGA implants prepared by hot-melt extrusion. However, balancing initial and sustained release profiles remains challenging.

Over the years, significant progress has been made in developing cost-effective and ethical in vitro bioengineered skin substitutes to study cutaneous wound healing processes. Rodents and small animal models are not optimal due to physiological differences in their skin compared to human skin. The generation of reproducible and precise wounds is key to obtaining comparable results. We created a three-dimensional skin wounding model by growing a fully differentiated, stratified squamous epithelium from human keratinocytes at an air–liquid interface on a type I collagen scaffold concealed with human dermal BJ fibroblasts. To generate the wounds, a stamp with incorporated needles with a length of 250 µm was used to puncture the epidermis to produce standardized wounds. The stamping needle technique is a practical and inexpensive method for creating length-tailored wounds on three-dimensional skin models. The effectiveness of this technique in treating 3D skin models was demonstrated, accompanied by an evaluation of the model’s functionality in terms of cell proliferation, differentiation, and immunological characteristics.

Although therapeutic nucleic acids reached the clinical application for a decade, the success of these new drugs is dependent on their delivery strategies, which are still a challenge. In particular, local delivery of nucleic acids is a promising approach to develop therapies with a spatially controlled site of action. However, compared to techniques for systemic administration, local nucleic acid delivery systems are still rarely described. In this study, we present a promising approach to fill this gap by the design of surface coatings based on polysaccharides for local delivery of nucleic acids. An automatized Layer-by-Layer deposition approach was applied using hyaluronic acid and chitosan to form polyelectrolyte multilayer systems, into which lipid nanoparticles, more specific lipoplexes, were embedded as nucleic acid carriers. Different manufacturing parameters, in particular the number of deposited polyelectrolyte layers and the preparation buffer, were varied. The multilayer film characteristics were investigated systematically regarding their physical properties, with a focus on thickness and topology as well as lipoplex deposition, to identify a system with efficient transfection properties. The multilayer systems prepared in acetate buffer were characterized by a good lipoplex embedding with a more uniform distribution and lower tendency for formation of large lipoplex aggregates in the polyelectrolyte film. Additionally, we were able to demonstrate the functionality of the developed system for nucleic acid delivery. The nucleic acids were successfully transferred into cells in a contact-triggered manner. Furthermore, we could demonstrate the enzymatic degradation-based release of nucleic acid cargo from the delivery system caused by hyaluronidase, followed by successful in vitro transfection.

Expansion microscopy (ExM) enables super-resolution visualization using standard light microscopes. Recent developments have explored dimethylacrylamide (DMAA) to improve gel robustness, but compatibility with organic solvents remains a challenge. This study presents a novel hydrogel formulation based solely on DMAA that omits acrylamide (AA) and sodium acrylate (SA): This formulation achieves superior mechanical properties and is compatible with a range of solvents, including ethanol, isopropanol, and acetone. Using eosin-stained tropoelastin fibers as a model, we demonstrated that the gel preserves structural integrity and achieves tunable linear expansion factors of 2.0 +/- 0.1-fold in water and ethanol, 1.9 +/- 0.1-fold in isopropanol, and 1.6 +/- 0.1-fold in acetone. We validated the protocol using dense murine aortic tissue, achieving a 2.0-fold expansion, which successfully resolved fine architectural details that were unresolvable in the native tissue at the same magnification. Furthermore, we found that expanding ethanol-based eosin-stained samples improves imaging contrast compared to the aqueous protocol. By enabling high-fidelity imaging of biological samples in diverse solvent environments, this DMAA-based gel system substantially broadens the applicability of ExM, opening new possibilities for integrating it with complex labeling workflows requiring organic solvents and enabling future correlative microscopy studies across multiple imaging platforms.

