Influence of purified multiwalled carbon nanotubes on the mechanical and morphological behavior in poly (L-lactic acid) matrix.

Poly (L-latic acid) (PLLA) is a bioresorbable polymer widely used as a biomaterial, but its fragility can limit its use. An alternative is to produce polymer nanocomposites, which can enhance the mechanical properties of polymeric matrix, resulting in a material with differentiated properties. In this work, PLLA based nanocomposites containing 0.25, 0.5 and 1.0wt% of purified multiwalled carbon nanotubes (p-MWCNTs) were prepared by the solvent casting method. The morphology and mechanical properties results show an improvement in strain at break for 0.25 and 0.5wt% p-MWCNTs and an increase in stiffness and elastic modulus for all compositions. Nanocomposites presented a p-MWCNTs agglomeration; however, there was a good stress transfer between PLLA and p-MWCNTs, which was confirmed by the increase in the hardness and elastic modulus. Atomic force microscopy analysis indicated an increase in roughness after nanotube addition. The in vitro biological study showed that PLLA/p-MWCNTs nanocomposites are cytocompatible with osteoblasts cells. The capacity of PLLA nanocomposites to stimulate osteogenesis was investigated by alkaline phosphatase (ALP) activity assay. Higher ALP activity was found on osteoblasts cultured on nanocomposites with 0.25 and 0.5wt% p-MWCNT compared to neat PLLA, confirming that PLLA cytocompatibility was improved on these compositions. Finally, our results showed that by a simple and inexpensive solvent casting method, it is possible to manufacture biofunctional nanocomposites devices with potential for orthopedic applications.

Expression of basal lamina components by Schwann cells cultured on poly(lactic acid) (PLLA) and poly(caprolactone) (PCL) membranes.

The present in vitro study investigated the expression of basal lamina components by Schwann cells (SCs) cultivated on PCL and PLLA membranes prepared by solvent evaporation. Cultures of SCs were obtained from sciatic nerves from neonatal Sprague Dawley rats and seeded on 24 well culture plates containing the polymer membranes. The purity of the cultures was evaluated with a Schwann cell marker antibody (anti-S-100). After one week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, laminin I and II. Positive labeling against the studied molecules was observed, indicating that such biomaterials positively stimulate Schwann cell adhesion and proliferation. Overall, the present results provide evidence that membrane-derived biodegradable polymers, particularly those derived from PLLA, are able to provide adequate substrate and stimulate SCs to produce ECM molecules, what may have in turn positive effects in vivo, influencing the peripheral nerve regeneration process.

The microscopical characterization of membranes poly (L-glycolic-co-lactic acid) with and without added plasticizer: an in vivo study.

The development of biodegradable materials has lead to renewed interest in the study of their interactions with the host organism in order to make the resulting products appropriate for use as temporary materials in clinical research, as well as important therapeutic applications. The copolymer poly (L-lactic-co-glycolic acid) or PLGA membranes have been used for several purposes. The physical properties of these materials can be modified by the addition of a plasticizer, such as the triethylcitrate, to provide flexibility and porosity to the implants, and enhance control of the polymer degradation time. Membranes with 7% plasticizer and without plasticizer (triethylcitrate) were compared. Membranes without plasticizer were denser and more compact than those with plasticizer. Two days and 30 days after implantation, the membranes with and without plasticizer showed little degradation. Sixty days and 120 days after implantation, the membranes with 7% plasticizer showed more cell invasion, and tissue adherence, as well as rapid degradation when compared to membranes without plasticizer.

The influence of triethylcitrate on the biological properties of poly (L-lactic-co-glycolic acid) membranes.

Biodegradable polymers have a variety of uses in basic and clinical research, as well as important therapeutic applications. The most commonly used are poly (lactic acid), poly (glycolic acid) and their copolymer, poly (L-lactic-co-glycolic acid) or PLGA. The incorporation of a plasticizer into a polymer can be used to obtain a product with specific properties. In this work, we examined the influence of a plasticizer (triethylcitrate) on the properties of PLGA membrane implants for human clinical uses. Membranes with and without plasticizer were dense and compact and contained no pores. The incorporation of 7% plasticizer enhanced the degradation the polymer when compared to polymer without plasticizer. In membranes without plasticizer, the initiation of degradation was very slow and was seen only 60 days after implantation, should allow the use of this material in the repair of damage tissue. In both cases, macroscopic analysis showed that there was no adhesion of the membrane to capsule fibrous, and this adversely affected preservation of the polymer. With time, the adherence of the polymer to surrounding tissue increased. Overall there was little degradation of membranes without plasticizer compared to those containing plasticizer.

Morphometrical analysis of multinucleated giant cells in subdermal implants of poly-lactic acid in rats.

