PLLA Membranes Research Articles

Strategy to introduce an hydroxyapatite-keratin nanocomposite into a fibrous membrane for bone tissue engineering.

An hydroxyapatite (HA)-keratin nanocomposite was introduced into an electrospun poly(l-lactic acid) (PLLA) fibrous membrane with good electrospinnability. Biological in vitro cell culture demonstrated that the incorporation of HA-keratin into PLLA fibers led to significant bone formation compared to that of the pure electrospun PLLA membrane.

Microporous membranes of PLLA/PCL blends for periosteal tissue scaffold

Flexible microporous scaffold membranes of poly(l-lactic acid) (PLLA)/poly(ε-caprolactone) (PCL) blend were developed for bone regeneration. The new membranes overcome the fragility problems of PLLA membranes. When the PCL content in the blend was increased to 50%, the mechanical property for elongation was significantly improved without sacrificing the pores on the membrane surface that help tissue segments adhere the membrane. However, further increases in PCL content reduced the surface pores. In addition, spontaneous cell invasion and formation of cell-multilayers were observed in the membrane of PLLA/PCL blend (50:50). These results indicate that the porous membrane of PLLA/PCL blend (50:50) is useful for the preparation of periosteal sheets in tissue engineering.

Fabrication of Poly(lactic acid)-Poly(ethylene oxide) Electrospun Membranes with Controlled Micro to Nanofiber Sizes

Biodegradable poly(L-lactide acid) (PLLA) nanofiber membranes were prepared by electrospinning of PLLA and poly(ethylene oxide) (PEO). The selective removal of PEO by water allows to obtain smaller fiber diameters and to increase the porosity of the membranes in comparison to PLLA membranes obtained under the same electrospinning conditions. After removal of PEO membranes with fiber sizes of 260 nm and average porosity close to 80% are obtained. Thermal and infrared results confirm the poor miscibility of PLLA and PEO, with the PEO randomly distributed along the PLLA fibers. On the other, PLLA and PEO mixing strongly affect their respective degradation temperatures. The influence of the PEO in the electrospinning process is discussed and the results are correlated to the evolution of the PLLA fiber diameter.

Synthesis and Characterization of Biocompatible Poly(ethylene glycol)-b-Poly(L-lactide) and Study on Their Electrospun Scaffolds

A series of multiblock copolymer, Poly(L-lactide)-b-Poly(ethylene glycol) (PLLA-b-PEG) were synthesized and characterized by Fourier transform infrared spectra, differential scanning calorimetry and wide angle X-ray diffraction. PLLA-b-PEG fibrous scaffolds were prepared by electrospinning. The morphology of the fibers was affected by the solution concentration and different weight ratio of PLLA/PEG. In comparison with the electrospun PLLA membrane, the electrospun fibrous membranes of PLLA-b-PEG demonstrated an enhanced water absorption percentage and reductive water contact angle. The electrospun PLLA-b-PEG with weight ratio 90/10 and 75/25 fibrous membranes exhibited good flexibility and deformability to be beneficial for tissue engineering scaffolds.

Hydrolytic degradation of PLLA/PCL microporous membranes prepared by freeze extraction

Poly(l-lactic acid) – Poly(ε-caprolactone) blends (PLLA/PCL) porous membranes were prepared by freeze extraction (a modification of freeze drying) with ratios 100/0, 80/20, 60/40, 40/60, 20/80, 0/100 in weight. Degradation of the membranes in phosphate buffer solution (PBS) up to 65 weeks was studied using weight loss measurements, high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC), mechanical indentation, gel permeation chromatography (GPC), and scanning electron microscopy (SEM). Degradation rate as observed by weight loss and reduction of molecular weight and mechanical properties depended on the composition of the blends. In most blends the degradation was more prominent in the PLLA phase and was accompanied by consequent recrystallization that formed a crystalline phase with increased resistance to hydrolysis. Occurrence of such crystalline phases and degradation of intercrystalline domain led to formation of nearly monodisperse molecular weight populations.Membranes with only 20% PCL presented favorable behavior compared to pure PLLA membranes as reflected in a lower degradation rate and a limited loss of the mechanical properties. At the same time, degradation rate of 80/20 membranes was enhanced with respect to pure PCL, and membranes were stiffer than PCL membranes at all degradation times. This composition could thus be useful for use in tissue engineering for bone or cartilage applications.