Chronic skin wounds place a heavy burden on patients and healthcare systems. To address this problem, we have developed a novel composite material consisting of an electrospun fleece and a free-standing multilayer film that combines the wound healing benefits of both materials. In detail a combination of spray coating and electrospinning is used to create a layer-by-layer film on top of a gelatin fleece, with a final thickness of about 1 mm. A gelatin fleece is partially crosslinked in formaldehyde vapor and 30 pH-sensitive bonding bilayers of partially oxidized hyaluronic acid (HA) and chitosan, followed by 120 bilayers of alginate and chitosan are sprayed on top. The resulting composite is crosslinked with genipin. Uncrosslinked and genipin crosslinked composites are compared to the unprocessed fleece and free-standing multilayer film. The spray coating method produces a stable composite, allows a fast growth of the film part und most importantly retains the nano-topography of the fleece side as confirmed by electron microscopy, profilometry, nano-tomography and dynamic mechanical analysis. To test biocompatibility, cell proliferation experiments with human dermal fibroblasts and THP-1 derived macrophages are performed, proliferative assays are accompanied by immunohistochemical staining and a pro/anti-inflammatory cytokine assay. The composite shows no cytotoxicity and is biocompatible in vitro. Furthermore, the electrospun fibers of the fleece act as a scaffold to highly promote cell adhesion and proliferation, while the modular design of the multilayer free-standing film, in combination with genipin crosslinking, allows the tuning of the anti-inflammatory effect by HA. Overall, the composite seems to be a promising starting point for the design of a novel wound dressing.

Williams–Beuren syndrome (WBS) is a congenital multisystem disorder affecting the cardiovascular, central nervous, and musculoskeletal systems. Cardiovascular abnormalities, which consist principally of vascular stenoses, occur in approximately 80% of people with WBS and are the predominant cause of early morbidity and mortality. Supravalvar aortic stenosis and peripheral pulmonary artery stenosis are the most common stenotic lesions in WBS, though other stenoses often occur, including stenoses of the coronary arteries. Approximately one‐third of people with WBS undergo cardiovascular interventions. The risk of sudden cardiac death is markedly higher than the general population, with most events occurring in the periprocedural period. Because of the rarity of WBS and the often‐complex nature of the cardiovascular abnormalities, most physicians, including cardiologists, have limited experience in caring for patients with WBS. Further, heretofore, clinical cardiovascular management guidelines based on international expert consensus have not been available. This state‐of‐the‐art review provides a comprehensive synopsis of the cardiovascular abnormalities in WBS and presents clinical management guidelines based on the authors' expert consensus.

Drug delivery to the inner ear presents a unique challenge due to the complex inner ear anatomy and its tight physiological barriers. This study investigates the degradation behavior of intracochlear drug delivery implants (IDDI) composed of dexamethasone and poly(lactic-co-glycolic acid) (PLGA) or polyethylene glycol-poly(lacticco-glycolic acid) (PEG-PLGA), respectively. IDDI were incubated in artificial perilymph and implants' degradation kinetics, morphological changes, water uptake behavior, and pH alterations were assessed. Microscopy revealed significant changes in appearance, with PLGA IDDI exhibiting rapid expansion, reaching up to 183 % in diameter and 185 % in length. PEG-PLGA implants showed gradual expansion, reaching a maximum of 178 % in diameter and 144 % in length. Despite these morphological changes, the IDDIs could still be applicable in terms of cochlear dimensions in combination with cochlear implants (CI) in humans or in a domestic pig animal model. Scanning electron microscopy analysis demonstrated surface alterations of PLGA implants, while PEG-PLGA implants remained shape-stable. Gravimetric analysis and gel permeation chromatography revealed distinct degradation profiles, with PLGA implants displaying rapid water uptake and mass loss, while PEG-PLGA implants showed delayed water uptake and minimal mass reduction. pH measurements using the pH-sensitive fluorescent dye SNARFTM-1 showed initial pH reduction in artificial perilymph for PLGA implants while PEG-PLGA implants maintained pH stability.

Stiffening of the vascular network is associated with the early stages of vascular aging, leading to cardiovascular disorders (hypertension), renal failures, or neurodegenerative diseases (Alzheimer's). Unfortunately, many people remain undiagnosed because diagnostic methods are either unsuitable for a large population or unfamiliar to clinicians which favor the hypertension evaluation. In preclinical research, stiffness studies are often partially conducted. We think that the evaluation of aortic stiffness is essential as it would improve our understanding of aging diseases progression. We propose here a systematic method using decision trees in a multi-scale and multimodal approaches. Our method was evaluated by analyzing the aortic situation in old and young mice. We demonstrate that both the endothelial and smooth muscle cells exhibit pronounced functional alterations in favor of constriction. Additionally, there is significant remodeling of the extracellular matrix, leading to a drastic degradation of elastic fibers and the accumulation of collagen in the aortic wall. This series of changes contributes to the development of vascular rigidity, a preliminary stage of arterial hypertension. Our results suggest that our method should improve preclinical understanding and encourage clinicians to equip themselves with tools for assessing vascular function, as it is an essential issue for preventing numerous pathologies.

