Moema Hausen

Nanoneurobiophysics: new challenges for diagnosis and therapy of neurologic disorders

Nanoneurobiophysics Research Group Department of Physics Chemistry and Mathematics Federal University of Sao Carlos (UFSCar)

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface.

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine–glycine–aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk’s surface on the nanoscale and the disk’s surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.

Study of mesenchymal stem cells cultured on a poly(lactic-co-glycolic acid) scaffold containing simvastatin for bone healing

Background Tissue engineering is a promising alternative for the development of bone substitutes; for this purpose, three things are necessary: stem cells, a scaffold to allow tissue growth and factors that induce tissue regeneration. Methods To congregate such efforts, we used the bioresorbable and biocompatible polymer poly(lactic-co-glycolic acid) (PLGA) as scaffold. For the osteoinductive factor, we used simvastatin (SIM), a drug with a pleiotropic effect on bone growth. Mesenchymal stem cells (MSCs) were cultured in PLGA containing SIM, and the bone substitute of PLGA/SIM/MSC was grafted into critical defects of rat calvaria. Results The in vitro results showed that SIM directly interfered with the proliferation of MSC promoting cell death, while in the pure PLGA scaffold the MSC grew continuously. Scaffolds were implanted in the calvaria of rats and separated into groups: control (empty defect), PLGA pure, PLGA/SIM, PLGA/MSC and PLGA/SIM/MSC. The increase in bone growth was higher in the PLGA/SIM group. Conclusions We observed no improvement in the growth of bone tissue after implantation of the PLGA/SIM/MSC scaffold. As compared with in vitro results, our main hypothesis is that the microarchitecture of PLGA associated with low SIM release would have created an in vivo microenvironment of concentrated SIM that might have induced MSC death. However, our findings indicate that once implanted, both PLGA/SIM and PLGA/MSC contributed to bone formation. We suggest that strategies to maintain the viability of MSCs after cultivation in PLGA/SIM will contribute to improvement of bone regeneration.

Bilaminar Device of Poly(Lactic‐co‐Glycolic Acid)/Collagen Cultured With Adipose‐Derived Stem Cells for Dermal Regeneration

Several materials are commercially available as substitutes for skin. However, new strategies are needed to improve the treatment of skin wounds. In this study, we developed and characterized a new device consisting of poly(lactic-co-glycolic acid) (PLGA) and collagen associated with mesenchymal stem cells derived from human adipose tissue. To develop the bilaminar device, we initially obtained a membrane of PLGA by dissolving the copolymer in chloroform and then produced a collagen type I scaffold by freeze-drying. The materials were characterized physically by gel permeation chromatography, scanning electron microscopy, and mass loss. Biological activity was assessed by cell proliferation assay. A preliminary study in vivo was performed with a pig model in which tissue regeneration was assessed macroscopically and histologically, the commercial device Integra being used as a control. The PLGA/collagen bilaminar material was porous, hydrolytically degradable, and compatible with skin growth. The polymer complex allowed cell adhesion and proliferation, making it a potentially useful cell carrier. In addition, the transparency of the material allowed monitoring of the lesion when the dressings were changed. Xenogeneic mesenchymal cells cultured on the device (PLGA/collagen/ASC) showed a reduced granulomatous reaction to bovine collagen, down-regulation of α-SMA, enhancement in the number of neoformed blood vessels, and collagen organization as compared with normal skin; the device was superior to other materials tested (PLGA/collagen and Integra) in its ability to stimulate the formation of new cutaneous tissue.

Nanomechanical Cantilever-Based Sensor: An Efficient Tool to Measure the Binding Between the Herbicide Mesotrione and 4-Hydroxyphenylpyruvate Dioxygenase

Nanomechanical biosensors based on atomic force microscopy (AFM) cantilevers have garnered considerable attention. AFM cantilevers are devices that can detect a target either via a surface functionalization process based on immobilization through molecular adsorption, or through the selective chemical binding of a specific molecule, transforming the device into a specific biosensor. In this study, we demonstrate that functionalized AFM cantilevers could be used, in a process involving self-assembling layers, to create a homogeneous surface layer of the widely used herbicide mesotrione. Controlled experiments to evaluate its detection were performed, and binding between mesotrione and its target molecule, 4-hydroxyphenylpyruvate dioxygenase (HPPD), was evaluated using deflection curves of functionalized cantilevers interacting with mesotrione. The cantilevers worked as nanomechanical sensors inside a fluid cell device, under different concentrations of HPPD diluted in PBS. After evaluating increasing conce...

Effects of Albumin Adsorption on Cell Adhesion in Hydroxyapatite Modified Surfaces

Synthetic hydroxyapatite (HA) is a widely used ceramic biomaterial due to its well described biocompatibility. Some modifications in HA surface can be made to increase surface porosity. Likewise, HA can be modified by the coating with proteins, which may impact on biocompatibility. In this work, we aimed to evaluate the impact of two surface modifications – coating with albumin, a major serum protein, and augmented porosity - over osteoblast adhesion on stoichiometric HA discs. Dense HA discs were obtained by pressing HA powder at 30 KN and sinterization at 1000°C, while porous HA was molded after the addition of alginate (15:1), followed by thermal treatment. Protein adsorption was attained by incubation on 0.5mg/mL bovine serum albumin (BSA) for 24 h at 37°C. MC3T3 mouse preosteoblasts were seeded over both protein-coated and uncoated dense or porous tablets, and cell viability after 24 h was estimated by XTT and Neutral Red assays. Cell density was quantified by fluorescence microscopy. While both dense and porous discs presented altered surfaces after protein treatment, as observed by scanning electron microscopy, porous HA tablets presented significantly higher levels of adsorbed protein. There was a decrease in the concentration of calcium ions in all samples analyzed. Porous HA treated with protein presented significant higher mitochondrial dehydrogenase activity (XTT) than non treated tablets (p<0.001). Although the BSA adsorption didn`t affect cell adhesion, the results obtained in fluorescence quantification suggests that de dense surface was best for cellular adhesion and spread than the porous one. We conclude that differences in the topography of a biomaterial can directly influence their ability to adsorb proteins, while the dense surface was more favorable for both the adhesion and the spreading of pre-osteoblasts.

