A facile method to fabricate versatile keratin cryogels for tissue engineering applications.

Human hair keratin (HHK) has been extensively explored as a biomaterial for soft tissue regeneration due to their excellent bioactivity and biocompatibility. The possibility to fabricate HHK into three-dimensional (3D) hydrogels with physical properties resembling soft tissues has been well demonstrated. However, conventional keratin hydrogels often exhibit a dense architecture that could hinder cell filtration. In the present study, HHK-based cryogels were fabricated using a freeze-thaw (FT) method, where oxidized dopamine (ODA) was employed to covalently crosslink thiol/amine rich-keratin molecules at sub-zero temperatures. The obtained HHK-ODA cryogels have micron-sized pores ranging between 100 and 200 µm and mechanical properties that can be tuned by varying the crosslinking density between ODA and HHK. Through optimization of the weight content of ODA and the number of FT cycles, the compressive strengths and stiffnesses of these cryogels achieved 15-fold increments from ∼1.5 kPa to ∼22 kPa and ∼300 Pa to ∼5000 Pa, respectively. The HHK-ODA cryogels competently supported human dermal fibroblast spreading and proliferation. Overall, this study exhibited a facile method to fabricate mechanically superior keratin-based cryogels with cell compatible microarchitecture, circumventing the need for complicated chemical modifications and the use of cytotoxic crosslinkers.

Comparative secretome metabolic dysregulation by six engineered dietary nanoparticles (EDNs) on the simulated gut microbiota.

The potential use of engineered dietary nanoparticles (EDNs) in diet has been increasing and poses a risk of exposure. The effect of EDNs on gut bacterial metabolism remains largely unknown. In this study, liquid chromatography-mass spectrometry (LC-MS) based metabolomics was used to reveal significantly altered metabolites and metabolic pathways in the secretome of simulated gut microbiome exposed to six different types of EDNs (Chitosan, cellulose nanocrystals (CNC), cellulose nanofibrils (CNF) and polylactic-co-glycolic acid (PLGA); two inorganic EDNs including TiO2 and SiO2) at two dietary doses. We demonstrated that all six EDNs can alter the composition in the secretome with distinct patterns. Chitosan, followed by PLGA and SiO2, has shown the highest potency in inducing the secretome change with major pathways in tryptophan and indole metabolism, bile acid metabolism, tyrosine and phenol metabolism. Metabolomic alterations with clear dose response were observed in most EDNs. Overall, phenylalanine has been shown as the most sensitive metabolites, followed by bile acids such as chenodeoxycholic acid and cholic acid. Those metabolites might be served as the representative metabolites for the EDNs-gut bacteria interaction. Collectively, our studies have demonstrated the sensitivity and feasibility of using metabolomic signatures to understand and predict EDNs-gut microbiome interaction.

3D Airway Epithelial-Fibroblast Biomimetic Microfluidic Platform to Unravel Engineered Nanoparticle-Induced Acute Stress Responses as Exposome Determinants.

Insights into how biological systems respond to high- and low-dose acute environmental stressors are a fundamental aspect of exposome research. However, studying the impact of low-level environmental exposure in conventional in vitro settings is challenging. This study employed a three-dimensional (3D) biomimetic microfluidic lung-on-chip (µLOC) platform and RNA-sequencing to examine the effects of two model anthropogenic engineered nanoparticles (NPs): zinc oxide nanoparticles (Nano-ZnO) and copier center nanoparticles (Nano-CCP). The airway epithelium exposed to these NPs exhibited dose-dependent increases in cytotoxicity and barrier dysregulation (dominance of the external exposome). Interestingly, even nontoxic and low-level exposure (10 µg/mL) of the epithelium compartment to Nano-ZnO triggered chemotaxis of lung fibroblasts toward the epithelium. An increase in α smooth muscle actin (α-SMA) expression and contractile activity was also observed in these cells, indicating a bystander-like adaptive response (dominance of internal exposome). Further bioinformatics and network analysis showed that a low-dose Nano-ZnO significantly induced a robust transcriptomic response and upregulated several hub genes associated with the development of lung fibrosis. We propose that Nano-ZnO, even at a no observable effect level (NOEL) dose according to conventional standards, can function as a potent nanostressor to disrupt airway epithelium homeostasis. This leads to a cascade of profibrotic events in a cross-tissue compartment fashion. Our findings offer new insights into the early acute events of respiratory harm associated with environmental NPs exposure, paving the way for better exposomic understanding of this emerging class of anthropogenic nanopollutants.

