Enhanced Delivery of Biomolecules into Caco2 Cells Based on the Cell-Penetrating Ability of Keratin Peptides.

Keratin, as a promising bioresource, possesses significant potential for diverse biological applications due to its favorable biocompatibility, low toxicity, biodegradability, and cell adhesion ability. However, there are few studies on the cell-penetrating ability of keratin peptides (KEPs) for biomolecule delivery. Therefore, this study explored the cell-penetrating ability of KEPs with different molecular weights (Mw) on Caco2 cells using fluorescein-labeled insulin (FITC-INS) as the target intracellular biomolecule. The potential cell-penetrating mechanism was elaborated by combining cellular investigation with the physicochemical characterization of KEPs. The result shows that the KEPs <3 kDa (KEP1) exhibited the highest cell-penetrating ability at 2 mg/mL, allowing efficient delivery of FITC-INS into Caco2 cells without covalent bonding. The cellular uptake mechanism was energy-dependent, mainly involving macropinocytosis. The further fractionation of KEP1 reveals that the most effective components consisted of 8-19 amino acids, including specific hydrophobic peptides (e.g., RVVIEPSPVVV and IIIQPSPVVV), PPII amphipathic peptides (e.g., PPPVVVTFP and FIQPPPVVV), and Cys-rich peptides (e.g., LCAPTPCGPTPL and CLPCRPCGPTPL). Additionally, analysis of the secondary and tertiary structure and amino acid composition illustrated that KEP1 exhibited rich hydrophobic residues and disulfide bonds, which probably contributed to its cell-penetrating ability, as opposed to its small particle size and electrostatic interactions. This study reveals the cell-penetrating ability of KEPs, thus highlighting their potential as biomaterials for noncovalently delivering biomolecules.

Curcumin-loaded keratin-chitosan hydrogels for enhanced peripheral nerve regeneration.

Peripheral nerve injury often leads to symptoms of motor and sensory impairment, and slow recovery of nerves after injury and limited treatment methods will aggravate symptoms or even lead to lifelong disability. Curcumin can promote peripheral nerve regeneration, but how to accurately deliver the appropriate concentration of curcumin in the local peripheral nerve remains to be solved. In this study, we designed a human hair keratin/chitosan (C/K) hydrogel with sodium tripolyphosphate ions crosslinked to deliver curcumin topically. Chitosan improves the mechanical properties of hydrogels and keratin improves the biocompatibility of hydrogels. C/K hydrogel showed good cytocompatibility, histocompatibility and degradability. In vitro experiments showed that hydrogels can continuously release curcumin for up to 10 days. In addition, a comprehensive analysis of behavioral, electrophysiological, histology, and target organ recovery results in animal experiments showed that locally delivered curcumin can enhance nerve regeneration in addition to hydrogels. In short, we provide a new method that combines the advantages of human hair keratin, chitosan, and curcumin for nerve damage repair.

Additive-free Absorbable Keratin Sponge With Procoagulant Activity for Noncompressible Hemostasis.

The development of a natural, additive-free, absorbable sponge with procoagulant activity for noncompressible hemostasis remains a challenging task. In this study, we extracted high molecular weight keratin (HK) from human hair and transformed it into a hemostatic sponge with a well-interconnected pore structure using a foaming technique, freeze-drying, and oxidation cross-linking. By controlling the cross-linking degree, the resulting sponge demonstrated excellent liquid absorption ability, shape recovery characteristics, and robust mechanical properties. The HK10 sponge exhibited rapid liquid absorption, expanding up to 600% within 5 s. Moreover, the HK sponge showed superior platelet activation and blood cell adhesion capabilities. In SD rat liver defect models, the sponges demonstrated excellent hemostatic performance by sealing the wound and expediting coagulation, reducing the hemostatic time from 825 to 297 s. Furthermore, HK sponges have excellent biosafety, positioning them as a promising absorbable sponge with the potential for the treatment of noncompressible hemostasis.

Peptide recovery from chicken feather keratin and their anti-biofilm properties against methicillin-resistant Staphylococcus aureus (MRSA).