The use of bioabsorbable polymers in (bio)medical applications has increased greatly in recent years, mainly because of their good bioreabsorption and biocompatibility. In this work, we examined the development of foreign body giant cells in intimate contact with porous membranes of poly L-lactic acid containing 7% of plasticizer triethylcitrate implanted in the backs of rats. The membranes were removed 2, 7, 14, 21, 28, 60, 90 and 180 days after implantation, along with a portion of the tissue around the implant. Histological analysis of the implant and tissue revealed the formation of a fibrous capsule from the seventh day of implantation onwards. Foreign body giant cells appeared from the seventh day and increased in number up to the twenty-eighth day and then up to the ninetieth day of implantation, remaining constant up to the end of the study onwards, and increased in number up to the ninetieth day after implantation and then remained constant. The number of nuclei in these cells increased from the seventh day of implantation up to the ninetieth day and then up to the end of the study.

Subdermal implants of poly(L-lactic acid) with plasticizer: an ultrastructural study in rats.

Poly(L-lactic acid) (PLLA) membranes containing 7% triethylcitrate plasticizer were implanted in the subcutaneous tissue of rats, and the cellular reaction was evaluated over a period of 2-180 days. The samples were processed for conventional transmission electron microscopy. Polymorphonuclear-type cells and a fibrin network were seen within membrane pores 2 days after implantation. In subsequent samples, there was cellular infiltration, which consisted mainly of fibroblasts, macrophages and multinuclear giant cells embedded in an abundant extracellular matrix containing a network of collagen fibers and blood vessels. At 90 and 180 days after implantation, a high density of voluminous phagocytic cells with a large number of endocytic polymer fragments within their cytoplasm was seen. These results show that PLLA membranes can support connective tissue proliferation and remodeling, which are important properties for successful bio-protheses.

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.

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 percent fetal calf serum at 37°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.

Pins composed of poly(L-lactic acid)/poly(3- hydroxybutyrate-co-hydroxyvalerate) PLLA/PHBV blends: Degradation in vitro.

Bioabsorbable polymers have shown good clinical success in the fixation and stabilization of bone fractures. Understanding and controlling polymer prosthetic degradation and the effect of the degradation products in vivo are crucial for successful implant developments. In this study, pins made from blends of PLLA/PHBV of varying compositions were degraded in phosphate buffer and characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA) and scanning electron microscopy (SEM). The PLLA/PHBV blends were found to be immiscible. PLLA began to degrade after approximately 12 weeks, whereas PHBV showed some degradation only after 53 weeks. The crystallinity of the blends increased with degradation. In conclusion, PHBV improved the thermal properties of PLLA and reduced the brittleness of the blends tested. The 40/60 PLLA/PHBV blend had the best properties for use in orthopedics since it degraded quicker than pure PHBV, and yet maintained its crystallinity for longer than PLLA; in addition, this blend did not have the brittleness of PLLA. (Journal of Applied Biomaterials and Biomechanics 2005; 3: 50-60).

Porous and dense poly(L-lactic acid) and poly(D,L-lactic acid-co-glycolic acid) scaffolds: in vitro degradation in culture medium and osteoblasts culture.

The use of bioresorbable polymers as a support for culturing cells has received special attention as an alternative for the treatment of lesions and the loss of tissue. The aim of this work was to evaluate the degradation in cell culture medium of dense and porous scaffolds of poly(L-lactic acid) (PLLA) and poly(D,L-lactic acid-co-glycolic acid) (50:50) (PLGA50) prepared by casting. The adhesion and morphology of osteoblast cells on the surface of these polymers was evaluated. Thermal analyses were done by differential scanning calorimetry and thermogravimetric analysis and cell morphology was assessed by scanning electron microscopy. Autocatalysis was observed in PLGA50 samples because of the concentration of acid constituents in this material. Samples of PLLA showed no autocatalysis and hence no changes in their morphology, indicating that this polymer can be used as a structural support. Osteoblasts showed low adhesion to PLLA compared to PLGA50. The cell morphology on the surface of these materials was highly dispersed, which indicated a good interaction of the cells with the polymer substrate.

Use of triethylcitrate plasticizer in the production of poly-L-lactic acid implants with different degradation times.

Bioabsorbable materials have been widely used in the repair of damaged tissue as well as in the controlled release of drugs and as a supports for cultured cells. The degradation time of poly-L-(lactic acid) (PLLA) may be controlled by altering the polymer porosity through the addition of the plasticizer triethylcitrate. This in turn influences the extent cellular infiltration. In this study, we examined the degradation of PLLA membranes containing different concentrations of plasticizer. PLLA discs were implanted subcutaneouly in rats and withdrawn 2, 14 and 60 days after implantation. The samples were processed for light microscopy and scanning electron microscopy (SEM). Polymer degradation was proportional to the concentration of plasticizer, indicating that triethylcitrate could affect the degradation time of the implants, without damaging the polymer biocompatibility.