Recent omics advances in hair aging biology and hair biomarkers analysis.

Aging is a complex natural process that leads to a decline in physiological functions, which is visible in signs such as hair graying, thinning, and loss. Although hair graying is characterized by a loss of pigment in the hair shaft, the underlying mechanism of age-associated hair graying is not fully understood. Hair graying and loss can have a significant impact on an individual's self-esteem and self-confidence, potentially leading to mental health problems such as depression and anxiety. Omics technologies, which have applications beyond clinical medicine, have led to the discovery of candidate hair biomarkers and may provide insight into the complex biology of hair aging and identify targets for effective therapies. This review provides an up-to-date overview of recent omics discoveries, including age-associated alterations of proteins and metabolites in the hair shaft and follicle, and highlights the significance of hair aging and graying biomarker discoveries. The decline in hair follicle stem cell activity with aging decreased the regeneration capacity of hair follicles. Cellular senescence, oxidative damage and altered extracellular matrix of hair follicle constituents characterized hair follicle and hair shaft aging and graying. The review attempts to correlate the impact of endogenous and exogenous factors on hair aging. We close by discussing the main challenges and limitations of the field, defining major open questions and offering an outlook for future research.

Profiling of hair proteome revealed individual demographics.

Human hair is often found at crime scenes, persists for a long time, and is a valuable biological specimen in forensic investigations. Hair contains minimal intact nuclear DNA for the discrimination of individual identity. In such cases, proteomics evaluation of hair proteins could provide an attractive alternative for protein-based human identification. Therefore, this study adopted a proteomic approach to profile hair shafts from both males and females across different ethnic populations including Chinese, Indians, Malays, and Filipinos in their 20-80 s. First, hair proteins were extracted by different methods to adopt the most suitable protocol that produced the highest extraction efficiency based on most significant enrichment of keratins and keratin-associated proteins. Abundance of hair keratins including both types I and II, and keratin-associated proteins, estimated using label-free quantification, showed distinguishable profiles, and the possibilities of distinguishing individuals within each ethnic origin. Similarly, several protein candidates and their abundances could be used to distinguish sex and age of individuals. This study explored the possibility of utilizing hair proteomics phenotyping in forensic science to differentiate individuals across various ethnic groups, sex and age.

Cellulose Nanofiber Platform for Pesticide Sequestration in the Gastrointestinal Tract.

Exploitation of nature-derived materials is an important approach to promote environmental sustainability. Among these materials, cellulose is of particular interest due to its abundance and relative ease of access. As a food ingredient, cellulose nanofibers (CNFs) have found interesting applications as emulsifiers and modulators of lipid digestion and absorption. In this report, we show that CNFs can also be modified to modulate the bioavailability of toxins, such as pesticides, in the gastrointestinal tract (GIT) by forming inclusion complexes and promoting interaction with surface hydroxyl groups. CNFs were successfully functionalized with (2-hydroxypropyl)-ß-cyclodextrin (HPBCD) using citric acid as a crosslinker via esterification. Functionally, the potential for pristine and functionalized CNFs (FCNFs) to interact with a model pesticide, boscalid, was tested. Based on direct interaction studies, adsorption of boscalid saturated at around 3.09% on CNFs and at 12.62% on FCNFs. Using an in vitro GIT simulation platform, the adsorption of boscalid on CNFs/FCNFs was also studied. The presence of a high-fat food model was found to have a positive effect in binding boscalid in a simulated intestinal fluid environment. In addition, FCNFs were found to have a greater effect in retarding triglyceride digestion than CNFs (61% vs 30.6%). Overall, FCNFs were demonstrated to evoke synergistic effects of reducing fat absorption and pesticide bioavailability through inclusion complex formation and the additional binding of the pesticide onto surface hydroxyl groups on HPBCD. By adopting food-compatible materials and processes for production, FCNFs have the potential to be developed into a functional food ingredient for modulating food digestion and the uptake of toxins.

Human hair proteins as natural reactive oxygen species scavengers for in vitro applications.