Biocompatibility of Carbonated Hydroxyapatite Nanoparticles with Different Crystallinities

Carbonated apatite (CHA) is commonly considered a promising synthetic material for biomedical applications in orthopedic and dental surgery due to its biocompatibility, bioresorption and bioactivity. CHA5, CHA37 and CHA90 powders were synthesized from wet method and the DRX patterns showed that the crystallinity and particle size of CHA samples increased proportionally with the synthesis temperature. Powder extracts medium were obtained from each sample to interact with MC3T3-E1 osteoblastics cells. It was evaluated morphology, citotoxicity, pH and Ca2+ concentration. Citotoxicity assays showed high metabolic activity on all samples when compared to control. The polygonal shaped and the confluent monolayer observed in control cells progressively changed according to the crystallinity increase of samples. Cells under mitosis and spindle-like shaped where the main alterations observed. In addition the cell viability could be sensitive to the acid reactivity and crystallinity of carbonated apatite samples.

Addition of Wollastonite Fibers to Calcium Phosphate Cement Increases Cell Viability and Stimulates Differentiation of Osteoblast-Like Cells

Calcium phosphate cement (CPC) that is based on α-tricalcium phosphate (α-TCP) is considered desirable for bone tissue engineering because of its relatively rapid degradation properties. However, such cement is relatively weak, restricting its use to areas of low mechanical stress. Wollastonite fibers (WF) have been used to improve the mechanical strength of biomaterials. However, the biological properties of WF remain poorly understood. Here, we tested the response of osteoblast-like cells to being cultured on CPC reinforced with 5% of WF (CPC-WF). We found that both types of cement studied achieved an ion balance for calcium and phosphate after 3 days of immersion in culture medium and this allowed subsequent long-term cell culture. CPC-WF increased cell viability and stimulated cell differentiation, compared to nonreinforced CPC. We hypothesize that late silicon release by CPC-WF induces increased cell proliferation and differentiation. Based on our findings, we propose that CPC-WF is a promising material for bone tissue engineering applications.

Development of a membrane of poly (L-co-D,L lactic acid-co-trimethylene carbonate) with aloe vera: An alternative biomaterial designed to improve skin healing:

The search for new therapies and drugs that act as topical agents to relieve pain and control the infectious processes in burns always attracted interest in clinical trials. As an alternative to synthetic drugs, the use of natural extracts is useful in the development of new strategies and formulations for improving the life quality. The aim of this study was to develop a wound dressing using Poly(L-co-D,L lactic acid-co-TMC) (PLDLA-co-TMC) containing aloe vera (AV). This natural plant extract is known for its modulatory effects under healing process. The membrane of PLDLA-co-TMC+aloe vera was prepared at different concentrations of AV (5, 10, 15 and 50%). The FTIR showed no change in the PLDLA-co-TMC spectrum after AV addition, while the swelling test showed changes only in PLDLA-co-TMC+AV at 50%. The wettability measurements showed decrease in the contact angle in all samples after the AV addition in the polymer, while the AV release test showed that PLDLA-co-TMC+50%AV sample has higher AV release rate than the sample with other AV concentrations. The SEM analysis showed that AV was homogeneously distributed at 5% only. Tensile tests demonstrated an increase in the Youngs modulus and a reduction in the elongation till rupture of the PLDLA-co-TMC after the addition of AV. Biocompatibility in vitro evaluation with fibroblast cells seeded in the membranes of PLDLA-co-TMC+AV showed that the cells were able to adhere, proliferate and maintain mitochondrial activity in all AV concentrations tested. Due to the known skin medicinal properties attributed to AV and the results here obtained, we suggest that after in vivo trials, the PLDLA-co-TMC+AV should be a promising biomaterial for application as a device for skin curative and healing agent.

Caracterização morfológica de nanocristais de celulose por microscopia de força atômica

O isolamento de nanocristais de celulose (CNCs) de fibras vegetais e uma alternativa promissora para sua aplicacao como reforco em matrizes polimericas. A caracterizacao dos CNCs e fundamental para a confiabilidade da tecnica, alem de determinar as aplicacoes possiveis a partir de cada tipo de fibra. A partir da tecnica de microscopia de forca atomica, um estudo da morfologia e distribuicao dos CNCs de semente de manga, vagem de algaroba, pseudocaule da bananeira e fibra do mesocarpo de dende foi realizado neste trabalho. Os CNCs foram obtidos via reacao hidrolitica com acido sulfurico em concentracoes que variaram de acordo com a fonte da fibra. Os resultados obtidos revelaram dimensoes variando de 300 a 500 nm em comprimento e 4 a 16 nm em diâmetro. A apresentacao morfologica em forma de agulha demonstrou que o isolamento das fibras de celulose em CNCs foi efetiva. A razao de aspecto associada a formacao cilindrica em agulha dos CNCs isolados evidenciou o alto potencial das fontes de dende e de vagem de algaroba para o reforco de bionanocompositos.