Patricia Valério

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.

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.

Biocompatibility evaluation of hydroxyapatite/collagen nanocomposites doped with Zn+2

In this work, novel composites based on calcium phosphates (CaP)/collagen (COL) doped with Zn(+2) have been synthesized. They were characterized by SEM coupled to EDS microprobe in order to evaluate their morphology and chemical composition, respectively. The biocompatibility of these synthetic CaP/COL nanocomposites doped and undoped with Zn(+2) was investigated through osteoblast cell culture assay. Calcium phosphates were produced via aqueous precipitation routes where two different phases were obtained, hydroxyapatite (HAP) and biphasic hydroxyapatite-betatricalcium phosphate (HAPbetaTCP). In the sequence, the type-I collagen (COL) was added to the inorganic phase based on calcium phosphate and the mixture was blended until a homogenous composite was obtained. Zn(+2) aqueous solution (1.0 wt%) was used as the doping reagent. The cell viability and the alkaline phosphatase production of osteoblasts in the presence of the composites were evaluated and compared to control osteoblasts. Also, the biocompatibility of the composite was investigated through cell morphological analysis using optical microscopy of osteoblasts. All experiments were performed in triplicates (n = 3) from three different experiments. They were analyzed by variance test (ANOVA) and Bonferronis post-test with differences statistically significant at p < 0.05. The results showed that the CaP/COL composites doped and undoped with Zn(+2) did not present alterations in cell morphology in 72 h and had similar cell viability and alkaline phosphatase activity to the control. All the tested CaP/COL composites showed adequate biological properties with the potential to be used in bone tissue replacement applications.

Synthesis, bioactivity and preliminary biocompatibility studies of glasses in the system CaO–MgO–SiO2–Na2O–P2O5–CaF2

New compositions of bioactive glasses are proposed in the CaO–MgO–SiO2–Na2O–P2O5–CaF2 system. Mineralization tests with immersion of the investigated glasses in simulated body fluid (SBF) at 37°C showed that the glasses favour the surface formation of hydroxyapatite (HA) from the early stages of the experiments. In the case of daily renewable SBF, monetite (CaHPO4) formation competed with the formation of HA. The influence of structural features of the glasses on their mineralization (bioactivity) performance is discussed. Preliminary in vitro experiments with osteoblasts’ cell-cultures showed that the glasses are biocompatible and there is no evidence of toxicity. Sintering and devitrification studies of glass powder compacts were also performed. Glass-ceramics with attractive properties were obtained after heat treatment of the glasses at relatively low temperatures (up to 850°C).

The effect of a chitosan-gelatin matrix and dexamethasone on the behavior of rabbit mesenchymal stem cells.

Cartilage tissue has poor capability of 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 defects in adult cartilage tissue. Mesenchymal stem cells (MSC) and chondrocytes are the two major cell sources for cartilage tissue engineering. The present study combined culture conditions of MSC in a chitosan-gelatin matrix in chondrogenic media to evaluate their effects on MSC viability and chondrogenesis for cartilage tissue engineering. MSC were harvested from rabbit bone marrows and cultured in chondrogenic media supplemented, or not, with dexamethasone in a chitosan-gelatin film (C-GF). The association of C-GF and dexamethasone promoted significant increase in cell adhesivity, viability and proliferation when compared to MCS cultured in media without dexamethasone or C-GF. In addition, dexamethasone promoted increase in the collagen concentration of MSC cultures. A reduction of alkaline phosphatase activity after three weeks of culture in chondrogenic media was verified. No influence of the C-GF or of dexamethasone was observed in this matter. Therefore, it is reasonable to suggest that biomaterial-based chitosan-gelatin and chondrogenic media supplemented with dexamethasone may stimulate the proliferation and differentiation of MSC according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the chondrogenesis of MSC suitable for cartilage tissue engineering.