The aim of this study was to investigate the suitability of selected essential oils as more environmentally friendly, sustainable antimicrobial agents in photographic films for long-term data storage. For this purpose, essential oils of oregano, thyme, and lemongrass were added in concentrations ranging from 0.4% to 4% to the gelatin comprising a protective layer for photographic microfilms. The emulsions were analyzed via UV-Vis for their optical transparency, then tested for their antimicrobial efficacy against Bacillus subtilis, Staphylococcus hominis and Staphylococcus epidermidis. Thin coatings generated by spreading and drying the emulsions onto photographic film substrates were then characterized morphologically via SEM before and after standardized artificial aging procedures. It was found that oregano oil at a concentration of 0.4% maintained the optical and physical properties of the emulsion and proved highly effective against all tested bacteria. Thyme and lemongrass oil also exhibited some antimicrobial activity, however, were not able to inhibit the bacterial growth completely. At higher concentrations of the oils, the gelatin coatings cracked after artificial aging, which makes them unsuitable for the application. To conclude, selected essential oils like oregano oil can be used as an effective antimicrobial agent without compromising the quality of the photographic films.Practical applications: The results of our research can be applied directly to the manufacturing of photographic films, specifically ones intended for long-term data storage. Furthermore, many applications of gelatin or related materials which require antimicrobial activity, such as food packaging, could be enhanced by the addition of essential oils. Selected essential oils were incorporated into photographic gelatin and tested for their suitability as antimicrobial agents, as well as their influence on the optical properties and aging behavior of the gelatin. image

AbstractReverse osmosis (RO) is the most common method for treating salt and brackish water. As a membrane‐driven process, a key challenge for RO systems is their susceptibility to scaling and biofouling. To address these issues, functional coatings utilizing metal nanoparticles (MNPs) are developed. In this study, silver, gold, and copper nanoparticles are applied onto thin‐film composite (TFC) membranes using plasma‐enhanced magnetron sputtering. The elemental composition, surface morphology, and hydrophilicity of the coatings are analyzed using X‐ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurements. The antimicrobial properties and the filtration efficiency of the coated membranes are assessed through application‐specific experimental setups. Silver and copper nanoparticles exhibit superior antimicrobial properties, reducing microorganism adhesion by a factor of 103 compared to uncoated membranes. Under appropriate coating conditions, no deterioration in filtration performance is observed. Enhancing the adhesion of MNPs is necessary for achieving sustained release of metal ions.

Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions. Here, a bioactive polyelectrolyte multilayer (PEM) is engineered by the combination of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and glycosaminoglycans to be used as reservoir for GF storage. PNIPAM-grafted-chitosan (PChi) with two degrees of substitution (DS) are synthesized, namely LMW* (DS 0.14) and HMW (DS 0.03), by grafting low (2 kDa) and high (10 kDa) molecular weight of PNIPAM on the backbone of chitosan (Chi) to be employed as polycations to form PEM with the polyanion heparin (Hep) at pH 4. Subsequently, PEMs are chemically crosslinked to improve their stability at physiological pH 7.4. Resulting surface and mechanical properties indicate that PEM containing HMW is responsive to temperature at 20 °C and 37 °C, while LMW is not. More importantly, Hep as terminal layer combined with HMW allows not only a better retention of the adhesive protein vitronectin but also a sustained release of FGF-2 at 37 °C. With the synergistic effect of vitronectin and matrix-bound FGF-2, significant promotion on adhesion, proliferation, and migration of 3T3 mouse embryonic fibroblasts is achieved on HMW-containing PEM compared to Chi-containing PEM and exogenously added FGF-2. Thus, PEM containing PNIPAM in combination with bioactive glycosaminoglycans like Hep represents a versatile approach to fabricate a GF delivery system for efficient cell culture, which can be potentially served as cell culture substrate for production of (stem) cells and bioactive wound dressing for tissue regeneration.

