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.

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.

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.

A keratin/chitosan sponge with excellent hemostatic performance for uncontrolled bleeding.

Uncontrolled bleeding leads to a higher fatality rate in the situation of surgery, traffic accidents and warfare. Traditional hemostatic materials such as bandages are not ideal for uncontrolled or incompressible bleeding. Therefore, it is of great significance to develop a new medical biomaterial with excellent rapid hemostatic effect. Keratin is a natural, biocompatible and biodegradable protein which contains amino acid sequences that induce cell adhesion. As a potential biomedical material, keratin has been developed and paid attention in tissue engineering fields such as promoting wound healing and nerve repair. Herein, a keratin/chitosan (K/C) sponge was prepared to achieve rapid hemostasis. The characterizations of K/C sponge were investigated, including SEM, TGA, liquid absorption and porosity, showing that the high porosity up to 90.12 ± 2.17 % resulted in an excellent blood absorption. The cytotoxicity test and implantation experiment proved that the K/C sponge was biocompatible and biodegradable. Moreover, the prepared K/C sponge showed better hemostatic performance than chitosan sponge (CS) and the commercially available gelatin sponge in both rat tail amputation and liver trauma bleeding models. Further experiments showed that K/C sponge plays a hemostatic role through the endogenous coagulation pathway, thus shortening the activated partial thromboplastin time (APTT) effectively. Therefore, this study provided a K/C sponge which can be served as a promising biomedical hemostatic material.

Keratose hydrogel for tissue regeneration and drug delivery.

Keratin (KRT), a natural fibrous structural protein, can be classified into two categories: "soft" cytosolic KRT that is primarily found in the epithelia tissues (e.g., skin, the inner lining of digestive tract) and "hard" KRT that is mainly found in the protective tissues (e.g., hair, horn). The latter is the predominant form of KRT widely used in biomedical research. The oxidized form of extracted KRT is exclusively denoted as keratose (KOS) while the reduced form of KRT is termed as kerateine (KRTN). KOS can be processed into various forms (e.g., hydrogel, films, fibers, and coatings) for different biomedical applications. KRT/KOS offers numerous advantages over other types of biomaterials, such as bioactivity, biocompatibility, degradability, immune/inflammatory privileges, mechanical resilience, chemical manipulability, and easy accessibility. As a result, KRT/KOS has attracted considerable attention and led to a large number of publications associated with this biomaterial over the past few decades; however, most (if not all) of the published review articles focus on KRT regarding its molecular structure, biochemical/biophysical properties, bioactivity, biocompatibility, drug/cell delivery, and in vivo transplantation, as well as its applications in biotechnical products and medical devices. Current progress that is directly associated with KOS applications in tissue regeneration and drug delivery appears an important topic that merits a commentary. To this end, the present review aims to summarize the current progress of KOS-associated biomedical applications, especially focusing on the in vitro and in vivo effects of KOS hydrogel on cultured cells and tissue regeneration following skin injury, skeletal muscle loss, peripheral nerve injury, and cardiac infarction.

Vibrational Study on Structure and Bioactivity of Protein Fibers Grafted with Phosphorylated Methacrylates.