Bacteria have the potential to adhere to abiotic surfaces, which has an undesirable effect in the food industry because they can survive for sustained periods through biofilm formation. In this study, an antibacterial peptide (ABP), with a molecular mass of 3861 Da, was purified from hydrolyzed chicken feathers using a locally isolated keratinolytic bacterium, namely Rhodococcus erythropolis, and its antibacterial and antibiofilm potential were investigated against planktonic and biofilm cells of Methicillin-Resistant Staphylococcus Aureus (MRSA). The results demonstrated that purified ABP showed the growth inhibition of MRSA cells with the minimum inhibitory concentration (MIC) of 45 µg/ml and disrupted MRSA biofilm formation at a concentration of 200 ug/ml, which results were confirmed by scanning electron micrograph (SEM). Moreover, the secondary structures of the peptide were assessed as part of the FTIR analysis to evaluate its mode of action. ExPASy tools were used to predict the ABP sequence, EPCVQUQDSRVVIQPSPVVVVTLPGPILSSFPQNTA, from a chicken feather keratin sequence database following in silico digestion by trypsin. Also, ABP had 54.29% hydrophobic amino acids, potentially contributing to its antimicrobial activity. The findings of toxicity prediction of the peptide by the ToxinPred tool revealed that ABP had non-toxic effects. Thus, these results support the potential of this peptide to be used as an antimicrobial agent for the treatment or prevention of MRSA biofilm formation in feed, food, or pharmaceutical applications.

Rhizoctonia solani disease suppression: addition of keratin-rich soil amendment leads to functional shifts in soil microbial communities.

Promoting soil suppressiveness against soil borne pathogens could be a promising strategy to manage crop diseases. One way to increase the suppression potential in agricultural soils is via the addition of organic amendments. This microbe-mediated phenomenon, although not fully understood, prompted our study to explore the microbial taxa and functional properties associated with Rhizoctonia solani disease suppression in sugar beet seedlings after amending soil with a keratin-rich waste stream. Soil samples were analyzed using shotgun metagenomics sequencing. Results showed that both amended soils were enriched in bacterial families found in disease suppressive soils before, indicating that the amendment of keratin-rich material can support the transformation into a suppressive soil. On a functional level, genes encoding keratinolytic enzymes were found to be abundant in the keratin-amended samples. Proteins enriched in amended soils were those potentially involved in the production of secondary metabolites/antibiotics, motility, keratin-degradation, and contractile secretion system proteins. We hypothesize these taxa contribute to the amendment-induced suppression effect due to their genomic potential to produce antibiotics, secrete effectors via the contractile secretion system, and degrade oxalate-a potential virulence factor of R. solani-while simultaneously possessing the ability to metabolize keratin.

Thermoresponsive keratin-methylcellulose self-healing injectable hydrogel accelerating full-thickness wound healing by promoting rapid epithelialization.

Chronic wounds suffer from impaired healing due to microbial attack and poor vascular growth. Thermoresponsive hydrogels gained attention in wound dressing owing to their gelation at physiological temperature enabling them to take the shape of asymmetric wounds. The present study delineates the development of thermoresponsive hydrogel (MCK), from hair-derived keratin (K) and methylcellulose (MC) in the presence of sodium sulfate. The gelation temperature (Tg) of this hydrogel is in the range of 30 °C to 33 °C. Protein-polymer interaction leading to thermoreversible sol-gel transition involved in MCK blends has been analyzed and confirmed by FTIR, XRD, and thermal studies. Keratin, has introduced antioxidant properties to the hydrogel imparted cytocompatibility towards human dermal fibroblasts (HDFs) as evidenced by both MTT and live dead assays. In vitro wound healing assessment has been shown by enhanced migration of HDFs in the presence of MCK hydrogel compared to the control. Also, CAM assay and CD31 expression by the Wistar rat model has shown increased blood vessel branching after the implantation of MCK hydrogel. Further, in vivo study, demonstrated MCK efficacy of hydrogel in accelerating full-thickness wounds with minimal scarring in Wistar rats, re-epithelialization, and reinstatement of the epidermal-dermal junction thereby exhibiting clinical relevance for chronic wounds.

Keratin Microspheres as Promising Tool for Targeting Follicular Growth.