Rietveld structure and in vitro analysis on the influence of magnesium in biphasic (hydroxyapatite and β-tricalcium phosphate) mixtures

The structure of two different Mg-substituted biphasic (HAP and beta-TCP) mixtures along with the biphasic mixtures without substituted Mg(2+) was investigated using Rietveld refinement technique. The substituted Mg(2+) was found in the beta-TCP phase and its influence on the composition has led to an increase in HAP content of Mg-containing biphasic mixtures when compared with the HAP content detected in pure biphasic mixtures. The refined structural parameters of Ca(10)(PO(4))(6)(OH)(2) and beta-Ca(3)(PO(4))(2) confirmed that all the investigated compositions have crystallized in the corresponding hexagonal (space group P6(3)/m) and rhombohedral (space group R3c) structures. The substitution of lower sized magnesium was found preferentially incorporated at the sixfold-coordinated Ca (5) site of beta-TCP, which is due to the strong Ca (5).O interaction among all the five different Ca sites of beta-Ca(3)(PO(4))(2). The in vitro tests using primary culture of osteoblasts showed that all the tested samples are biocompatible and promising materials for in vivo studies.

Biocompatibility Evaluation of Three Different Titanium-Hydroxyapatite Composites

Titanium reinforced with hydroxyapatite (TiHA) prepared using 15% of titanium and 3 different sinterizing temperatures 1100, 1200 and 1300 oC showed a significant increase in cell proliferation, when compared to the control.

Attachment and Proliferation of Osteoblasts on Lithium-Hydroxyapatite Composites

The biocompatibility and bioactivity properties of hydroxyapatites (HAs) modified through lithium addition were investigated. Hydroxyapatites obtained from bovine bone were mixed with lithium carbonate (Li), in the proportions of 0.25, 0.50, 1.00, and 2.00% wt, and sintered at 900°, 1000°, 1100°, 1200°, and 1300°C, creating LiHA samples. The osteoblast culture behavior was assessed in the presence of these LiHA compositions. The cellular interactions were analyzed by evaluating the viability and cellular proliferation, ALP production and collagen secretion. The cytotoxic potential was investigated through measurement of apoptosis and necrosis induction. The process of cellular attachment in the presence of the product of dissolution of LiHA, was evaluated trough fluorescence analysis. The physical characteristics of these materials and their cellular interactions were examined with SEM and EDS. The results of this study indicate that the LiHA ceramics are biocompatible and have variable bioactivities, which can be tailored by different combinations of the concentration of lithium carbonate and the sintering temperature. Our findings suggest that LiHA 0.25% wt, sintered at 1300°C, combines the necessary physical and structural qualities with favorable biocompatibility characteristics, achieving a bioactivity that seems to be adequate for use as a bone implant material.

Evaluation of Osteoblasts Viability, Alkaline Phosphatase Production and Collagen Secretion in the Presence of TiHA

Biocomposite materials have been developed in order to combine bioactivity of cera mics and mechanical properties of metals (1). Hydroxyapatite (HA) is known for its weakness and brittles. When titanium is added to HA, an improvement of the biomaterial m echanical properties occurs (2). However, the proportion of Ti and HA in each biocomposite can alt er its characteristics. The sintered temperatures can also alter the biological properties and therefore, they need to be characterized. Considering that calvarie osteoblast primary cultur e is a well stabilished model to investigate biocompatibility, in this study it is evaluated the os teoblast viability, alkaline phosphatase production and collagen secretion in the presence of four diffe rent TiHA biocomposites.

Biocompatibility Evaluation of Lithium-Hydroxyapatite Composites

Fifteen different lithium-hydroxyapatite composites were tested in this work. LiHA were prepared using 0.25 %, 1 % and 2 % of lithium and five different sintering temperature 900, 1000, 1100, 1200 and 1300oC. Primary culture of osteoblasts were used to evaluate the biocompatibility of the samples, concerning to cell viability and alkaline phosphatase production. The 1% LiHA samples sintered at 1100, 1200 and 13000C showed the best results.