In the last decades, silk fibroin and wool keratin have been considered functional materials for biomedical applications. In this study, fabrics containing silk fibers from Bombyx mori and Tussah silk fibers from Antheraea pernyi, as well as wool keratin fabrics, were grafted with phosmer CL and phosmer M (commercial names, i.e., methacrylate monomers containing phosphate groups in the molecular side chain) with different weight gains. Both phosmers were recently proposed as flame retarding agents, and their chemical composition suggested a possible application in bone tissue engineering. IR and Raman spectroscopy were used to disclose the possible structural changes induced by grafting and identify the most reactive amino acids towards the phosmers. The same techniques were used to investigate the nucleation of a calcium phosphate phase on the surface of the samples (i.e., bioactivity) after ageing in simulated body fluid (SBF). The phosmers were found to polymerize onto the biopolymers efficiently, and tyrosine and serine underwent phosphorylation (monitored through the strengthening of the Raman band at 1600 cm-1 and the weakening of the Raman band at 1400 cm-1, respectively). In grafted wool keratin, cysteic acid and other oxidation products of disulphide bridges were detected together with sulphated residues. Only slight conformational changes were observed upon grafting, generally towards an enrichment in ordered domains, suggesting that the amorphous regions were more prone to react (and, sometimes, degrade). All samples were shown to be bioactive, with a weight gain of up to 8%. The most bioactive samples contained the highest phosmers amounts, i.e., the highest amounts of phosphate nucleating sites. The sulphate/sulphonate groups present in grafted wool samples appeared to increase bioactivity, as shown by the five-fold increase of the IR phosphate band at 1040 cm-1.

Synergistic effect of sericin and keratin in gelatin based nanofibers for in vitro applications.

Protein-based nanomaterials are gaining growing interest in biomedical field. The present paper evaluates the physico-chemical properties of electrospun nanofibers resulting from the combination of gelatin with keratin (from wool) and sericin (from silk) to validate their use for in vitro interaction studies. We demonstrated that that presence of sericin influences the fiber morphology at macroscopic level - i.e., wide diameter distributions by SEM and image analysis - with effects on chemical - i.e., a decrease of hydrogen bonds of NH groups verified by infrared spectroscopy - and thermal behavior of electrospun nanofibers, in comparison with gelatin-based ones. Moreover, we verified that sericin, in combination with keratin macromolecules, can amplify the biochemical signal of gelatin, improving the in-vitro stability of gelatin-based nanofibers. In vitro results confirm a synergistic effect of sericin and keratin on human Mesenchymal Stem Cells (hMSC) proliferation - increase over 50% respect to other types - associated to the enhancement of in vitro stability directly ascribable to the peculiar physical interaction among the proteins. These findings suggest the use of sericin/keratin/gelatin enriched electrospun fibers as nanostructured platforms for interface tissue engineering.

Keratin Scaffolds Containing Casomorphin Stimulate Macrophage Infiltration and Accelerate Full-Thickness Cutaneous Wound Healing in Diabetic Mice.

Impaired wound healing is a major medical challenge, especially in diabetics. Over the centuries, the main goal of tissue engineering and regenerative medicine has been to invent biomaterials that accelerate the wound healing process. In this context, keratin-derived biomaterial is a promising candidate due to its biocompatibility and biodegradability. In this study, we evaluated an insoluble fraction of keratin containing casomorphin as a wound dressing in a full-thickness surgical skin wound model in mice (n = 20) with iatrogenically induced diabetes. Casomorphin, an opioid peptide with analgesic properties, was incorporated into keratin and shown to be slowly released from the dressing. An in vitro study showed that keratin-casomorphin dressing is biocompatible, non-toxic, and supports cell growth. In vivo experiments demonstrated that keratin-casomorphin dressing significantly (p < 0.05) accelerates the whole process of skin wound healing to the its final stage. Wounds covered with keratin-casomorphin dressing underwent reepithelization faster, ending up with a thicker epidermis than control wounds, as confirmed by histopathological and immunohistochemical examinations. This investigated dressing stimulated macrophages infiltration, which favors tissue remodeling and regeneration, unlike in the control wounds in which neutrophils predominated. Additionally, in dressed wounds, the number of microhemorrhages was significantly decreased (p < 0.05) as compared with control wounds. The dressing was naturally incorporated into regenerating tissue during the wound healing process. Applied keratin dressing favored reconstruction of more regular skin structure and assured better cosmetic outcome in terms of scar formation and appearance. Our results have shown that insoluble keratin wound dressing containing casomorphin supports skin wound healing in diabetic mice.

Stability and cytotoxicity of DPPH inhibitory peptides derived from biodegradation of chicken feather.

