Raquel Farias Weska

Effect of Freezing Methods on the Properties of Lyophilized Porous Silk Fibroin Membranes

Silk fibroin is a fibrous protein that has been extensively studied for application in the biomedical field, and has been used as a scaffold for bone tissue engineering. Biomaterials made of proteins are prone to physical and chemical degradation during storage; lyophilization, a drying method that consists of freezing and drying steps, is known to promote minimal changes in structure and biological activity of biomaterials. This study evaluates the effect of freezing methods on the properties of lyophilized porous silk fibroin membranes. The membranes were obtained from silk fibroin solution, frozen in liquid nitrogen or ultrafreezer, lyophilized, and then characterized by XRD, FTIR, TGA, DSC and SEM. Although the membranes presented similar physical, chemical and microstructural characteristics, quench freezing with liquid nitrogen, followed by lyophilization, promoted collapse of the membranes, while slow cooling performed by ultrafreezer preserved membrane integrity.

Effects of sterilization methods on the physical, chemical, and biological properties of silk fibroin membranes.

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. Sterilization is a fundamental step in biomaterials processing and it must not jeopardize the functionality of medical devices. The aim of this study was to analyze the influence of different sterilization methods in the physical, chemical, and biological characteristics of dense and porous silk fibroin membranes. Silk fibroin membranes were treated by several procedures: immersion in 70% ethanol solution, ultraviolet radiation, autoclave, ethylene oxide, and gamma radiation, and were analyzed by scanning electron microscopy, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, tensile strength and in vitro cytotoxicity to Chinese hamster ovary cells. The results indicated that the sterilization methods did not cause perceivable morphological changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and FTIR results.

Natural and prosthetic heart valve calcification: morphology and chemical composition characterization.

Calcification is the most common cause of damage and subsequent failure of heart valves. Although it is a common phenomenon, little is known about it, and less about the inorganic phase obtained from this type of calcification. This article describes the scanning electron microscopy (SEM)/energy dispersive X-ray spectroscopy and Ca K-edge X-ray absorption near edge structure (XANES) characterization performed in natural and bioprosthetic heart valves calcified in vivo (in comparison to in vitro-calcified valves). SEM micrographs indicated the presence of deposits of similar morphology, and XANES results indicate, at a molecular level, that the calcification mechanism of both types of valves are probably similar, resulting in formation of poorly crystalline hydroxyapatite deposits, with Ca/P ratios that increase with time, depending on the maturation state. These findings may contribute to the search for long-term efficient anticalcification treatments.

Silk fibroin and sodium alginate blend: miscibility and physical characteristics.

Films of silk fibroin (SF) and sodium alginate (SA) blends were prepared by solution casting technique. The miscibility of SF and SA in those blends was evaluated and scanning electron microscopy (SEM) revealed that SF/SA 25/75 wt.% blends underwent microscopic phase separation, resulting in globular structures composed mainly of SF. X-ray diffraction indicated the amorphous nature of these blends, even after a treatment with ethanol that turned them insoluble in water. Thermal analyses of blends showed the peaks of degradation of pristine SF and SA shifted to intermediate temperatures. Water vapor permeability, swelling capacity and tensile strength of SF films could be enhanced by blending with SA. Cell viability remained between 90 and 100%, as indicated by in vitro cytotoxicity test. The SF/SA blend with self-assembled SF globules can be used to modulate structural and mechanical properties of the final material and may be used in designing high performance wound dressing.

Porous Silk Fibroin Membrane as a Potential Scaffold for Bone Regeneration

The requirements for scaffolds for bone tissue engineering include appropriate chemistry, morphology and structure to promote cell adhesion and synthesis of new bone matrix. Silk fibroin (SF) represents an important biomaterial for biomedical application, due to its suitable mechanical properties, biodegradability, biocompatibility, and versatility in processing. Our group has developed a new method to obtain a porous SF membrane, and the study of its potential for use as a scaffold for bone regeneration was the aim of this study. Porous membranes were obtained from SF solution, through the compression of a material generated by phase separation. For in vitro calcification experiments, porous SF membrane samples were immersed in SBF at pH 7.4 placed in polyethylene flasks. The experiments were carried out for seven days, at 36.5±0.5 °C. After 48 and 96h, the solutions were changed for fresh SBF with the ion concentration 1.5-fold higher than that of the standard one, to accelerate the calcification process. The characterization of morphology and composition of samples was performed by using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), respectively. The SEM micrographs indicated that the porous SF membranes presented calcium phosphate deposits after undergoing in vitro calcification. These results were confirmed by EDS spectra, which showed a stoichiometric molar Ca/P ratio ranging from 1.27 to 1.52. This fact may suggest that calcification deposits consisted of mixtures of HAP (Ca/P ratio = 1.67) and transient HAP precursor phases, such as octacalcium phosphate (Ca/P = 1.33) and dicalcium phosphate dehydrate (Ca/P = 1), indicating early stage mineralization. The porous silk fibroin membrane analysed in the current study is a promising material to be used as scaffolds for bone regeneration.