Skin is the body barrier that constrains the infiltration of particles and exogenous aggression, in which the hair follicle plays an important role. Recent studies have shown that small particles can penetrate the skin barrier and reach the hair follicle, making them a potential avenue for delivering hair growth-related substances. Interestingly, keratin-based microspheres are widely used as drug delivery carriers in various fields. In this current study, we pursue the effect of newly synthesized 3D spherical keratin particles on inducing hair growth in C57BL/6 male mice and in human hair follicle dermal papilla cells. The microspheres were created from partially sulfonated, water-soluble keratin. The keratin microspheres swelled in water to form spherical gels, which were used for further experiments. Following topical application for a period of 20 days, we observed a regrowth of hair in the previously depleted area on the dorsal part of the mice in the keratin microsphere group. This observation was accompanied by the regulation of hair-growth-related pathways as well as changes in markers associated with epidermal cells, keratin, and collagen. Interestingly, microsphere keratin treatment enhanced the cell proliferation and the expression of hair growth markers in dermal papilla cells. Based on our data, we propose that 3D spherical keratin has the potential to specifically target hair follicle growth and can be employed as a carrier for promoting hair growth-related agents.

Engineered keratin/bFGF hydrogel to promote diabetic wound healing in rats.

Keratin materials are promising in wound healing acceleration, however, it is a challenge for the keratin to efficiently therapy the impaired wound healing, such as diabetic foot ulcers. Here, we report a keratin/bFGF hydrogel for skin repair of chronic wounds in diabetic rats based on their characteristics of extracellular matrix and growth factor degradation in diabetic ulcer. Recombinant keratin 31 (K31), the most abundant keratin in human hair, exhibited the highly efficient performances in cell adhesion, proliferation and migration. More importantly, the introduction of bFGF into K31 hydrogel significantly enhances the properties of cell proliferation, wound closure acceleration, angiogenesis and skin appendages regeneration. Furthermore, the combination of K31 and bFGF can promote epithelial-mesenchymal transition by inhibiting the expression of E-cadherin and promoting the expression of vimentin and fibronectin. These findings demonstrate the engineered K31/bFGF hydrogel as a promising therapeutic agent for diabetic wound healing.

Fabrication of bioinspired keratin/sodium alginate based biopolymeric mat loaded with herbal drug and green synthesized zinc oxide nanoparticles as a dual drug antimicrobial wound dressing.

Dual drug antibacterial wound dressings with biological materials possess crucial wound healing characteristics including biocompatibility, non-toxicity, degradability, mechanical strength and antibacterial properties. The study focusses on fabricating keratin (K)­sodium alginate (A) based wound dressings by loading green synthesized zinc oxide nanoparticles (ZnO NPs) using C. roseus (leaf extract) and M. recutita (Chamomile flower part) herbal drug (CH) as a bioactive dual antibacterial wound dressing for the first time. The optimized ZnO NPs and CH exhibits strong physiochemical and electrostatic interactions (FT-IR, XRD and SEM) on the fabricated K-A-CH-ZnO biopolymeric mats. Moreover, the tiny porous network of the biopolymeric mat enhances thermal stability, hydrophilicity, mechanical strength and explores the water vapor transmission (2538.07 g/m2/day) and oxygen permeability (7.38 ± 0.31 g/m2) to maintain moist environment and cell-material interactions. During enzymatic degradation studies, ZnO NPs and CH of biopolymeric mat not only retains structural integrity but also increases the characteristic of swelling with sustained drug release (57 %) in 144 h which accelerates wound healing process. Also, K-A-CH-ZnO mat exhibited excellent antibacterial effects against B. subtilis and E. coli. Furthermore, NIH 3T3 fibroblast cell behavior using MTT assay and in vivo evaluations of biopolymeric mat depicted enhanced biocompatibility with increased collagen deposition at the wound site as a prominent dual drug medicated antimicrobial wound dressing.

Keratin- and VEGF-Incorporated Honey-Based Sponge-Nanofiber Dressing: An Ideal Construct for Wound Healing.

To overcome the drawbacks of single-layered wound dressings, bilayer dressings are now introduced as an alternative to achieve effective and long-term treatment. Here, a bilayer dressing composed of electrospun nanofibers in the bottom layer (BL) and a sponge structure as the top layer (TL) is presented. Hydrophilic poly(acrylic acid) (PAAc)-honey (Hny) with interconnected pores of 76.04 µm was prepared as the TL and keratin (Kr), Hny, and vascular endothelial growth factor (VEGF) were prepared as the BL. VEGF indicates a gradual release over 7 days, promoting angiogenesis, as proven by the chick chorioallantoic membrane assay and in vivo tissue histomorphology observation. Additionally, the fabricated dressing material indicated a satisfactory tensile profile, cytocompatibility for human keratinocyte cells, and the ability to promote cell attachment and migration. The in vivo animal model demonstrated that the full-thickness wound healed faster when it was covered with PAAc-Hny/Hny-Kr-VEGF than in other groups. Additionally, faster blood vessel formation, collagen synthetization, and epidermal layer generation were also confirmed, which have proven efficient healing acceleration in wounds treated with synthesized bilayer dressings. Our findings indicated that the fabricated material can be promising as a functional wound dressing.

Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Ceramide AD™ Restores Skin Integrity and Function following Exposure to House Dust Mite.

Ceramides are epidermal lipids important for normal skin barrier function. Reduced Ceramide content is associated with atopic dermatitis (AD). House dust mite (HDM) has been localized in AD skin where it plays an exacerbator role. We set to examine the impact of HDM on skin integrity and the effect of three separate Ceramides (AD™, DS, Y30) on HDM-induced cutaneous damage. The effect was tested in vitro on primary human keratinocytes and ex vivo on skin explants. HDM (100 µg/mL) decreased the expression of adhesion protein E-cadherin, supra-basal (K1, K10) and basal (K5, K14) keratins and increased matrix metallopeptidase (MMP)-9 activity. The presence of Ceramide AD™ in topical cream inhibited HDM-induced E-cadherin and keratin destruction and dampened MMP-9 activity ex vivo which was not seen for the control cream or cream containing DS or Y30 Ceramides. The efficacy of Ceramide AD™ was tested in a clinical setting on moderate to very dry skin (as surrogate for environment-induced skin damage). When applied topically for 21 days, Ceramide AD™ significantly reduced transepidermal water loss (TEWL) in patients with very dry skin compared to their TEWL baseline data. Our study demonstrates Ceramide AD™ cream to be effective in restoring skin homeostasis and barrier function in damaged skin and warrants testing in larger clinical trials for possible treatment of AD and xerosis.

Feather keratin-montmorillonite nanocomposite hydrogel promotes bone regeneration by stimulating the osteogenic differentiation of endogenous stem cells.

Bone defects caused by bone trauma, infection, surgery, or other systemic diseases remain a severe challenge for the medical field. To address this clinical problem, different hydrogels were exploited to promote bone tissue regrowth and regeneration. Keratins are natural fibrous proteins found in wool, hair, horns, nails, and feather. Due to their unique characteristics of outstanding biocompatibility, great biodegradability, and hydrophilic, keratins have been widely applicated in different fields. In our study, the feather keratin-montmorillonite nanocomposite hydrogels that consist of keratin hydrogels serving as the scaffold support to accommodate endogenous stem cells and montmorillonite is synthesized. The introduction of montmorillonite greatly improves the osteogenic effect of the keratin hydrogels via bone morphogenetic protein 2 (BMP-2)/phosphorylated small mothers against decapentaplegic homolog 1/5/8 (p-SMAD 1/5/8)/runt-related transcription factor 2 (RUNX2) expression. Moreover, the incorporation of montmorillonite into hydrogels can improve the mechanical properties and bioactivity of the hydrogels. The morphology of feather keratin-montmorillonite nanocomposite hydrogels was shown by scanning electron microscopy (SEM) to have an interconnected porous structure. The incorporation of montmorillonite into the keratin hydrogels was confirmed by the energy dispersive spectrum (EDS). We prove that the feather keratin-montmorillonite nanocomposite hydrogels enhance the osteogenic differentiation of BMSCs. Furthermore, micro-CT and histological analysis of rat cranial bone defect demonstrated that feather keratin-montmorillonite nanocomposite hydrogels dramatically stimulated bone regeneration in vivo. Collectively, feather keratin-montmorillonite nanocomposite hydrogels can regulate BMP/SMAD signaling pathway to stimulate osteogenic differentiation of endogenous stem cells and promote bone defect healing, indicating their promising candidate in bone tissue engineering.

Biological evaluation and osteogenic potential of polyhydroxybutyrate-keratin/Al2O3 electrospun nanocomposite scaffold: A novel bone regeneration construct.