The antioxidant activity and cell viability of feather hydrolysates obtained with the Bacillus licheniformis were evaluated using an in-vitro model. The results indicate that feathers-derived peptides under 3 kDa have antioxidant activity with IC50 values of 5.03 ± 0.215 mg/mL by using DPPH antioxidant assay. Although the antioxidant activity of the peptides under 3 kDa preserved after applying diverse heating (from 20 to 100 °C), they lost their activity under strongly acidic or alkaline conditions. Antioxidant activity of the mixed feather bioactive peptides (MFBPs) obtained with partial purification of peptides under 3 kDa was with IC50 amount of 0.169 mg/mL ± 0.004 using DPPH radical scavenging assay. Also, MFBPs within an amount range of from 0.0048 to 5.0 mg/mL, illustrated no cytotoxicity to gingival fibroblast blood cell lines. In light of our results, the obtained value-added peptides could be useful in different food products as a future functional ingredient with antioxidant potency.

A keratin-based microparticle for cell delivery.

Keratin-based biomaterials represent an attractive opportunity in the fields of wound healing and tissue regeneration, not only for their chemical and physical properties, but also for their ability to act as a delivery system for a variety of payloads. Importantly, keratins are the only natural biomaterial that is not targeted by specific tissue turnover-related enzymes, giving it potential stability advantages and greater control over degradation after implantation. However, in-situ polymerization chemistry in some keratin systems are not compatible with cells, and incorporation within constructs such as hydrogels may lead to hypoxia and cell death. To address these challenges, we envisioned a pre-formed keratin microparticle on which cells could be seeded, while other payloads (e.g. drugs, growth factors or other biologic compounds) could be contained within, although studies investigating the potential partitioning between phases during emulsion polymerization would need to be conducted. This study employs well-established water-in-oil emulsion procedures as well as a suspension culture method to load keratin-based microparticles with bone marrow-derived mesenchymal stem cells. Fabricated microparticles were characterized for size, porosity and surface structure and further analyzed to investigate their ability to form gels upon hydration. The suspension culture technique was validated based on the ability for loaded cells to maintain their viability and express actin and vinculin proteins, which are key indicators of cell attachment and growth. Maintenance of expression of markers associated with cell plasticity was also investigated. As a comparative model, a collagen-coated microparticle (Sigma) of similar size was used. Results showed that an oxidized form of keratin ("keratose" or "KOS") formed unique microparticle structures of various size that appeared to contain a fibrous sub-structure. Cell adhesion and viability was greater on keratin microparticles compared to collagen-coated microparticles, while marker expression was retained on both.

The adaptive immune response to porous regenerated keratin as a bone graft substitute in an ovine model.

Reconstituted keratin is a novel bone graft material when prepared as a rigid scaffold. Understanding the immunogenicity of this material is important to determine whether this substance is a viable surgical option. Previous studies have shown no innate immune system activation in response to reconstituted keratin implants. To examine antibody-mediated immune responses to reconstituted keratin implants, bone and blood samples were taken from twelve sheep with surgically created tibial defects containing such implants. RT-PCR was used to detect mRNA of the inflammatory marker SOCS 3 in local bony tissue, and a novel immunohistochemistry assay developed to detect antikeratin antibodies in serum. Two animals were sacrificed per time-point at weeks 1, 2, 4, 6, 8 and 12. Time points for serum analysis included baseline (pre-surgery) and all other time points; mRNA analysis examined samples from all time points. No upregulation in antikeratin antibodies or SOCS 3 mRNA was observed at any time point, indicating that reconstituted keratin implants do not trigger an adaptive immune response in vivo in an ovine model. These findings provide the platform for further development of keratin implants in other mammalian models to define its immunogenic profile and safety.

Fabrication of an expandable keratin sponge for improved hemostasis in a penetrating trauma.