Improvement of Collagen Hydrogel Scaffolds Properties by the Addition of Konjac Glucomannan

Collagen gels have been investigated for a number of applications in tissue engineering because of their excellent biological properties. However, their limited mechanical behavior represents a major bottleneck for clinical use, especially for vascular tissue engineering. The targeting of their mechanical properties may be envisaged by the addition of other biopolymers, such as konjac glucomannan (KGM), a neutral high-molecular weight polysaccharide extracted from the tubers of Amorphophallus konjac, which has already been studied for biomedical applications due to its biocompatibility and biodegradable activity. In the present study, reconstituted collagen gels were prepared at pH 10 and room temperature, by mixing collagen with NaOH, NaCl and 0.05 to 0.2% of KGM. Collagen fibrillogenesis was monitored by spectrophotometric analysis at 310 nm. Gel samples were analyzed by compression tests, FTIR and SEM. Comparing to the control, the addition of KGM reduced the half-time (t1/2) of gelation from ca. 3 h to 2 h and the mechanical tests showed increases in the compressive strain energy of up to 3 times, and in compressive modulus of almost 4 times. Scanning electron images of collagen gel samples with KGM revealed the presence of micro-domains of KGM in the collagen matrix, revealing a phase separated scaffold for vascular tissue engineering.

In Vitro Calcification of Silk Fibroin Hydrogel

Silk fibroin hydrogels were prepared and their potential to deposit calcium phosphates in vitro was observed. Pristine and lyophilized samples were tested in 1xSBF and 1.5xSBF. The results showed that silk fibroin hydrogels can induce calcium phosphate deposits both in the pristine and lyophilized form. However, the pristine silk fibroin hydrogel after calcification presented a fragile structure making it difficult to handle, while the lyophilized samples presented better resistance to handling. Calcium phosphates deposits were intense in samples submitted to tests in 1.5xSBF, however, few and isolated deposits were observed on samples submitted to tests in 1xSBF. The 3-D porous structure and the ability to deposit calcium phosphates, turn silk fibroin hydrogel a potential material suitable to use in biomimetic processes.

Silk Fibroin: A Promising Biomaterial

Silk fibroin (SF) is a protein fiber spun by Bombyx mori silkworm. SF fibers are about 10-25 μm wide in diameter and a single cocoon may provide over 1000 m of SF fibers. SF can present several conformations regarding protein secondary structure which ultimately define the structural properties of SF-based materials. For this reason, a rigorous control on its processing conditions shall be performed. It is known that SF has excellent properties to be used in biomaterials field, controlled release and scaffolds for tissue engineering. In addition, SF can be processed in several forms, such as films, fibers, hydrogels or microparticles. This work seeks to provide an overview on SF processing conditions, regarding the preparation of SF membranes (dense and porous), hydrogels and biocomposites, focusing on biomaterials application.

Study of Morphology of Cardiac Valves (Human and Bovine Pericardium) after In Vivo Calcification

Pathologic calcification can lead to failure or deterioration of cardiac valves. Several researchers have tried alternatives to construct these devices, such as the incorporation or utilization of new biomaterials able to inhibit or decrease the calcification process. In vitro calcification tests can be used to screen new biomaterials regarding their potential to calcify in vivo. However, the mechanisms involved in both cases are not completely understood. In order to collect more information about the calcification process of implanted materials, morphology and elemental analyses of calcified cardiac valve fragments explanted from different patients were investigated and compared to previous reports of in vitro calcification tests. Scanning Electron Microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses indicated that the calcium phosphate deposits from both bovine pericardium and human cardiac valves calcified in vivo were similar to the deposits obtained from in vitro calcification samples as previously reported in the literature.