Human hair proteins are recognized for their intrinsically high cysteine content. They can be solubilized while preserving their highly reductive thiol groups for free radical scavenging applications. The presence of aromatic and nucleophilic amino acids such as methionine, serine, phenylalanine, and threonine further contribute to the antioxidative potential of this material. Herein, utilizing the DPPH (2,2-diphenyl-1-picrylhydrazyl) and acellular 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) assays, keratins are demonstrated to possess the highest radical scavenging activity among the studied hair proteins. Consequently, protection against hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs) cultured in human hair keratin supplemented media is demonstrated. Quenching of reactive oxygen species in the HDF is observed using the CellROX Green dye and the expression levels of antioxidant (HMOX1, SOD2, GPX1) and tumor suppressor (TP53) genes is analyzed using qPCR. Collectively, this study presents further evidence and demonstrates the in vitro application potential of hair proteins, especially keratins, as an antioxidizing supplement.

Nanoparticle-induced chemoresistance: the emerging modulatory effects of engineered nanomaterials on human intestinal cancer cell redox metabolic adaptation.

The widespread use of engineered nanomaterials (ENMs) in food products necessitates the understanding of their impact on the gastrointestinal tract (GIT). Herein, we screened several representative food-borne comparator ENMs (i.e. ZnO, SiO2 and TiO2 nanoparticles (NPs)) and report that human colon cancer cells can insidiously exploit ZnO NP-induced adaptive response to acquire resistance against several chemotherapeutic drugs. By employing a conditioning and challenge treatment regime, we demonstrate that repeated exposure to a non-toxic dose of ZnO NPs (20 µM) could dampen the efficacy of cisplatin, paclitaxel and doxorubicin by 10-50% in monolayer culture and 3D spheroids of human colon adenocarcinoma cells. Structure-activity relationship studies revealed a complex interplay between nanoparticle surface chemistry and cell type in determining the chemoresistance-inducing effect, with silica coated ZnO NPs having a negligible influence on the anticancer treatment. Mechanistically, we showed that the pro-survival paracrine signaling was potentiated and propagated by a subset of ZnO NP "stressed" (Zn2++/ROS+) cells to the surrounding "bystander" (Zn2++/ROS-) cells. Transcriptome profiling, bioinformatics analysis and siRNA gene knockdown experiments revealed the nuclear factor erythroid 2-related factor 2 (Nrf2) as the key modulator of the ZnO NP-induced drug resistance. Our findings suggest that a ROS-inducing ENM can emerge as a nano-stressor, capable of regulating the chemosensitivity of colon cancer cells.

Printer center nanoparticles alter the DNA repair capacity of human bronchial airway epithelial cells.

Nano-enabled, toner-based printing equipment emit nanoparticles during operation. The bioactivity of these nanoparticles as documented in a plethora of published toxicological studies raises concerns about their potential health effects. These include pro-inflammatory effects that can lead to adverse epigenetic alterations and cardiovascular disorders in rats. At the same time, their potential to alter DNA repair pathways at realistic doses remains unclear. In this study, size-fractionated, airborne particles from a printer center in Singapore were sampled and characterized. The PM0.1 size fraction (particles with an aerodynamic diameter less than 100 nm) of printer center particles (PCP) were then administered to human lung adenocarcinoma (Calu-3) or lymphoblastoid (TK6) cells. We evaluated plasma membrane integrity, mitochondrial activity, and intracellular reactive oxygen species (ROS) generation. Moreover, we quantified DNA damage and alterations in the cells' capacity to repair 6 distinct types of DNA lesions. Results show that PCP altered the ability of Calu-3 cells to repair 8oxoG:C lesions and perform nucleotide excision repair, in the absence of acute cytotoxicity or DNA damage. Alterations in DNA repair capacity have been correlated with the risk of various diseases, including cancer, therefore further genotoxicity studies are needed to assess the potential risks of PCP exposure, at both occupational settings and at the end-consumer level.

Enhancing Agrichemical Delivery and Plant Development with Biopolymer-Based Stimuli Responsive Core-Shell Nanostructures.