Effective hemostasis improvements for penetrating traumas remain a research priority for civilian and noncivilian applications. Herein, we fabricated an expandable keratin sponge (EKS) for the hemostatic treatment of a penetrating trauma based on the excellent hemostatic ability of keratin and the expandable property of polyacrylamide (PAM). EKSs with semi-interpenetrating networks were fabricated by radical polymerization of keratin and PAM, and the EKS showed rapid expansion upon blood absorption. This sponge exhibited effective hemostasis on a rat penetrating liver hemorrhage, and the expansion of the EKS was dependent on the bleeding volume. In addition, the results of a shear wave elastography analysis showed that the elasticity of the liver tissue increased from 12.5 kPa to 21.2 kPa after the penetrating liver trauma treated by the EKS, and the mechanical strength of the liver tissue was maintained after 1 h of the EKS application. Further in vivo tests indicated the effectiveness of the EKS for hemostasis in a swine femoral artery transection hemorrhage model. This EKS is promising for hemostatic applications.

Highly polydisperse keratin rich nanofibers: Scaffold design and in vitro characterization.

The use of bioactive proteins such as keratin has been successfully explored to improve the biological interface of scaffolds with cells during the tissue regeneration. In this work, it is optimized the fabrication of nanofibers combining wool keratin extracted by sulfitolysis, with polycaprolactone (PCL) in order to design bicomponent fibrous matrices able to exert a self-adapting pattern of signals-morphological, chemical, or physical-confined at the single fiber level, to influence cell and bacteria interactions. It is demonstrated that the blending of highly polydisperse keratin with PCL (50:50) improves the stability of the electrospinning process, promoting the formation of nanofibers-144.1 ± 43.9 nm-without the formation of defects (i.e., beads, ribbons) typically recognized in the fabrication of keratin ones. Moreover, keratin drastically increases the fiber hydrophilicity-compared with PCL fiber alone-thus improving the hMSC adhesion and in vitro proliferation until 14 days. Moreover, the growth of bacterial strains (i.e., Escherichia coli and Staphylococcus aureus) seems to be not specifically inhibited by the contribution of keratin, so that the integration of further selected compounds (i.e., metal ions) is suggested to more efficiently fight against bacteria resistance, to make them suitable for the regeneration of different interfaces and soft tissues (i.e., skin and cornea). © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1803-1813, 2019.

Using of keratin substrate for enrichment of HT29 colorectal cancer stem-like cells.

Eradication of cancer stem-like cells (CSLCs) are becoming increasingly an important target for new cancer therapies. The ability to study their behavior in vitro will provide the opportunity for high-throughput testing of more effective treatments. In this study, spheroid-like structures' formation and enrichment of HT29 CSLCs were evaluated on a wool keratin-based substrate as a bio-mimic of natural extracellular matrix (ECM) proteins. The results indicated that culturing on keratin substrate increased spheroid formation ability and radio-/chemoresistance of HT29 cells. Moreover, cell surface expression of CD133 CSLCs' marker and the mRNA level of stemness genes such as Nanog, Oct4, and c-MYC were increased. These data suggest that keratin can potentially be used for spheroid-like structure formation and enrichment of HT29 CSLCs. In addition, it seems that the induction of stemness characteristics on keratin substrate is probably because of the activation of α2 ß1 integrin signaling pathway. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1264-1271, 2019.

Development of a novel keratin dressing which accelerates full-thickness skin wound healing in diabetic mice: In vitro and in vivo studies.