Engineered neural tissues serve as models for studying neurological conditions and drug screening. Besides observing the cellular physiological properties, in situ monitoring of neurochemical concentrations with cellular spatial resolution in such neural tissues can provide additional valuable insights in models of disease and drug efficacy. In this work, we demonstrate the first three-dimensional (3D) tissue cultures with embedded optical dopamine (DA) sensors. We developed an alginate/Pluronic F127 based bio-ink for human dopaminergic brain tissue printing with tetrapodal-shaped-ZnO microparticles (t-ZnO) additive as the DA sensor. DA quenches the autofluorescence of t-ZnO in physiological environments, and the reduction of the fluorescence intensity serves as an indicator of the DA concentration. The neurons that were 3D printed with the t-ZnO showed good viability, and extensive 3D neural networks were formed within one week after printing. The t-ZnO could sense DA in the 3D printed neural network with a detection limit of 0.137 μM. The results are a first step toward integrating tissue engineering with intensiometric biosensing for advanced artificial tissue/organ monitoring.

Elastin is an essential extracellular matrix protein that enables tissues and organs such as arteries, lungs, and skin, which undergo continuous deformation, to stretch and recoil. Here, an approach to fabricating artificial elastin with close-to-native molecular and mechanical characteristics is described. Recombinantly produced tropoelastin are polymerized through coacervation and allysine-mediated cross-linking induced by pyrroloquinoline quinone (PQQ). A technique that allows the recovery and repeated use of PQQ for protein cross-linking by covalent attachment to magnetic Sepharose beads is developed. The produced material closely resembles natural elastin in its molecular, biochemical, and mechanical properties, enabled by the occurrence of the cross-linking amino acids desmosine, isodesmosine, and merodesmosine. It possesses elevated resistance against tryptic proteolysis, and its Young's modulus ranging between 1 and 2 MPa is similar to that of natural elastin. The approach described herein enables the engineering of mechanically resilient, elastin-like materials for biomedical applications.

Surface coatings prepared by layer-by-layer technique permit loading of growth factors (GFs) and their spatially controlled release. Here, native chondroitin sulfate (nCS), oxidized CS (oCS100), or mixture of both (oCS50) are combined with collagen I (Col I) to fabricate polyelectrolyte multilayers (PEMs) that exhibit structural, mechanical, and biochemical cues like the natural extracellular-matrix. The use of oCS enables intrinsic cross-linking of PEM that offers higher stability, stiffness, and better control of bone morphogenetic protein-2 (BMP-2) release compared to nCS. oCS100 PEMs have enhanced stiffness, promote Col I fibrillization, and present BMP-2 in a matrix-bound manner. oCS50 PEMs show intermediate effects on osteogenesis, soft surface, high water content but also moderately slow BMP-2 release profile. C2C12 myoblasts used for osteogenesis studies show that oCS PEMs are more stable and superior to nCS PEMs in supporting cell adhesion and spreading as well as in presenting BMP-2 to the cells. oCS PEMs are triggering more osteogenesis as proved by the quantitative real-time polymerase chain reaction, immune and histochemical staining. These findings show that intrinsic cross-linking in oCS/Col I multilayers provides a successful tool to control GFs delivery and subsequent cell differentiation which opens new opportunities in regenerative therapies of bone and other tissues.

The vascular wall is a composite environment made of a myriad of different cell types and a complex extracellular matrix (ECM). The latter has a direct impact on vessel function and pathophysiological remodeling of ECM is associated with alterations of vessel structure and modifications of cell phenotype. In this context, elastin plays a fundamental role. Elastin is a protein with is one of the longest half-lives in humans, and its biosynthesis is virtually completed before adolescence, thereby suffering from several alterations during its biological life. These modifications are fundamentally linked to appearance of vascular rigidity and hypertension. Generation of elastin-derived peptides (EDPs) can trigger dramatic changes in cell phenotype through binding on the elastin receptor complex (ERC). EDPs and ERC signaling are involved in the development of several vascular wall-related diseases and can amplify elastin degradation through stimulation of elastases secretion, thereby actively contributing to the development of arterial stiffness.