Protein Paper from Exfoliated Eri Silk Nanofibers.

The exfoliation of silk fiber is an attractive method to produce silk micro- and nanofibers that retain the secondary structure of native silk. However, most fibrillation methods used to date require the use of toxic and/or expensive solvents and the use of high energy. This study describes a low cost, scalable method to produce microfibrillated silk nanofibers without the use of toxic chemicals by controlling the application of shear using commercially scalable milling and homogenization equipment. Manipulation of the degumming conditions (alkaline concentration and degumming temperature) and the shear in milling and/or homogenization enabled control over the degree of fibrillation. The microfibrillated silk was then characterized to determine structural change during processing and the stability of the resulting suspensions at different pH. Silk nanofibers obtained from milling degummed silk were characterized using atomic force microscopy. Nanofibers obtained both with and without high-pressure homogenization were then used to produce silk "protein paper" through casting. Silk degumming conditions played a critical role in determining the degree of microfibrillation and the properties of the cast silk papers. The silk papers produced from homogenized nanofibers showed excellent mechanical properties, high water absorption, and wicking properties. The silk papers were excellent for supporting the attachment and growth of human skin keratinocytes, demonstrating application possibilities in healthcare such as wound healing.

Scaffolds for tympanic membrane regeneration in rats.

Tympanic membrane (TM) perforations lead to significant hearing loss and result in possible infection of the middle ear. Myringoplasty is commonly performed to repair chronic perforations. Although various grafts and materials have been used to promote TM regeneration, all have associated limitations. The aim of this study was to evaluate the efficacy and feasibility of two graft materials, silk fibroin scaffold (SFS) and porcine-derived acellular collagen type I/III scaffold (ACS), compared with two commonly used graft materials (paper patch and Gelfoam) for the promotion of TM regeneration. These scaffolds were implanted using on-lay myringoplasty in an acute TM perforation rat model. Surface morphology of the scaffolds was observed with scanning electron microscopy. The morphology of the TM was assessed at various time points postimplantation using otoscopy, light and electron microscopy, and functional outcomes by auditory brainstem responses. We found that SFS and ACS significantly accelerated the TM perforation closure, obtained optimal TM thickness, and resulted in better trilaminar morphology with well-organized collagen fibers and early restoration of hearing. However, paper patch and Gelfoam lost their scaffold function in the early stages and showed an inflammatory response, which may have contributed to delayed healing. This study indicates that compared with paper patch and Gelfoam, SFS and ACS are more effective in promoting an early TM regeneration and an improved hearing, suggesting that these scaffolds may be potential substitutes for clinical use.

Age related changes in gene expression within the cochlea of C57BL/6J mice.

BACKGROUND AND AIMS: Hearing loss as a unique symptom is highly prevalent in the elderly and while there exists several pathologies that would result in age related hearing loss (ARHL), most agree it is multifactorial with environmental, metabolic and genetic components. However, no research to date has discovered a definitive genetic cause. This paper describes the use of microarray to address this issue. METHODS: The left cochlea of laboratory aged C57BL6J mice at 4, 15 and 45 weeks was extracted from pooled, agematched animals and the RNA extracted for use in microarray analysis. The contralateral cochlea was also removed and used for immunohistochemical analysis. RESULTS: Microarray revealed 116 genes to be up or down regulated between young and old animals, the most prominent being prolactin (108.2 fold increase) and growth hormone (43.94 fold increase). The prolactin inhibitor calcitonin was also down regulated over 2 fold. Immunohistochemistry revealed prolactin to be weakly expressed within the spiral ganglion, whilst its receptor was widely distributed throughout the cochlea. CONCLUSION: This is the first study demonstrating the cochlea as a site for extrapituitary prolactin expression and that this expression is related to age. Combined with the widespread distribution of prolactin receptor, there may be implications for prolactin's role in ARHL.

Histology of the healing tympanic membrane following perforation in rats.

OBJECTIVES/HYPOTHESIS: The aim of this study was to provide a detailed cytological account on the healing tympanic membrane (TM) over 14 days and to complement existing research into TM wound healing. STUDY DESIGN: The study is a prospective cohort study of 19 male Sprague-Dawley (Rattus norvegicus) rats. METHODS: Rat TMs were perforated using a sterile needle and sacrificed at time points during the 14 days following perforation. RESULTS: The healing of the TM resembles cutaneous wound healing except that the TM is unique in the lack of a supportive matrix beneath the regenerating epithelia. This prevents the influx of reparative cells and nutrients and the in growth of the usual fibroblastic reaction. CONCLUSIONS: TM wound healing contrasts with cutaneous wound healing in that keratinocytes are the first cells to close the wound and not the last. A keratin scaffold may not be important in the healing process. The malleus plays a crucial role in the healing of the TM and is the site of significant mitotic activity during the healing process. Migration across layers of the TM appears to account for the closure of the perforation.

Preliminary results of the application of a silk fibroin scaffold to otology.

The surgical treatment to repair chronic tympanic membrane perforations is myringoplasty. Although multiple autologous grafts, allografts, and synthetic graft materials have been used over the years, no single graft material is superior for repairing all perforation types. Recently, the remarkable properties of silk fibroin protein have been studied, with biomedical and tissue engineering applications in mind, across a number of medical and surgical disciplines. The present study examines the use of silk fibroin for its potential suitability as an alternative graft in myringoplasty surgery by investigating the growth and proliferation of human tympanic membrane keratinocytes on a silk fibroin scaffold in vitro. Light microscopy, immunofluorescent staining, and confocal imaging all reveal promising preliminary results. The biocompatibility, transparency, stability, high tensile strength, and biodegradability of fibroin make this biomaterial an attractive option to study for this utility.

Grafts in myringoplasty: utilizing a silk fibroin scaffold as a novel device.

Chronic perforations of the eardrum or tympanic membrane represent a significant source of morbidity worldwide. Myringoplasty is the operative repair of a perforated tympanic membrane and is a procedure commonly performed by otolaryngologists. Its purpose is to close the tympanic membrane, improve hearing and limit patient susceptibility to middle ear infections. The success rates of the different surgical techniques used to perform a myringoplasty, and the optimal graft materials to achieve complete closure and restore hearing, vary significantly in the literature. A number of autologous tissues, homografts and synthetic materials are described as graft options. With the advent and development of tissue engineering in the last decade, a number of biomaterials have been studied and attempts have been made to mimic biological functions with these materials. Fibroin, a core structural protein in silk from silkworms, has been widely studied with biomedical applications in mind. Several cell types, including keratinocytes, have grown on silk biomaterials, and scaffolds manufactured from silk have successfully been used in wound healing and for tissue engineering purposes. This review focuses on the current available grafts for myringoplasty and their limitations, and examines the biomechanical properties of silk, assessing the potential benefits of a silk fibroin scaffold as a novel device for use as a graft in myringoplasty surgery.