The inefficient delivery of agrichemicals in agrifood systems is among the leading cause of serious negative planetary and public health impacts. Such inefficiency is mainly attributed to the inability to deliver the agrichemicals at the right place (target), right time, and right dose. In this study, scalable, biodegradable, sustainable, biopolymer-based multistimuli responsive core-shell nanostructures were developed for smart agrichemical delivery. Three types of responsive core/shell nanostructures incorporated with model agrichemicals (i.e., CuSO4 and NPK fertilizer) were synthesized by coaxial electrospray, and the resulting nanostructures showed spherical morphology with an average diameter about 160 nm. Tunable agrichemical release kinetics were achieved by controlling the surface hydrophobicity of nanostructures. The pH and enzyme responsiveness was also demonstrated by the model analyte release kinetics (up to 7 days) in aqueous solution. Finally, the efficacy of the stimuli responsive nanostructures was evaluated in soil-based greenhouse studies using soybean and wheat in terms of photosynthesis efficacy and linear electron flow (LEF), two important metrics for seedling development and health. Findings confirmed plant specificity; for soybean, the nanostructures resulted in 34.3% higher value of relative chlorophyll content and 41.2% higher value of PS1 centers in photosystem I than the ionic control with equivalent agrichemical concentration. For wheat, the nanostructures resulted in 37.6% higher value of LEF than the ionic agrichemicals applied at 4 times higher concentration, indicating that the responsive core-shell nanostructure is an effective platform to achieve precision agrichemical delivery while minimizing inputs. Moreover, the Zn and Na content in the leaves of 4-week-old soybean seedlings were significantly increased with nanostructure amendment, indicating that the developed nanostructures can potentially be used to modulate the accumulation of other important micronutrients through a potential biofortification strategy.

Association of nanoparticle exposure with serum metabolic disorders of healthy adults in printing centers.

Printers are everyday devices in both our homes and workplaces. We have previously found high occupational exposure levels to toner-based printer emitted nanoparticles (PEPs) at printing centers. To elucidate the potential health effects from exposure to PEPs, a total of 124 human serum samples were collected from 32 workers in the printing centers during the repeated follow-up measurements, and global serum metabolomics were analyzed in three ways: correlation between metabolic response and personal exposure (dose response exposure); metabolite response changes between Monday and Friday of a work week (short-term exposure), and metabolite response in relation to length of service in a center (long-term exposure). A total of 52 key metabolites changed significantly in relation to nanoparticle exposure levels. The primary dysregulated pathways included inflammation and immunity related arginine and tryptophan metabolism. Besides, some distinct metabolite expression patterns were found to occur during the transition from short-term to long-term exposures, suggesting cumulative effect of PEPs exposure. These findings, for the first time, highlight the inhalation exposure responses to printer emitted nanoparticles at the metabolite level, potentially serving as pre-requisites for whole organism and population responses, and are inline with emerging findings on potential health effects.

Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances.

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.

Anisotropic hair keratin-dopamine composite scaffolds exhibit strain-stiffening properties.

Human hair keratin (HHK) has been successfully explored as raw materials for three-dimensional scaffolds for soft tissue regeneration due to its excellent biocompatibility and bioactivity. However, none of the reported HHK based scaffolds is able to replicate the strain-stiffening capacity of living tissues when responding to large deformations. In the present study, strain-stiffening property was achieved in scaffolds fabricated from HHK via a synergistic effect of well-defined, aligned microstructure and chemical crosslinking. Directed ice-templating method was used to fabricate HHK-based scaffolds with highly aligned (anisotropic) microstructure while oxidized dopamine (ODA) was used to crosslink covalently to HHKs. The resultant HHK-ODA scaffolds exhibited strain-stiffening behavior characterized by the increased gradient of the stress-strain curve after the yield point. Both ultimate tensile strength and the elongation at break were enhanced significantly (~700 kPa, ~170%) in comparison to that of HHK scaffolds lacking of aligned microstructure or ODA crosslinking. In vitro cell culture studies indicated that HHK-ODA scaffolds successfully supported human dermal fibroblasts (HDFs) adhesion, spreading and proliferation. Moreover, anisotropic HHK-ODA scaffolds guided cell growth in alignment with the defined microstructure as shown by the highly organized cytoskeletal networks and nuclei distribution. The findings suggest that HHK-ODA scaffolds, with strain-stiffening properties, biocompatibility and bioactivity, have the potential to be applied as biomimetic matrices for soft tissue regeneration.

Biomarkers of oxidative stress in urine and plasma of operators at six Singapore printing centers and their association with several metrics of printer-emitted nanoparticle exposures.

