Marivalda M. Pereira

Mechanisms of hydroxyapatite formation on porous gel-silica substrates

The variables affecting the nucleation and crystallization of biological hydroxy carbonate apatite (HCA) on porous gel-silica substrates are examined. Texture is the critical variable with the rate of HCA formation increasing as pore size and pore volume increase, with pore sizes >2 nm required to achieve rapid kinetics (4–6 days) of fully crystallized HCA. The concentration of silanol groups on the silica surface does not control the rate of HCA formation although metastable surface defects, such as trisiloxane rings, may be involved in HCA nucleation.

Synthesis and characterization of biodegradable polyurethane films based on HDI with hydrolyzable crosslinked bonds and a homogeneous structure for biomedical applications.

Synthetic biodegradable polymers are considered strategic in the biomaterials field and are used in various applications. Among the polymers used as biomaterials, polyurethanes (PUs) feature prominently due to their versatility and the ability to obtain products with a wide range of physical and mechanical properties. In this work, new biodegradable polyurethane films were developed based on hexamethylene diisocyanate (HDI) and glycerol as the hard segment (HS), and poly(caprolactone) triol (PCL triol) and low-molecular-weight poly(ethylene glycol) PEG as the soft segment (SS) without the use of a catalyst. The films obtained were characterized by structural, mechanical and biological testing. A highly connected network with a homogeneous PU structure was obtained due to crosslinked bonds. The films showed amorphous structures, high water uptake, hydrogel behavior, and susceptibility to hydrolytic degradation. Mechanical tests indicated that the films reached a high deformation at break of up to 425.4%, an elastic modulus of 1.6 MPa and a tensile strength of 3.6 MPa. The materials presented a moderate toxic effect on MTT assay and can be considered potential materials for biomedical applications.

Effect of a Three-Dimensional Chitosan Porous Scaffold on the Differentiation of Mesenchymal Stem Cells into Chondrocytes

Cartilage tissue has a poor capacity for self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of cartilage tissue. The present work demonstrates that a three-dimensional (3D) chitosan scaffold increases the efficiency of the adhesion and differentiation of mesenchymal stem cells (MSCs) after the addition of a chondrogenic medium. These culture conditions promoted MSC differentiation into chondrocytes during the first 9 weeks of monolayer or 3D culture in a scaffold composed of chitosan or chitosan/gelatin. The results demonstrated that a chitosan scaffold caused a reduction in alkaline phosphatase production and an increase in the collagen concentration indicating phenotypic changes in the cells. In support of these results, the production of collagen type II by the MSCs cultured in the chitosan scaffold increased after 3 weeks of culture, indicating the beginning of differentiation. However, the addition of gelatin to the chitosan scaffold did not improve on the results obtained with chitosan alone. These results suggest that this 3D chitosan scaffold is a promising candidate for biomaterial implants designed to promote MSC colonization and has applications in regenerative medicine.

Thermogelling chitosan-collagen-bioactive glass nanoparticle hybrids as potential injectable systems for tissue engineering.

Recently, stimuli-responsive nanocomposite-derived hydrogels have gained prominence in tissue engineering because they can be applied as injectable scaffolds in bone and cartilage repair. Due to the great potential of these systems, this study aimed to synthesize and characterize novel thermosensitive chitosan-based composites, chemically modified with collagen and reinforced by bioactive glass nanoparticles (BG) on the development of injectable nanohybrids for regenerative medicine applications. Thus, the composite hydrogels were extensively characterized by structural, morphological, rheological, and biological testing. The composites showed thermosensitive response with the gelation temperature at approximately 37 °C, which is compatible with the human body temperature. In addition, scanning electron microscopy (SEM) analysis indicated that the chitosan hydrogels exhibited 3D-porous structures, and the incorporation of collagen in the system caused increase on the average pore size. Fourier transform infrared spectroscopy (FTIR) analysis indicated the main functional groups of each component of the composite system and their chemical interactions forming the scaffold. Moreover, rheological measurements were employed to assess the viscoelastic behavior of the hydrogels as a function of the temperature. The results demonstrated that the addition of collagen and bioactive glass increases the mechanical properties after the gelation process. The addition of 2 wt.% of BG nanoparticles caused an increase of approximately 39% on stiffness compared to pure chitosan and the addition of 30 wt.% collagen caused a further increase on the stiffness by 95%. The cytotoxicity and cell viability of the hydrogels were assessed by MTT and LIVE/DEAD® assays, where the results demonstrated no toxic effect of the composites on the human osteosarcoma cell culture (SAOS) and kidney cells line of human embryo (HEK 293 T). Hence, it can be stated that innovative composites were successfully designed and synthesized in this research with promising potential to be used as thermoresponsive biomaterials for bone-tissue bioapplications.

