Maria Lucia Furlan Wada

Polímeros biorreabsorvíveis como substrato para cultura de células e engenharia tecidual

Polymeric biomaterials are used as replacements for damaged tissue or/and for stimulating its regeneration. A class of polymeric biomaterials is the bioreabsorbable ones, which degrade in vitro as well as in vivo. They are used in tissues that need a temporary support for its tissue regeneration. Among the several bioabsorbable polymers are included a-hydroxy acids, in different forms of poly(lactic acid) (PLA), such as the poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA), poly(DL-lactic acid) (PDLLA), in addition to poly(glycolic acid) (PGA) and poly(caprolactone) (PCL). These polymers are well-known because their decomposition products are eliminated from the body via metabolic routes and due to their good biocompatibility. Many results show that the different PLA based substrates do not present toxicity, once the cells could differentiate over the different polymers, which was demonstrated by the production of extracellular matrix components by many cell types. In this work, we reviewed the employment of a-hydroxy acid, with emphasis on different forms of PLA scaffolds used as cell culture substrates and their applications.

Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37 degrees C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.

ALTERATIONS IN THE GROWTH AND ADHESION PATTERN OF VERO CELLS INDUCED BY NUTRITIONAL STRESS CONDITIONS

The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44‐kDa protein was observed in stressed cells and was absent in non‐stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell—cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.

Differential Schwann cell migration in adult and old mice: an in vitro study

The influence of aging on Schwann cell (SC) proliferation, migration and viability was studied in vitro. SCs were cultured in Ham F-10 medium enriched with 20% fetal calf serum (FCS), 40% FCS or collagen I gel plus 20% FCS. The migration of adult mice derived SCs was stimulated with FCS and collagen. With aging, SC migration, multiplication and viability decreased, indicating that ideal culturing conditions should be adjusted.

Analysis of the growth pattern of Vero cells cultured on dense and porous poly (L-Lactic Acid) scaffolds

Poly (L-lactic acid) (PLLA) polymers are the most frequently used substrates for cell culture, tissue regeneration and orthopedic prostheses, mainly because of their atoxic characteristics and good biocompatibility. The objective of this study was to evaluate whether a higher density or different pore diameters (less than 45, 180-250, and 250-350 µm) would change the growth pattern of cultured cells. The cells were found to adhere to and spread over all PLLA scaffolds studied. The cells also showed similar proliferation on dense and porous PLLA scaffolds, except for PLLA scaffolds with a smaller pore diameter. The cytochemical data showed high metabolic cellular activity on the various substrates. Overall, the results indicated satisfactory cell growth and proliferation on the different PLLA scaffolds studied, especially for those with pore diameters of 180-250 µm and 250-350 µm.