Inhalation of nanoparticles emitted from toner-based printing equipment (TPE), such as laser printers and photocopiers, also known as PEPs, has been associated with systemic inflammation, hypertension, cardiovascular disease, respiratory disorders, and genotoxicity. Global serum metabolomics analysis in 19 healthy TPE operators found 52 dysregulated biomolecules involved in upregulation of inflammation, immune, and antioxidant responses and downregulation of cellular energetics and cell proliferation. Here, we build on the metabolomics study by investigating the association of a panel of nine urinary OS biomarkers reflecting DNA/RNA damage (8OHdG, 8OHG, and 5OHMeU), protein/amino acid oxidation (o-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine), and lipid oxidation (8-isoprostane, 4-hydroxy nonenal, and malondialdehyde [MDA]), as well as plasma total MDA and total protein carbonyl (TPC), with several nanoparticle exposure metrics in the same 19 healthy TPE operators. Plasma total MDA, urinary 5OHMeU, 3-chlorotyrosine, and 3-nitrotyrosine were positively, whereas o-tyrosine inversely and statistically significantly associated with PEPs exposure in multivariate models, after adjusting for age and urinary creatinine. Urinary 8OHdG, 8OHG, 5OHMeU, and total MDA in urine and plasma had group mean values higher than expected in healthy controls without PEPs exposure and comparable to those of workers experiencing low to moderate levels of oxidative stress (OS). The highest exposure group had OS biomarker values, most notably 8OHdG, 8OHG, and total MDA, that compared to workers exposed to welding fumes and titanium dioxide. Particle number concentration was the most sensitive and robust exposure metric. A combination of nanoparticle number concentration and OS potential of fresh aerosols is recommended for larger scale future studies.

Enzyme- and Relative Humidity-Responsive Antimicrobial Fibers for Active Food Packaging.

Active food packaging materials that are sustainable, biodegradable, and capable of precise delivery of antimicrobial active ingredients (AIs) are in high demand. Here, we report the development of novel enzyme- and relative humidity (RH)-responsive antimicrobial fibers with an average diameter of 225 ± 50 nm, which can be deposited as a functional layer for packaging materials. Cellulose nanocrystals (CNCs), zein (protein), and starch were electrospun to form multistimuli-responsive fibers that incorporated a cocktail of both free nature-derived antimicrobials such as thyme oil, citric acid, and nisin and cyclodextrin-inclusion complexes (CD-ICs) of thyme oil, sorbic acid, and nisin. The multistimuli-responsive fibers were designed to release the free AIs and CD-ICs of AIs in response to enzyme and RH triggers, respectively. Enzyme-responsive release of free AIs is achieved due to the degradation of selected polymers, forming the backbone of the fibers. For instance, protease enzyme can degrade zein polymer, further accelerating the release of AIs from the fibers. Similarly, RH-responsive release is obtained due to the unique chemical nature of CD-ICs, enabling the release of AIs from the cavity at high RH. The successful synthesis of CD-ICs of AIs and incorporation of antimicrobials in the structure of the multistimuli-responsive fibers were confirmed by X-ray diffraction and Fourier transform infrared spectrometry. Fibers were capable of releasing free AIs when triggered by microorganism-exudated enzymes in a dose-dependent manner and releasing CD-IC form of AIs in response to high relative humidity (95% RH). With 24 h of exposure, stimuli-responsive fibers significantly reduced the populations of foodborne pathogenic bacterial surrogates Escherichia coli (by ∼5 log unit) and Listeria innocua (by ∼5 log unit), as well as fungi Aspergillus fumigatus (by >1 log unit). More importantly, the fibers released more AIs at 95% RH than at 50% RH, which resulted in a higher population reduction of E. coli at 95% RH. Such biodegradable, nontoxic, and multistimuli-responsive antimicrobial fibers have great potential for broad applications as active and smart packaging systems.

Fate, cytotoxicity and cellular metabolomic impact of ingested nanoscale carbon dots using simulated digestion and a triculture small intestinal epithelial model.

Carbon dots (CDs) are a promising material currently being explored in many industrial applications in the biomedical and agri-food areas; however, studies supporting the environmental health risk assessment of CDs are needed. This study focuses on various CD forms including iron (FeCD) and copper (CuCD) doped CDs synthesized using hydrothermal method, their fate in gastrointestinal tract, and their cytotoxicity and potential changes to cellular metabolome in a triculture small intestinal epithelial model. Physicochemical characterization revealed that 75% of Fe in FeCD and 95% of Cu in CuCD were dissolved during digestion. No significant toxic effects were observed for pristine CDs and FeCDs. However, CuCD induced significant dose-dependent toxic effects including decreases in TEER and cell viability, increases in cytotoxicity and ROS production, and alterations in important metabolites, including D-glucose, L-cysteine, uridine, citric acid and multiple fatty acids. These results support the current understanding that pristine CDs are relatively non-toxic and the cytotoxicity is dependent on the doping molecules.