FTIR and UV‒vis study of chemically engineered biomaterial surfaces for protein immobilization

The biomaterials research field has broadened in the last 3 decades, including replacement of diseased or damaged parts, assist in healing, correct and improve functional abnormality, drug delivery systems, immunological kits and biosensors. Proteins play crucial role in almost every biological system. They are involved in enzymatic catalysis, transport and storage, coordinated motion, mechanical support, immune protection, control of growth and cell differentiation among many others. The immobilization of proteins onto surface functionalized substrates has been one of the most promising areas in bioengineering field. It is important to note that the term immobilization can refer either to a temporary or to a permanent localization of the biomolecule on or within a support. Proteins have very particular chain configurations and conformations that promote high levels of specificity during chemical interactions. In the present work, we aimed to study the phenomenon of protein immobilization onto biomaterial with chemically engineered surface. We have tailored the surface of the porous gels of SiO2 with 5 different silane surface modifying agents: tetraethoxysilane (TEOS), 3‒mercaptopropyltrimethoxysilane (MPTMS) and 3‒aminopropyltriethoxysilane (APTES), 3‒glycidoxypropyltrimethoxysilane (GPTMS) and 3‒isocyanatopropyltriethoxysilane (ICPES). Fourier Transform Infrared Spectroscopy (FTIR) was used to characterize the presence of all specific chemical groups in the materials. The surface functionalized gels were then immersed in porcine insulin (PI) solutions for protein immobilization. The incorporation of protein within the gels was also monitored by FTIR spectroscopy. The kinetics of protein adsorption and desorption from the gel matrix in vitro tests were monitored by UV‒visible spectroscopy. We could not observe any evidence of denaturation of insulin after its desorption from gel matrices using UV‒visible spectroscopy technique. In vivo tests with adult male rats were used to verify the immobilized insulin bioactivity after implantation of different biomaterial with functionalized surfaces. Plasma glucose levels were obtained by using the Glucose GOD‒ANA Colorimetric Assay. All surface modified materials have presented acute hypoglycemic peak response associated with the insulin bioactivity.

Synthesis, characterization and cytocompatibility of spherical bioactive glass nanoparticles for potential hard tissue engineering applications

Nanotechnology offers a new strategy to develop novel bioactive materials, given that nano-scaled biomaterials exhibit an enhanced biocompatibility and bioactivity. In this work, we developed a method for the synthesis of spherical bioactive glass nanoparticles (BGNP) aimed at producing biomaterials for potential use in the repair of hard tissues. The BGNP were prepared using the sol-gel process based on the reaction of alkoxides and other precursors in aqueous media for obtaining the oxide-ternary system with the stoichiometric proportion of 60% SiO2, 36% CaO and 4% P2O5. The system was extensively characterized by Fourier transform infrared, x-ray diffraction and scanning electron microscope/energy-dispersive x-ray spectroscopy with regard to chemical composition, crystallinity and morphology. Moreover, the results suggested the BGNP to be highly bioactive, which was confirmed by the formation of a hydroxyapatite biomimetic layer on the material surfaces upon immersion in simulated body fluid solution. In addition, the bioactivity response toward the developed BGNPs was assessed by direct contact of osteoblast cells using resazurin and alkaline phosphatase assays. The new BGNP have presented a significant increase in the osteoblast in vitro cytocompatibility behavior as compared to similar micro-sized bioactive glass particles. Such improvement in the overall bioactive behavior of BGNP was attributed to the much higher surface area causing enhanced interactions at the cell-nanomaterial interfaces. Hence, based on the results, the BGNP produced are the biomaterials to be potentially utilized in hard tissue engineering applications.

Effects of extracellular calcium concentration on the glutamate release by bioactive glass (BG60S) preincubated osteoblasts

Glutamate released by osteoblasts sharing similarities with its role in neuronal transmission is a very new scientific concept which actually changed the understanding of bone physiology. Since glutamate release is a calcium (Ca(2+))-dependent process and considering that we have previously demonstrated that the dissolution of bioactive glass with 60% of silicon (BG60S) can alter osteoblast Ca(2+)-signaling machinery, we investigated whether BG60S induces glutamate secretion in osteoblasts and whether it requires an increase in intracellular Ca(2+). Here we showed that the extracellular Ca(2+) increase due to BG60S dissolution leads to an intracellular Ca(2+) increase in the osteoblast, through the activation of an inositol 1,4,5-triphosphate receptor (InsP(3)R) and a ryanodine receptor (RyR). Additionally, we also demonstrated that glutamate released by osteoblasts can be profoundly altered by BG60S. The modulation of osteoblast glutamate released by the extracellular Ca(2+) concentration opens a new window in the field of tissue engineering, since many biomaterials used for bone repair are able to increase the extracellular Ca(2+) concentration due to their dissolution products.