In this study, the effect of alumina nanowire on the physical and biological properties of polyhydroxybutyrate-keratin (PHB-K) electrospun scaffold was investigated. First, PHB-K/alumina nanowire nanocomposite scaffolds were made with an optimal concentration of 3 wt% alumina nanowire by using the electrospinning method. The samples were examined in terms of morphology, porosity, tensile strength, contact angle, biodegradability, bioactivity, cell viability, ALP activity, mineralization ability, and gene expression. The nanocomposite scaffold provided a porosity of >80 % and a tensile strength of about 6.72 MPa, which were noticeable for an electrospun scaffold. AFM images showed an increase in surface roughness with the presence of alumina nanowires. This led to an improvement in the degradation rate and bioactivity of PHB-K/alumina nanowire scaffolds. The viability of mesenchymal cells, alkaline phosphatase secretion, and mineralization significantly increased with the presence of alumina nanowire compared to PHB and PHB-K scaffolds. In addition, the expression level of collagen I, osteocalcin, and RUNX2 genes in nanocomposite scaffolds increased significantly compared to other groups. In general, this nanocomposite scaffold could be a novel and interesting construct for osteogenic induction in bone tissue engineering.

Chlorhexidine-loaded polysulfobetaine/keratin hydrogels with antioxidant and antibacterial activity for infected wound healing.

Multifunctional hydrogel dressings are promising for wound healing. In the study, chlorhexidine(CHX) loaded double network hydrogels were prepared by free radical polymerization of sulfobetaine and oxidative self-crosslinking of reduced keratin. The introduced keratin and CHX endowed hydrogels with cytocompatibility, antioxidant capability as well as enhanced antibacterial activity due to the antifouling property of polysulfobetaine. These hydrogels exhibited acidity, glutathione(GSH), and trypsin triple-responsive release behaviors, resulting in the accelerated release of CHX under wound microenvironments. Intriguingly, the freeze-drying hydrogels could be ground to powders and sprinkled on the irregular wound bed, followed by absorbing wound fluid to reform hydrogel in situ. These xerogel powders were more convenient for sterilization, formulation, and storage. Further, these xerogel powders could be rejected after being mixed with an appropriate amount of water. In vivo infected wound healing confirmed that the xerogel powder dressing significantly promoted collagen deposition and reduced inflammation, thereby accelerating the closure and regeneration of skin wounds. Taken together, these degradable xerogel powders have great potential applications for wound healing.

Novel PLCL nanofibrous/keratin hydrogel bilayer wound dressing for skin wound repair.

In this study, a novel poly(L-lactate-caprolactone) copolymer (PLCL) nanofibrous/keratin hydrogel bilayer wound dressing loaded with fibroblast growth factor (FGF-2) was prepared by the low-pressure filtration-assisted method. The ability of the keratin hydrogel in the bilayer dressing to mimic the dermis and that of the nanofibrous PLCL to mimic the epidermis were discussed. Keratin hydrogel exhibited good porosity and maximum water absorption of 874.09%. Compared with that of the dressing prepared by the coating method, the interface of the bilayer dressing manufactured by the low-pressure filtration-assisted method (filtration time: 20 min) was tightly bonded, and its bilayer dressing interface could not be easily peeled off. The elastic modulus of hydrogel was about 44 kPa, which was similar to the elastic modulus of the dermis (2-80 kPa). Additionally, PLCL nanofibers had certain toughness and flexibility suitable for simulating the epidermal structures. In vitro studies showed that the bilayer dressing was biocompatible and biodegradable. In vivo studies indicated that PLCL/keratin-FGF-2 bilayer dressing could promote re-epithelialization, collagen deposition, skin appendages (hair follicles) regeneration, microangiogenesis construction, and adipose-derived stem cells (ADSCs) recruitment. The introduction of FGF-2 resulted in a better repair effect. The bilayer dressing also solved the problems of poor interface adhesion of hydrogel/electrospinning nanofibers. This paper also explored the preliminary role and mechanism of bilayer dressing in promoting skin healing, showing that its potential applications as a biomedical wound dressing in the field of skin tissue engineering.

Nitric oxide releasing poly(vinyl alcohol)/S-nitrosated keratin film as a potential vascular graft.

Nitric oxide (NO) releasing vascular graft is promising due to its merits of thromboembolism reduction and endothelialization promotion. In this study, keratin-based NO donor of S-nitrosated keratin (KSNO) was blended with poly(vinyl alcohol) (PVA) and further crosslinked with sodium trimetaphosphate (STMP) to afford PVA/KSNO biocomposite films. These films could release NO sustainably for up to 10 days, resulting in the promotion of HUVECs growth and the inhibition of HUASMCs growth. In addition, these films displayed good blood compatibility and antibacterial activity. Taken together, these films have potential applications in vascular grafts.

