Skin grafts : local quest for viable alternatives to autologous grafts using silk and acellular dermal matrices

The gold standard with regards to skin transplantation is the use of the patient’s own skin obtained from a healthy donor site. Such grafts can be either full thickness skin or more commonly nowadays, split thickness skin. Various materials, having either natural and or synthetic origins, have been used in the engineering of skin substitutes to-date and these grafts are then confronted against autologous skin grafts. If proven to be successful, such matrices could be utilised in clinical applications such as in the treatment of burn wounds and in cases of skin ulcers amongst others. In this study the primary cells used, keratinocytes and fibroblast, were obtained from donor skin and cultured. Scaffolds of xenogenic (raw silk) as well as of allogenic (acellular dermal matrices) origins were obtained via low-cost methods and seeded using the fibroblasts and keratinocytes so as to determine which gave the closest mimic to skin grafts. Out of the matrices assessed, the raw silk matrix allowed the best colonisation with skin cells in our hands. The ADM matrice also showed some cell colonisation, but will need further experimentation.peer-reviewe

De novo bone formation on macro/microporous silk and silk/nano-sized calcium phosphate scaffolds

Macro/micro porous silk/nano-sized calcium phosphate scaffolds (SC16) with bioactive and superior physicochemical properties have been recently developed. In this study, we aim at evaluating the new bone formation ability of the SC 16 scaffolds in vivo, using silk fibroin scaffolds (S16) as control. The CaP distribution profile in the scaffolds was characterized by Micro-Computed Tomography. The in vitro mineralization behavior was examined by immersion in Simulated Body Fluid solution from 1 to 14 days. The long-term hydration degree and weight loss ratio of the scaffolds were evaluated by immersion in an Isotonic Saline Solution from 1 month to 1 year. In vivo osteogenesis properties of the scaffolds were screened by implantation into the rat femur defects for 3 weeks. The results showed that the CaP phase distributed homogeneously in the SC16 scaffolds. Mineralization was only observed in SC16 scaffolds, and both scaffolds gradually degraded with time. The staining of the explants showed that new bone formation with higher density was observed in the SC16 scaffolds as compared to S16 scaffolds, guiding the growth of new bone directly onto its surface. These results demonstrated that the SC16 hybrid scaffolds are osteoconductive and can be good candidates for bone tissue engineering as promoted superior de novo bone formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) projects OsteoCart (PTDC/CTM-BPC/115977/2009) and Tissue2Tissue (PTDC/CTM/105703/2008). Research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS. Le-Ping Yan is an FCT PhD scholarship holder (SFRH/BD/64717/2009)

A silk composite fiber reinforced by telechelic-type polyalanine and its strengthening mechanism

Antiparallel β-sheets play a key role in determining the physical properties of fibroins, e.g., degradation and mechanical properties, and are typically formed by poly(A) motifs from spider dragline silks and GAGAGS motifs from Bombyx mori silkworm silks. To explore the interaction between these two motifs within the same system, a telechelic-type polyalanine (TPA) was prepared through chemoenzymatic synthesis and doped in silkworm silk fibroins to fabricate silk composite fibers. Interestingly, when TPA was added at suitable ratios of 1 and 3 wt%, the mechanical properties of the composite fibers were largely improved by approximately 42% and 51% compared with those of silk-only fibers in terms of tensile strength and toughness, respectively. As revealed by wide-angle X-ray diffraction (WAXD), silk composite fibers achieved the highest crystallinity at a TPA ratio of 1 wt%, largely contributing to their tensile strength. Evidenced by simultaneous stretching during WAXD measurement, TPA did not compete with the silk matrix by forming its own crystallization. Ultimately, a strengthening mechanism of nucleus-dependent crystallization was discussed to show the favorable heterogeneous nucleation created from TPA molecules for the promotion of crystallization in silk fibroins. Interestingly, regularly packed and aligned granules within composite fibers were detected by AFM to further support the enhanced mechanical performance. This work envisions sophisticated control of β-sheet crystals to better understand the structure–property relationship