Preparation of hybrid biomaterials for bone tissue engineering

Tissue engineering has evolved from the use of biomaterials for bone substitution that fulfill the clinical demands of biocompatibility, biodegradability, non-immunogeneity, structural strength and porosity. Porous scaffolds have been developed in many forms and materials, but few reached the need of adequate physical, biological and mechanical properties. In the present paper we report the preparation of hybrid porous polyvinyl alcohol (PVA)/bioactive glass through the sol-gel route, using partially and fully hydrolyzed polyvinyl alcohol, and perform structural characterization. Hybrids containing PVA and bioactive glass with composition 58SiO2-33CaO-9P2O5 were synthesized by foaming a mixture of polymer solution and bioactive glass sol-gel precursor solution. Sol-gel solution was prepared from mixing tetraethoxysilane (TEOS), triethylphosphate (TEP), and calcium chloride as chemical precursors. The hybrid composites obtained after aging and drying at low temperature were chemically and morphologically characterized through infrared spectroscopy and scanning electron microscopy. The degree of hydrolysis of PVA, concentration of PVA solution and different PVA-bioglass composition ratios affect the synthesis procedure. Synthesis parameters must be very well combined in order to allow foaming and gelation. The hybrid scaffolds obtained exhibited macroporous structure with pore size varying from 50 to 600 µm.

Development of biodegradable polyurethane and bioactive glass nanoparticles scaffolds for bone tissue engineering applications.

The development of polymer/bioactive glass has been recognized as a strategy to improve the mechanical behavior of bioactive glass-based materials. Several studies have reported systems based on bioactive glass/biopolymer composites. In this study, we developed a composite system based on bioactive glass nanoparticles (BGNP), obtained by a modified Stöber method. We also developed a new chemical route to obtain aqueous dispersive biodegradable polyurethane. The production of polyurethane/BGNP scaffolds intending to combine biocompatibility, mechanical, and physical properties in a material designed for tissue engineering applications. The composites obtained were characterized by structural, biological, and mechanical tests. The films presented 350% of deformation and the foams presented pore structure and mechanical properties adequate to support cell growth and proliferation. The materials presented good cell viability and hydroxyapatite layer formation upon immersion in simulated body fluid.

In vitro and in vivo osteogenic potential of bioactive glass-PVA hybrid scaffolds colonized by mesenchymal stem cells

Bioactive glass/polymer composites are promising materials for bone tissue engineering. The present research group has developed porous hybrid scaffolds comprised of 50% polyvinyl alcohol/50% bioactive glass with a 70%SiO(2)-30%CaO composition. Prior studies have also shown the adequate structural and mechanical behavior of these scaffolds. As such, the present study investigates the in vitro and in vivo osteogenic potential of the scaffold, using mesenchymal stem cells (MSC) from the bone marrow of female rats. MTT, alkaline phosphatase activity, collagen secretion and Von Kossa staining were conducted to evaluate the differentiation ability of MSC in an osteogenic medium. The in vitro results indicate an increase in both cell proliferation and osteogenic differentiation when the hybrid material is present. Von Kossa staining showed a progressive increase in mineralization nodules, coupled with time differentiation. For the in vivo evaluation, three groups were studied: (1) group implanted with the hybrid scaffold, (2) group implanted with scaffold colonized by non-differentiated MSC and (3) group implanted with scaffold colonized by differentiated MSC. The scaffolds were subcutaneously implanted on the back of Wistar rats for 1-8 weeks, and histological and histomorphometric analyses were performed. The tissue ingrowth proved to be higher in the groups colonized by MSC in the first week. In the second week, only the hybrid colonized by differentiated MSC presented a larger percentage of connective tissue. In the third, fourth and eighth weeks, all groups presented 70% of the hybrid scaffold filled with tissue. However, only the group with differentiated MSC presented some form of osteoid tissue, indicating that the hybrid scaffold with differentiated MSC does indeed present osteogenic potential.