Impaired wound healing is a major medical problem in diabetes. The objective of this study was to determine the possible application of an insoluble fraction of fur-derived keratin biomaterial as a wound dressing in a full thickness surgical skin wound model in mice ( n = 20) with iatrogenically induced diabetes. The obtained keratin dressing was examined in vitro and in vivo. In vitro study showed the keratin dressing is tissue biocompatible and non-toxic for murine fibroblasts. Antimicrobial examination revealed the keratin dressing inhibited the growth of S. aureus and E. coli. In vivo studies showed the obtained dressing significantly ( p < 0.05) accelerated healing during the first week after surgery compared to control wounds. Keratin dressings were incorporated naturally into granulation and regenerating tissue without any visible signs of inflammatory response, which was confirmed by clinical and histopathological analysis. It is one of the first studies to show application of insoluble keratin proteins and its properties as a wound dressing. The obtained keratin dressing accelerated wound healing in mice with iatrogenically induced diabetes. Therefore, it can be considered as a safe and efficient wound dressing. Although future studies are needed to explain the molecular mechanism behind fur-derived keratin effect during the multilayer wound healing process, our findings may open the way for a new class of insoluble fur keratin dressings in chronic difficult to heal wounds treatment.

Keratin biomaterials augment anti-inflammatory macrophage phenotype in vitro.

Tissue regeneration following injury is mediated by macrophage recruitment and differentiation in response to environmental signals. In general, macrophages adopt either a classically M1 (M[IFN-γ, LPS]) or alternatively activated M2 (M[IL-4, IL-13] or M[IL-10]) phenotype. Recent studies have highlighted the importance of alternatively activated macrophages in tissue remodeling and repair as well as the contribution of an imbalance of classically and alternatively activated macrophages to tissue degeneration and disease progression. Keratin biomaterials have recently demonstrated their ability to promote alternatively activated macrophage polarization in an in vitro model using a monocytic cell line. In the present study, the ability of extracted human hair keratins to influence alternative activation of human primary monocytes in vitro is assessed by evaluating changes in surface receptor expression, inflammatory cytokine secretion, and phagocytic activity. The impact of keratin molecular weight fractionation on these outcomes was also investigated. High and low molecular weight fractions of the oxidized form of extractable human hair keratins - referred to as keratose (KOSH and KOSP, respectively) - were characterized by size exclusion chromatography, mass spectrometry, and Western blot. Primary macrophages underwent traditional differentiation to the M[IFN-γ, LPS], M[IL-4, IL-13], or M[IL-10]) phenotypes or were plated on different molecular weight keratin coatings (KOSH and KOSP). Macrophages plated on keratin and analyzed via flow cytometry yielded the largest CD163+ cell populations and CD163 mean fluorescence intensities. Cells in the KOSP group were significantly more phagocytic than all other cell types at the 1.5 and 3 h time points and exhibited behavior and a cytokine production profile most similar to the M[IL-10] treated group. These findings may have important implications for understanding and evaluating the ability of keratin biomaterials to influence inflammation and tissue regeneration in disease and injury models. STATEMENT OF SIGNIFICANCE: Biomaterials made from human hair keratins have previously been shown to elicit anti-inflammatory responses from naïve macrophages and polarize them toward an M2 phenotype. In this work we show for the first time that primary human cells respond similarly, that it is the M2c phenotype that predominates, that a sub-fraction of hydrolyzed keratin peptides are most likely responsible for the response, and that immobilization of the keratin peptides to a surface is required. Keratin biomaterials have been used to regenerate several tissues such as skin, muscle, bone, nerve, and cornea, in vitro and in animal studies. Our current findings will help guide the development of keratin-based biomaterials that seek to direct responses toward regenerative outcomes by attenuating inflammation.

Hair growth promoting activity of discarded biocomposite keratin extract.

Keratin biomaterial has been used in regenerative medicine owing to its in-vivo and in-vitro biocompatibility. The present study was aimed to investigate the hair growth promoting activity of keratin extract and its mechanism of action. Keratin extract was topically applied on the synchronized depilated dorsal skin of telogenic C57BL/6 mice and promoted hair growth by inducing the anagen phase. The histomorphometric observation indicated significantly increases the number, shaft of hair follicles and deep subcutis area in the keratin extract treated group in contrast to the control group, which was considered an indication of anagen phase induction. Subsequently, the quantitative real-time polymerase chain reaction analysis revealed that fibroblast growth factor-10, vascular endothelial growth factor, insulin-like growth factor-1, ß-catenin, and Shh were expressed earlier in the keratin extract-treated group than in the control group. Besides, keratin extract has been observed to be biocompatible when analyzed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 4',6-diamidino-2-phenylindole staining using immortalized human keratinocyte cells, showing more than 90% cell viability. Our study demonstrated that keratin extract stimulating hair follicle growth by inducing the growth phase; anagen in telogenic C57BL/6 mice and thus the topical application of keratin extract may represent a promising biomaterial for the management and applications of hair follicle disorder.