Silk Fibroin/Nano-CaP Bilayered scaffolds for osteochondral tissue engineering

In this study, bilayered silk and silk/nano-CaP scaffolds were developed for osteochondral (OC) tissue engineering. Aqueous silk solution (16 wt.%) was used for preparation of the cartilage-like layer and, for generation of the silk/nano-CaP suspension and the bottom layer (CaP/Silk: 16 wt.%). The scaffolds were formed by using salt-leaching/lyophilization approach. The scanning electron microscopy revealed that the both layers presented porous structure and integrated well. Micro-computed tomography images confirmed that the CaP phase was only retained in the silk/nano-CaP layer. The hydration degree and mechanical properties of the bilayered scaffold were comparable to the ones of each single layer. The apatite crystal formation was limited to the silk/nano-CaP layer, when soaking the scaffold in a simulated body fluid solution, which is a must for the application of the developed scaffolds in OC tissue engineerin

Into the groove:instructive conductive silk films with topological guidance cues

Instructive biomaterials capable of controlling the behaviour of the cells are particularly interesting scaffolds for tissue engineering and regenerative medicine. Novel biomaterials are particularly important in societies with rapidly aging populations, where demand for organ/tissue donations is greater than their supply. Herein we describe the preparation of electrically conductive silk film-based nerve tissue scaffolds that are manufactured using all aqueous processing. Aqueous solutions of Bombyx mori silk were cast on flexible polydimethylsiloxane substrates with micrometer-scale grooves on their surfaces, allowed to dry, and annealed to impart β-sheets to the silk which assures that the materials are stable for further processing in water. The silk films were rendered conductive by generating an interpenetrating network of polypyrrole and polystyrenesulfonate in the silk matrix. Films were incubated in an aqueous solution of pyrrole (monomer), polystyrenesulfonate (dopant) and iron chloride (initiator), after which they were thoroughly washed to remove low molecular weight components (monomers, initiators, and oligomers) and dried, yielding conductive films with sheet resistances of 124 ± 23 kΩ square-1. The micrometer-scale grooves that are present on the surface of the films are analogous to the natural topography in the extracellular matrix of various tissues (bone, muscle, nerve, skin) to which cells respond. Dorsal Root Gangions (DRGs) adhere to the films and the grooves in the surface of the films instruct the aligned growth of processes extending from the DRGs. Such materials potentially enable the electrical stimulation of cells cultured on them, and future in vitro studies will focus on understanding the interplay between electrical and topographical cues on the behaviour of cells cultured on them

Mimicking Hierarchical Complexity of the Osteochondral Interface Using Electrospun Silk-Bioactive Glass Composites

The anatomical complexity and slow regeneration capacity of hyaline cartilage at the osteochondral interface pose a great challenge in the repair of osteochondral defects (OCD). In this study, we utilized the processing feasibility offered by the sol derived 70S bioactive glass and silk fibroin (mulberry Bombyx mori and endemic Indian non-mulberry Antheraea assama), in fabricating a well-integrated, biomimetic scaffolding matrix with a coherent interface. Differences in surface properties such as wettability and amorphousness between the two silk groups resulted in profound variations in cell attachment and extracellular matrix protein deposition. Mechanical assessment showed that the biphasic composites exhibited both an elastic region pertinent for cartilage tissue and a stiff compression resistant region simulating the bone phase. In vitro biological studies revealed that the biphasic mats presented spatial confinement for the growth and maturation of both osteoblasts and chondrocytes, marked by increased alkaline phosphatase (ALP) activity, osteopontin (OPN), sulfated glycosaminoglycan (sGAG) and collagen secretion in the cocultured mats. The non-mulberry silk based biphasic composite mats performed better than their mulberry counterpart, as evidenced by enhanced expression levels of key cartilage and bone specific marker genes. Therefore, the developed biphasic scaffold show great promise for improving the current clinical strategies for osteochondral tissue repair

Water-based spider silk films

Spider silk proteins represent exceptional natural polymers due to their mechanical properties in combination with biocompatibility. As both hydrophobic and slowly biodegrading biopolymers, recombinant spider silk proteins fulfill the required properties for a drug delivery system. The recombinant production of spider silk proteins allows the cost-effective fabrication of these biopolymers. The main focus of this thesis was to obtain a reproducible all-aqueous protein film production process in order to cast spider silk films characterized by biocompatibility, water insolubility and good mechanical properties. The evaluation of spider silk films as novel carrier for various pharmaceuticals was also part of this work. Ideally, the film preparation process has to be compatible with protein encapsulation and the obtained matrix should be able to release a therapeutic protein for a prolonged time