Injectable Human Hair Keratin-Fibrinogen Hydrogels for Engineering 3D Microenvironments to Accelerate Oral Tissue Regeneration.

Traumatic injury of the oral cavity is atypical and often accompanied by uncontrolled bleeding and inflammation. Injectable hydrogels have been considered to be promising candidates for the treatment of oral injuries because of their simple formulation, minimally invasive application technique, and site-specific delivery. Fibrinogen-based hydrogels have been widely explored as effective materials for wound healing in tissue engineering due to their uniqueness. Recently, an injectable foam has taken the spotlight. However, the fibrin component of this biomaterial is relatively stiff. To address these challenges, we created keratin-conjugated fibrinogen (KRT-FIB). This study aimed to develop a novel keratin biomaterial and assess cell-biomaterial interactions. Consequently, a novel injectable KRT-FIB hydrogel was optimized through rheological measurements, and its injection performance, swelling behavior, and surface morphology were investigated. We observed an excellent cell viability, proliferation, and migration/cell-cell interaction, indicating that the novel KRT-FIB-injectable hydrogel is a promising platform for oral tissue regeneration with a high clinical applicability.

Human hair keratins promote the regeneration of peripheral nerves in a rat sciatic nerve crush model.

Axon regeneration and functional recovery after peripheral nerve injury remains a clinical challenge. Injury leads to axonal disintegration after which Schwann cells (SCs) and macrophages re-engage in the process of regeneration. At present, biomaterials are regarded as the most promising way to repair peripheral nerve damage. As a natural material, keratin has a wide range of sources and has good biocompatibility and biodegradability. Here, a keratin was extracted from human hair by reducing method and a keratin sponge with porous structure was obtained by further processing. The results suggested that keratin can promote cell adhesion, proliferation, migration as well as the secretion of neurotrophic factors by SCs and the regulation of the expression of macrophage inflammatory cytokines in vitro. We report for the first time that human hair keratin can promote the extension of axon in DRG neurons. The motor deficits caused by a sciatic nerve crush injury were alleviated by keratin sponge dressing in vivo. Thus, keratin has been identified as a valuable biomaterial that can enhance peripheral nerve regeneration.

Insight into the Regulatory Function of Human Hair Keratins in Wound Healing Using Proteomics.

Keratins derived from human hair possess excellent wound healing qualities. However, their functional contribution to this process is poorly understood. In this study, the regulatory function of human hair keratins in wound healing is investigated using proteomic analysis by dividing keratins into different groups based on their molecular weight distributions: low molecular weight keratins (LMWK, 10-30 kDa), medium molecular weight keratins (MMWK, 30-50 kDa), and high molecular weight keratins (HMWK, >50 kDa). Keratin hydrogels with different molecular weights exhibit various morphologies, rheological properties, degradation rates, and wound healing activities. Using proteomic analysis, LMWK and HMWK hydrogels exhibit a stronger regulatory ability for wound healing at days 1 and 7, respectively. The major functions of LMWK during wound healing are regulation of cells communication and function. In contrast, proteins associated with energy metabolism are significantly expressed after HMWK hydrogel treatment at day 1, and these play an important role in cellular growth and reactive oxygen species scavenging at day 7. These results demonstrate that the wound healing qualities of human hair keratins are influenced by their molecular weight distribution, and the proteomic analysis sheds new light on the regulatory function of human hair keratins during wound healing.

Recombinant Human Hair Keratin Nanoparticles Accelerate Dermal Wound Healing.

In recent years, favorable enhanced wound-healing properties and excellent biocompatibility of keratin derived from human hair have attracted considerable attention. Recombinant keratin proteins can be produced by recombinant DNA technology and have higher purity than extracted keratin. However, the wound-healing properties of recombinant keratin proteins remain unclear. Herein, two recombinant trichocyte keratins including human type I hair keratin 37 and human type II hair keratin 81 were expressed using a bacterial expression system, and recombinant keratin nanoparticles (RKNPs) were prepared via an ultrasonic dispersion method. The molecular weight, purity, and physicochemical properties of the recombinant keratin proteins and nanoparticles were assessed using gel electrophoresis, circular dichroism, mass spectrometry, and scanning electron microscope analyses. The RKNPs significantly enhanced cell proliferation and migration in vitro, and the treatment of dermal wounds in vivo with RKNPs resulted in improved wound healing associated with improved epithelialization, vascularization, and collagen deposition and remodeling. In addition, the in vivo biocompatibility test revealed no systemic toxicity. Overall, this work demonstrates that RKNPs are a promising candidate for enhanced wound healing, and this study opens up new prospects for the development of keratin biomaterials.

Evaluating the antioxidant effects of human hair protein extracts.