Characterization of keratin microparticles from feather biomass with potent antioxidant and anticancer activities.

In the present study chicken feathers were hydrolyzed by chemical treatment in alkaline conditions. The pH value of feather hydrolyzed solution was amended accordingly the iso-electric precipitation. Two types of keratin microparticles KM1, KM2 were synthesized under acidic conditions at 3.5 and 5.5pH respectively. The synthesized keratin microparticles possessed uniform and round surface by scanning electron microscopy (SEM). The thermal degradation of microparticles were examined by thermogravimetry (TGA). Fourier transform infrared spectroscopy (FTIR) revealed that the extracted keratin retained the most of protein backbone. The microparticles were screened for their in vitro anticancer activities by SRB bioassay towards HeLa, SK-OV-3 and A549 cancer cell lines. Futhermore, their cytotoxicity towards healthy cell lines was analyzed having Malin Darby canine kidney (MDCK) cell lines along with in vitro antioxidant activity using DPPH and ABTS methods KM1 and KM2 showed 200.31±1.01 and 139.73±0.94, 214.16±0.29 and 153.92±0.61, 328.92±3.46 and 200.33±2.48µg/mL of IC50 levels against HeLa, SK-OV-3, and A549 cell lines, respectively. Moreover, KM1 and KM2 demonstrated significant antioxidant potency with IC50 levels 13.15 and 9.02µg/mL as well as 8.96 and 5.60µg/mL in DPPH and ABTS radical scavenging bioassay, respectively.

Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model.

Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injuries in which tissue loss results in permanent functional impairment, is common among these injuries. The present standard of care entails the use of muscle flap transfers, which suffer from the need for additional surgery when using autografts or the risk of rejection when cadaveric grafts are used. Tissue engineering (TE) strategies for skeletal muscle repair have been investigated as a means to overcome current therapeutic limitations. In that regard, human hair-derived keratin (KN) biomaterials have been found to possess several favorable properties for use in TE applications and, as such, are a viable candidate for use in skeletal muscle repair. Herein, KN hydrogels with and without the addition of skeletal muscle progenitor cells (MPCs) and/or insulin-like growth factor 1 (IGF-1) and/or basic fibroblast growth factor (bFGF) were implanted in an established murine model of surgically induced VML injury to the latissimus dorsi (LD) muscle. Control treatments included surgery with no repair (NR) as well as implantation of bladder acellular matrix (BAM). In vitro muscle contraction force was evaluated at two months postsurgery through electrical stimulation of the explanted LD in an organ bath. Functional data indicated that implantation of KN+bFGF+IGF-1 (n = 8) enabled a greater recovery of contractile force than KN+bFGF (n = 8)***, KN+MPC (n = 8)**, KN+MPC+bFGF+IGF-1 (n = 8)**, BAM (n = 8)*, KN+IGF-1 (n = 8)*, KN+MPCs+bFGF (n = 9)*, or NR (n = 9)**, (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent with the physiological findings, histological evaluation of retrieved tissue revealed much more extensive new muscle tissue formation in groups with greater functional recovery (e.g., KN+IGF-1+bFGF) when compared with observations in tissue from groups with lower functional recovery (i.e., BAM and NR). Taken together, these findings further indicate the general utility of KN biomaterials in TE and, moreover, specifically highlight their potential application in the treatment of VML injuries.