Degradation of electrospun poly(L‐lactide) membranes under cyclic loading

AbstractBecause of the great effect of mechanical loads on degradation of biodegradable biomaterials, investigation on degradation of electrospun membranes under dynamic loading is of importance. In this article, degradation of electrospun poly(L‐lactide) (PLLA) membranes was carried out in a Tris‐HCl buffer solution containing proteinase K under a cyclic stretch loading (dynamic condition) in comparison with that in shaking water bath (static condition). The evolutions of mass loss, morphology, thermal properties, and relative molecular weight of PLLA were monitored during degradation. The results showed that after degradation for 10 h, the mass of the electrospun PLLA membranes dropped by 38.7% and 31.3%, respectively, under dynamic and static conditions. And the electrospun PLLA membranes showed higher value of crystallinity during degradation under both cyclic loading and static condition. In addition, benzyl triethyl ammonium chloride (BTEAC) was introduced during electrospinning, and the effects of electrospun membranes characteristic and crystallinity on degradation were also investigated. The degradation study results are expected to be a basic study in mimicking actually mechanical environments. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci 124:258–266, 2012

Fibrous membrane of nano‐hybrid poly‐L‐lactic acid/silica xerogel for guided bone regeneration

Nanofibrous membranes, consisting of a poly(L-lactic acid) (PLLA)-silica xerogel hybrid material, were successfully fabricated from a hybrid sol using the electrospinning technique for guided bone regeneration (GBR) application. These hybrid nanofibers exhibited a homogeneous and continuous morphology, with a nano-sized dispersed silica xerogel phase in the PLLA fiber matrix. The mechanical properties, such as the tensile strength and the elastic modulus, were improved as the silica xerogel content increased up to 40%. All of the hybrid membranes exhibited highly hydrophilic surfaces and good proliferation levels. After culturing for 13 days, the cells that were cultured on the hybrid membranes exhibited a significantly higher ALP activity compared to the pure PLLA membrane. Moreover, the in vivo animal experiments that used the rat calvarial defect model revealed a remarkably improved bone regeneration ability for the hybrid membrane compared to pure PLLA. These results demonstrated the feasibility of these hybrid membranes for efficient GBR.

Formation of Highly Aligned, Single‐Layered, Hollow Fibrous Assemblies and the Fabrication of Large Pieces of PLLA Membranes

AbstractLarge pieces of paper‐thin PLLA membranes are prepared and characterized as single‐layered hollow fibrous assemblies with an extremely high degree of fiber alignment. The diameter of the electrospun hollow fibers is in the range of some tens of micrometers with a wall thickness of a few micrometers. They are best described as 2D arrays of highly aligned microtubes. The mechanical properties of the membranes are studied. A processing map is constructed using the applied electric field strength and the concentration (viscosity) of the electrospinning dope. The influence of factors such as the field strength and weight and viscosity of the jetted solutions is discussed. The results will help in the future fabrication of highly anisotropic scaffolds for tissue engineering applications.magnified image

Heat and compression molded electrospun poly(l-lactide) membranes: Preparation and characterization

Abstract The fibrous membranes prepared by electrospinning have great advantages, such as high porosity and high specific surface area. However, low mechanical strength of electrospun membranes has been one of the most difficult technical problems to overcome, resulting in negative impact on the application. In this paper, the heat-assisted compression approach was employed to improve the mechanical performances of electrospun poly( l -lactide)(PLLA) membranes, especially in tensile strength. It is found that the electrospun PLLA membranes crystallize in α form and strong fiber-to-fiber linkages occurred with the aid of heat and compression. The tensile properties including tensile strength and modulus of membranes treated with a press at 6 MPa and a temperature at 60 °C (80 °C and 100 °C) increased by more than 100% compared with those of the as-electrospun membranes.

Synthesis and Characterization of Biodegradable Poly(ϵ-caprolactone)-b-Poly(L-lactide) and Study on Their Electrospun Scaffolds

A series of multi-block copolymers, poly(L-lactide)-b-poly (ϵ-caprolactone) (PLLA-b-PCL) were synthesized. The first step of the synthesis consisted of the transesterification between the PLLA and 1,4-Butanediol, followed by the copolymerization of PLLA-diols and PCL, using isophorone diisocyanate (IPDI) as a coupling agent. The synthesized polymers were characterized by Fourier transform infrared (FTIR) spectra, differential scanning calorimetry (DSC) and wide angle X-ray diffraction (WAXD). PLLA/PCL block copolymers were electrospun into ultrafine fibers. The morphology of the electrospun fibrous scaffolds were investigated by Scanning Electron Microscopy (SEM). Results showed that the morphology and diameter of the fibers were affected by the electrospinning solution concentrationan and different weight ratio of PLLA/PCL. These electrospun PLLA-b-PCL fibrous membranes exhibited good flexibility and deformability. In comparison with the electrospun PLLA membrane, the electrospun fibrous membranes of PLLA-b-PCL demonstrated an enhanced elongation with still high tensile strength and Young's modulus to be beneficial for tissue engineering scaffolds.