Liposome interaction with macrophages and foam cells for atherosclerosis treatment: effects of size, surface charge and lipid composition.

Liposomes are potential drug carriers for atherosclerosis therapy due to low immunogenicity and ease of surface modifications that allow them to have prolonged circulation half-life and specifically target atherosclerotic sites to increase uptake efficiency. However, the effects of their size, charge, and lipid compositions on macrophage and foam cell behaviour are not fully understood. In this study, liposomes of different sizes (60 nm, 100 nm and 180 nm), charges (-40 mV, -20 mV, neutral, +15 mV and +30 mV) and lipid compositions (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, L-a-phosphatidylcholine, and egg sphingomyelin) were synthesized, characterized and exposed to macrophages and foam cells. Compared to 100 nm neutral 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes, flow cytometry and confocal imaging indicated that cationic liposomes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPC) liposomes were internalized more by both macrophages and foam cells. Through endocytosis inhibition, phagocytosis and clathrin-mediated endocytosis were identified as the dominant mechanisms of uptake. Anionic and DSPC liposomes induced more cholesterol efflux capacity in foam cells. These results provide a guide for the optimal size, charge, and lipid composition of liposomes as drug carriers for atherosclerosis treatment.

Keratin-Alginate Sponges Support Healing of Partial-Thickness Burns.

Deep partial-thickness burns damage most of the dermis and can cause severe pain, scarring, and mortality if left untreated. This study serves to evaluate the effectiveness of crosslinked keratin-alginate composite sponges as dermal substitutes for deep partial-thickness burns. Crosslinked keratin-alginate sponges were tested for the ability to support human dermal fibroblasts in vitro and to support the closure and healing of partial-thickness burn wounds in Sus scrofa pigs. Keratin-alginate composite sponges supported the enhanced proliferation of human dermal fibroblasts compared to alginate-only sponges and exhibited decreased contraction in vitro when compared to keratin only sponges. As dermal substitutes in vivo, the sponges supported the expression of keratin 14, alpha-smooth muscle actin, and collagen IV within wound sites, comparable to collagen sponges. Keratin-alginate composite sponges supported the regeneration of basement membranes in the wounds more than in collagen-treated wounds and non-grafted controls, suggesting the subsequent development of pathological scar tissues may be minimized. Results from this study indicate that crosslinked keratin-alginate sponges are suitable alternative dermal substitutes for clinical applications in wound healing and skin regeneration.

Synergistic Effect of PVDF-Coated PCL-TCP Scaffolds and Pulsed Electromagnetic Field on Osteogenesis.

Bone exhibits piezoelectric properties. Thus, electrical stimulations such as pulsed electromagnetic fields (PEMFs) and stimuli-responsive piezoelectric properties of scaffolds have been investigated separately to evaluate their efficacy in supporting osteogenesis. However, current understanding of cells responding under the combined influence of PEMF and piezoelectric properties in scaffolds is still lacking. Therefore, in this study, we fabricated piezoelectric scaffolds by functionalization of polycaprolactone-tricalcium phosphate (PCL-TCP) films with a polyvinylidene fluoride (PVDF) coating that is self-polarized by a modified breath-figure technique. The osteoinductive properties of these PVDF-coated PCL-TCP films on MC3T3-E1 cells were studied under the stimulation of PEMF. Piezoelectric and ferroelectric characterization demonstrated that scaffolds with piezoelectric coefficient d33 = -1.2 pC/N were obtained at a powder dissolution temperature of 100 °C and coating relative humidity (RH) of 56%. DNA quantification showed that cell proliferation was significantly enhanced by PEMF as low as 0.6 mT and 50 Hz. Hydroxyapatite staining showed that cell mineralization was significantly enhanced by incorporation of PVDF coating. Gene expression study showed that the combination of PEMF and PVDF coating promoted late osteogenic gene expression marker most significantly. Collectively, our results suggest that the synergistic effects of PEMF and piezoelectric scaffolds on osteogenesis provide a promising alternative strategy for electrically augmented osteoinduction. The piezoelectric response of PVDF by PEMF, which could provide mechanical strain, is particularly interesting as it could deliver local mechanical stimulation to osteogenic cells using PEMF.