The intrinsically high cysteine content in human hair keratins and keratin associated proteins confer hair its outstanding mechanical strength through the formation of strong intermolecular disulfide bonds. In addition, these proteins offer the potential to be exploited as potent antioxidants. This report presents our findings on the antioxidant effects of human hair protein extracts and their consequent protective role against oxidative stress in human dermal fibroblast (HDF) cultures. Protein extracts were obtained from human hair using sodium sulfide as the reducing agent, and characterized using SDS-PAGE, Western blotting, MALDI-ToF mass spectrometry and amino acid analysis. Cysteine was found to account for 11.2 mol % in the extracted fractions. By measuring 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, the hair protein fractions were shown to possess significant antioxidant ability (IC50 = 16.22 µM). As a supplement in cell culture media, the extracts protected HDFs from H2O2 induced oxidative stress, which was demonstrated by the maintenance of cell viability and reduced reactive oxygen species production. Besides offering mechanical support as a scaffolding material, the unique antioxidizing ability of human hair protein extracts may also be exploited in biomedical applications.

A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces.

OBJECTIVE: We aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro. METHODS: Keratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4. RESULTS: Keratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~106 cells per heart. Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin-coated substrates similar to fibronectin-coated surfaces. Phenylephrine (PE) treated cardiomyocytes grown on keratin-coated substrates exhibited increased cell size, sarcomere organization and perinuclear ANP expression indicating the development of cardiac hypertrophy. In addition, we observed increased activation of Akt and ERK pathways, induction of the fetal genes and increased protein synthesis upon PE treatment, which are characteristics of cardiomyocyte hypertrophy. The protocol was extended to mouse cardiomyocytes and found to show similar results upon examination. CONCLUSION: We demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Alkylation of human hair keratin for tunable hydrogel erosion and drug delivery in tissue engineering applications.

Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or "capping") of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system.

Brazilian keratin hair treatment: a review.

Brazilian keratin treatments are widely available products that are used by women all over the world to straighten hair. Marketers of these products claim that the keratin treatments render naturally curly hair more manageable and frizz-free while enhancing color and shine, giving the hair a healthier appearance. Although widely used, there have been virtually no reports of adverse side effects. Unfortunately, many of the products that are applied by salon professionals contain formaldehyde or its derivatives and are being marketed as safe.

Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration.

The cytocompatibility, in vivo biodegradation and wound healing of keratin biomaterials were investigated. For the purposes, three groups of keratin scaffolds were fabricated by freeze-drying reduced solutions at 2 wt.%, 4 wt.% and 8 wt.% keratins extracted from human hairs. These scaffolds exhibited evenly distributed high porous structures with pore size of 120-220 µm and the porosity >90%. NIH3T3 cells proliferated well on these scaffolds in culture lasting up to 22 days. Confocal micrographs stained with AO visually revealed cell attachment and infiltration as well as scaffold architectural stability. In vivo animal experiments were conducted with 4 wt.% keratin scaffolds. Early degradation of subcutaneously implanted scaffolds occurred at 3 weeks in the outermost surface, in concomitant with inflammatory response. At 5 weeks, the overall porous structure of scaffolds severely deteriorated while the early inflammatory response in the outermost surface obviously subsided. A faster keratin biodegradation was observed in repairing full-thickness skin defects. Compared with the blank control, keratin scaffolds gave rise to more blood vessels at 2 weeks and better complete wound repair at 3 weeks with a thicker epidermis, less contraction and newly formed hair follicles. These preliminary results suggest that human hair keratin scaffolds are promising dermal substitutes for skin regeneration.

The effect of human hair keratin hydrogel on early cellular response to sciatic nerve injury in a rat model.

Peripheral nerve injuries requiring surgery can be repaired by autograft, the clinical "gold standard", allograft, or nerve conduits. Most published clinical studies show the effectiveness of nerve conduits in small size defects in sensory nerves. Many preclinical studies suggest that peripheral nerve regeneration through conduits can be enhanced and repair lengths increased with the use of a biomaterial filler in the conduit lumen. We have previously shown that a luminal hydrogel filler derived from human hair keratin (HHK) can improve electrophysiological and histological outcomes in mouse, rabbit, and non-human primate nerve injury models, but insight into potential mechanisms has been lacking. Based on the premise that a keratin biomaterial (KOS) hydrogel provides an instantaneous structural matrix within the lumen, the current study compares the cellular behavior elicited by KOS hydrogel to Matrigel (MAT) and saline (SAL) conduit fillers in a 1 cm rat sciatic nerve injury model at early stages of regeneration. While there was little difference in initial cellular influx, the KOS group showed earlier migration of dedifferentiated Schwann cells (SC) from the proximal nerve end compared to the other groups. The KOS group also showed faster SC dedifferentiation and myelin debris clearance, and decreased macrophage infiltration during Wallerian degeneration of the distal nerve tissue.