Synthesis and characterization of poly(L-lactic acid) membranes: studies in vivo and in vitro.

The use of biodegradable polyesters as temporary structural supports in the recuperation of damaged live tissue is a promising area of research. Poly(L-lactic acid) (PLLA) membranes can act as a support for cell fixation and growth or as a barrier against soft tissues invasion in recuperating bone tissues. In this work, five different types of PLLA membranes, which varied in their polymer-solvent ratio and their content of plasticizer were studied. For the study in vivo, 6 mm diameter disks were inserted subcutaneously in the dorsal region of 15 Wistar rats, and the reactions on rats were studied 15 days later. In another series of experiments the samples were immersed in phosphate buffer, pH 7.4 at 37 degrees C, for 30 days. Membranes without plasticizer were morphologically dense and did not allow cell invasion nor tissue adherence, in contrast to membranes with plasticizer. While porosity enhanced cell fixation and growth, it made the membrane more fragile mechanically when compared to membranes without pores.

Adhesion and morphology of fibroblastic cells cultured on different polymeric biomaterials.

Cell adhesion is influenced by the physical and chemical characteristics of the materials used as substrate for cell culturing. In this work, we evaluated the influence of the morphological and chemical characteristics of different polymeric substrates on the adhesion and morphology of fibroblastic cells. Cell growth on poly (L-lactic acid) [PLLA] membranes and poly(2-hydroxy ethyl methacrylate) [polyHEMA], poly(2-hydroxy ethyl methacrylate)-cellulose acetate [polyHEMA-CA] and poly(2-hydroxy ethyl methacrylate)-poly(methyl methacrylate-co-acrylic acid) [polyHEMA-poly(MMA-co-AA)] hydrogels of different densities and pore diameters was examined. Cells adhered preferentially to more negatively charged substrates, with polyHEMA hydrogels being more adhesive than the other substractes. The pores present in PLLA membranes did not interfere with adhesion, but the cells showed a distinctive morphology on each membrane.

In vivo interaction of cells on poly L-(lactic acid) membranes containing plasticizer.

The development of biodegradable materials has lead to renewed interest in the study of their interactions with the host organism in order to make the resulting products appropriate for use as temporary materials in protheses. Poly L-(lactic acid)(PLLA)-based biodegradable devices have been used for several purposes. The physical properties of these materials can be modified by the addition of a plasticizer, such as the triethylcitrate, to provide flexibility and porosity to the implants and enhance control of the polymer degradation time. In this work we examined the biological properties of a PLLA porous membrane containing 7% triethylcitrate, by assessing the process of degradation and the interaction with dermal tissue. Samples of skin obtained from female Wistar rats 2-180 days after implantation with PLLA-based membrane were processed for light microscopy and scanning electron microscopy. The membranes became surrounded by a delicate network of connective tissue which gradually invaded the membrane structure. Polymer degradation began with the appearance of radial fractures in the globular units of the biodegradable membrane, especially by 90 and 180 days after implantation.

Vero cell growth and differentiation on poly(L-lactic acid) membranes of different pore diameters.

In the last few years, the demand has increased for research on polymeric materials, which can be used as substitutes for injured tissues and organs or to improve their regeneration. In this work, we studied poly(L-lactic acid) (PLLA) membranes, a resorbable biomaterial, which were either dense or had different pore diameters (less than 45 microm, between 180 and 250 microm, and between 250 and 350 microm), in relation to stimulation of cell adhesion, growth, and differentiation in vitro. We used Vero cells, a fibroblastic cell line, as the biological model of investigation. We found that cells attached slowly to all PLLA membranes studied. On the other hand, once the adhesion occurs, the cells are able to grow and differentiate on the different polymers. The cells grew to form a confluent monolayer and were capable of producing collagen Type IV and fibronectin on different PLLA membranes. This behavior indicates that cells try to create a better environment to stimulate their growth. This also indicates that Vero cells alter their differentiation pattern once they are producing extracellular matrix molecules related to epithelial differentiation.

Preparation of bioabsorbable nerve guide tubes.

The use of bioabsorbable polymers in applications as temporary structural function, recovering damage in live tissues, is a promising research area. Membranes of poly(lactic acid) (PLA) may act as support to adhesion and cellular invasion or as devices for guided tissue regeneration (GTR). In this study, the same casting technique used to prepare membranes was used to prepare PLA tubes. These tubes can be used for tests in nerve guided regeneration (NGR). To improve flexibility of the device, a bioabsorbable plasticizer was added to the polymer. The initial results showed that the proposed technique allowed the preparation of flexible tubes that can be used for NGR.