Papers by Author: Marisa Masumi Beppu

Abstract: Layer-by-layer (LbL) is a bottom-up technique used for construction of films with self-assembly and self-organizing properties. In most cases, the fundamental driving force for the formation of these films is originated from the electrostatic interaction between oppositely charged species. The charged segments of polyelectrolytes behave as small building units and their orientation and position can be designed to target structures of great complexity. Furthermore, the technique enables the use of various materials, including natural polymers. In this work, we chose the cationic biopolymer chitosan (CHI) and the negative polyelectrolytes sodium alginate (ALG) and hyaluronic acid (HA). The aim of this study was to evaluate the effect of ionic strength (0 versus 200 mM) and pH (3 versus 5) on ALG/CHI and HA/CHI nanostructured multilayered thin films properties. From profilometry and atomic force microscopy (AFM) analyses, changes in thickness and roughness of the coatings were monitored. The presence of salt in polyelectrolyte solutions induced the polymer chains to adopt conformations with more loops and tails and this arrangement in solution was transmitted to films, resulting in rougher surfaces. Furthermore, the film thickness can be precisely controlled by adjusting the pH of the polyelectrolyte solution. The variation of these parameters shows that it is possible to molecularly control chemical and structural properties of nanostructured coatings, thus opening up new possibilities of application (e.g. cell adhesion).

Abstract: The main objective of this work was to produce membranes of chitosan and collagen type I and check their ability to undergo “in vitro” calcification. The membranes of chitosan-collagen blends were characterized by TGA, infra-red spectroscopy and DSC. Samples of dense and porous membranes were immersed in solution SBF (Simulated Body Fluid) in order to verify their “in vitro” calcification. The membranes were observed by SEM. The production of chitosan-collagen membranes is possible, in dense and porous versions. We can conclude that the blend is less resistant to high temperatures, in comparison to pristine chitosan membranes shown in literature. Through the initial assays of calcification, we observe that it is possible to induce the calcium deposition on a chitosan-collagen membrane, as seen by SEM. Microscopy of fracture surfaces showed fibril structures, probably formed by collagen.

Abstract: The aim of this work was to study the phase transformation during the setting reaction of beta tricalcium phosphate (β-TCP) and phosphoric acid with chitosan solution added. To follow the kinetics of the phase transformation, two methods were used: x-ray diffraction (XRD) was used to study the phase evolution during the hardening process in real time, and was also used in samples where the reaction was supposedly stopped in different times using acetone, as indicated in literature. The setting reaction occurs so fast that the phase transformation could not be observed, but it was possible to invalidate the second mentioned method for this system, as it induces the final product dicalcium phosphate dihydrate DCPD (brushite) to be converted into his anhydrous form dicalcium phosphate DCP (monetite). The addition of chitosan in order to improve biocompatibility was successfully done, it could be observed that chitosan inhibits brushite crystallization in the first moment of the reaction, but the final product was not affected by it.

Abstract: Small caliber vascular replacement (<4 mm) still remains a challenge for medical and research teams, as no available vascular substitutes (VS) are suitable for small diameter bypass. Vascular engineering proposes new models of small diameter VS but rare are those that meet the biocompatibility and mechanical criteria. In this study, we developed a new scaffold made by the combination of two natural biomacromolecules: collagen and silk fibroin. The scaffold was further cellularised with porcine smooth muscle cells. First, the behavior of cells in the collagen-fibroin constructs was verified in order to evaluate the biocompatibility of the scaffold with the cells. Then, gel mass loss and cellular attachment, morphology, spreading and viability were analysed. The results showed an excellent interaction and biocompatibility between collagen, silk fibroin fibers and cells. Thus, the collagen-fibroin construct appears to be a very attractive material for vascular tissue engineering.

Abstract: Sterilization is very important for the use of biomaterials in the medical field. This work describes the preparation of chitosan/carboxymethylcellulose thin films with the layer-by-layer deposition technique, and the investigation on the effects that thermal treatments have on them during sterilization. The influence of different heating and sterilization methods on the chemical and physical structure of biopolymer thin films composed of chitosan and carboxymethylcellulose was evaluated. Films were heated in an oven at specified temperatures or autoclaved and their characteristics analyzed with contact angle, profilometry, FTIR, anionic dye uptake and UV-Vis measurements. Results show that, depending on the heating conditions, these thin films may undergo the Maillard reaction that turns the films from being transparent to brownish in color. This reaction may lead to a decrease in the free hydroxyl groups of both carboxymethylcellulose and chitosan and free ammonium groups of chitosan - consequently leading to changes in hydrophilicity and wettability of the film. Temperature effects on the characteristics of the synthetic pre-layer coating composed of poly (diallyldimethylammonium chloride) and poly (sodium 4-styrene-sulfonate) - used to provide a high cationic surface for the deposition of the biopolymer films - were also observed. These findings are of practical interest because biopolymer thin films find a great number of applications where sterilization is a must, such as clinical and medical applications and in the areas of materials science and biotechnology.

Abstract: 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 (t_1/2) 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.