Bioinspired Robust Keratin Hydrogels for Biomedical Applications.

Although keratins are robust in nature, hydrogels producing their extracts exhibit poor mechanical properties due to the complicated composition and ineffective self-assembly. Here we report a bioinspired strategy to fabricate robust keratin hydrogels based on mechanism study through recombinant proteins. Homotypic and heterotypic self-assembly of selected type I and type II keratins in different combinations was conducted to identify crucial domain structures for the process, their kinetics, and relationship with the mechanical strength of hydrogels. Segments with best performance were isolated and used to construct novel assembling units. The new design outperformed combinations of native proteins in mechanical properties and in biomedical applications such as controlled drug release and skin regeneration. Our approach not only elucidated the critical structural domains and underlying mechanisms for keratin self-assembly but also opens an avenue toward the rational design of robust keratin hydrogels for biomedical applications.

Zwitterionic keratin coating on silk-Laponite fibrous membranes for guided bone regeneration.

Implant-related infection is one of the main challenges in periodontal diseases. According to the zwitterionic properties of keratin, we aim to develop guided bone regeneration (GBR) membrane with antibacterial and bioactivity properties using a keratin coating. In this study, electrospun silk fibroin (SF)-Laponite (LAP) fibrous membranes were developed as GBR membranes, and keratin extracted from sheep wool was electrosprayed on them. Here, the role of electrospraying time (2, 3, and 4h) on the properties of the GBR membranes was investigated. After physicochemical characterization of the keratin-modified membranes, in vitro bioactivity and degradation rate of the membranes were studied in simulated body fluid and phosphate buffer saline, respectively. Moreover, proliferation and differentiation of mesenchymal stem cells were evaluated in contact with the keratin-modified SF-LAP membrane. Finally, the antibacterial activity of membranes against gram-positive bacteria (Staphylococcus aureus) was investigated. Results demonstrated the successful formation of homogeneous wool keratin coating on SF-LAP fibrous membranes using a simple electrospray process. While wool keratin coating significantly enhanced the elongation and hydrophilicity of the SF-LAP membrane, the mechanical strength was not changed. In addition, keratin coating significantly improved the bioactivity and degradation rate of SF-LAP membranes, owing to the carboxyl groups of amino acids in keratin coating. In addition, the synergic role of LAP nanoparticles and keratin coating drastically improved osteoblast proliferation and differentiation. Finally, the zwitterionic property of wool keratin coating originating from their equal positive (NH3 + ) and negative (COO- ) charges considerably improved the antibacterial activity of the SF-LAP membrane. Overall, keratin-coated SF-LAP fibrous membranes with significant mechanical and biological properties could have the potential for GBR membranes.

Investigation of combined effects of propyl paraben and methyl paraben on the hypothalamic-pituitary-adrenal axis in male rats.

The aim of this study was to investigate the endocrine-disrupting effects of methyl paraben (MeP) and propyl paraben (PrP) mixture on the hypothalamic-pituitary-adrenal axis (HPA). In this study, six experimental groups were designated. These groups included three control groups (control, corn oil control, and positive control (50 mg/kg/day BPA)) and three dose groups (10, 100, and 500 mg/kg/day MeP+PrP). MeP with PrP were mixed in a 1:1 ratio and administered to the 42-day-old male rats by oral gavage for 30 days. At the end of the experiment, adrenocorticotropic hormone (ACTH), corticosterone and aldosterone hormones were analyzed in serum. Effects of MeP+PrP on the adrenal glands were investigated by immunohistochemical staining of 11ß hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) enzymes involved in the synthesis steps of corticosterone and aldosterone. Also, pituitary and adrenal glands were examined histopathologically. In the histopathological findings, cortical nodule, congestion, and edema were found in the tissues. In the pituitary gland, cytokeratin rings were detected in all MeP+PrP dose groups, supporting the increase of corticosterone and ACTH. Serum corticosterone, aldosterone, and ACTH hormone levels were increased in the 100 mg/kg/day MeP+PrP and BPA groups. Results obtained from immunohistochemical staining showed that increased staining parallelled increased corticosterone and aldosterone hormone levels. In summary, the results showed that exposure to the MeP+PrP mixture caused a significant increase in ACTH and corticosterone. Also, the MeP+PrP mixture caused a significant increase of CYP11B1 and CYP11B2. MeP+PrP exposure disrupts the normal HPA axis.