Effect of the content of hydroxyapatite nanoparticles on the properties and bioactivity of poly(l-lactide) – Hybrid membranes

Poly(l-lactide)/hydroxyapatite, PLLA/HA, composite membranes for bone regeneration with different concentrations of nanoparticles have been prepared and their physicochemical properties and bioactivity have been determined. Hydroxyapatite nanoparticles act as nucleating agent of the poly(l-lactide) crystals, as detected by DSC, and as reinforcing filler, as proven by the monotonous increase of the elastic modulus of the microporous membranes with increasing nano-filler content. The bioactivity, which regards to the use of these materials in bone regeneration, was tested by immersing the samples in a simulated body fluid, SBF. A faster deposition of a biomimetic apatite layer was observed as increases the content of hydroxyapatite nanoparticles, thus membranes with a 15% (w/w) of hydroxyapatite particles (relative to PLLA weight) present a homogeneous layer of hydroxyapatite on the surface of their pores after 7days of immersion in SBF. An especial emphasis has been made on the influence of a plasma treatment on the bioactivity of the membranes. With this aim, the membranes were submitted to a plasma treatment previously to their immersion in a simulated body fluid. It has been observed that the surface of a PLLA membrane after 21days of immersion in SBF is still not completely covered by hydroxyapatite whereas the same sample treated with plasma show a smooth layer of biomimetic hydroxyapatite. The increase of bioactivity achieved with this treatment was less important in high hydroxyapatite content composites.

Influence of micro-patterned PLLA membranes on outgrowth and orientation of hippocampal neurites

In neuronal tissue engineering many efforts are focused on creating biomaterials with physical and chemical pathways for controlling cellular proliferation and orientation. Neurons have the ability to respond to topographical features in their microenvironment causing among others, axons to proliferate along surface features such as substrate grooves in micro-and nanoscales. As a consequence these neuronal elements are able to correctly adhere, migrate and orient within their new environment during growth. Here we explored the polarization and orientation of hippocampal neuronal cells on nonpatterned and micro-patterned biodegradable poly( l-lactic acid) (PLLA) membranes with highly selective permeable properties. Dense and porous nonpatterned and micro-patterned membranes were prepared from PLLA by Phase Separation Micromolding. The micro-patterned membranes have a three-dimensional structure consisting of channels and ridges and of bricks of different widths. Nonpatterned and patterned membranes were used for hippocampal neuronal cultures isolated from postnatal days 1–3 hamsters and the neurite length, orientation and specific functions of cells were investigated up to 12 days of culture. Neurite outgrowth, length plus orientation tightly overlapped the pattern of the membrane surface. Cell distribution occurred only in correspondence to membrane grooves characterized by continuous channels whereas on membranes with interconnected channels, cells not only adhered to and elongated their cellular processes in the grooves but also in the breaking points. High orientation degrees of cells were determined particularly on the patterned porous membranes with channel width of 20 μm and ridges of 17 μm whereas on dense nonpatterned membranes as well as on polystyrene culture dish (PSCD) controls, a larger number of primary developed neurites were distributed. Based on these results, PLLA patterned membranes may directly improve the guidance of neurite extension and thereby enhancing their orientation with a consequently highly ordered neuronal cell matrix, which may have strong bearings on the elucidation of regeneration mechanisms.

Determining the protein drug release characteristics and cell adhesion to a PLLA or PLGA biodegradable polymer membrane

Biodegradable polymers are of interest for developing controlled protein drug delivery platforms. In this study, two poly (alpha-hydroxy) esters were formulated with Aerosol-OT, a surfactant stabilizer, to encapsulate the protein keratinocyte growth factor (KGF) for controlled release KGF is involved in a number of crucial biologic processes, most notably epithelial growth and repair. The concentration of KGF that caused a biological response in vitro was determined (optimally 10 ng/mL) and compared with the release of KGF from the two biodegradable polymer membrane formulations. Each polymer formulation released biologically relevant levels, 10 ng/mL of active KGF, although with different times release kinetics. The membrane composed of PLGA/AOT/KGF exhibited a faster release rate of KGF into solution after 120 h of degradation time than the release rate of the PLLA/AOT/KGF matrices. Cell seeding assays showed that both polymer matrices, when formulated with AOT, sustained cell growth. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was used to characterize the distribution of AOT and KGF through the polymer membrane. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Microporous Poly(L-Lactic Acid) Membranes Fabricated by Polyethylene Glycol Solvent-Cast/Particulate Leaching Technique

With the eventual goal of developing a tissue-engineered tear secretory system, we found that primary lacrimal gland acinar cells grown on solid poly(L-lactic acid) (PLLA) supports expressed the best histiotypic morphology. However, to be able to perform vectorial transport functions, epithelia must be supported by a permeable substratum. In the present study, we describe the use of a solvent-cast/particulate leaching technique to fabricate microporous PLLA membranes (mpPLLAm) from PLLA/polyethylene glycol blends. Scanning electron microscopy revealed pores on both the air-cured ( approximately 4 microm) and glass-cured sides (<2 microm) of the mpPLLAm. Diffusion studies were performed with mpPLLAm fabricated from 57.1% PLLA/42.9% polyethylene glycol blends to confirm the presence of channelized pores. The data reveal that glucose, L-tryptophan, and dextran (a high molecular weight glucose polymer) readily permeate mpPLLAm. Diffusion of the immunoglobulin G through the mpPLLAm decreased with time, suggesting the possible adsorption and occlusion of the pores. Cells cultured on the mpPLLAm (57.1/42.9 wt%) grew to subconfluent monolayers but retained histiotypic morphological and physiological characteristics of lacrimal acinar cells in vivo. Our results suggest that mpPLLAm fabricated using this technique may be useful as a scaffold for a bioartificial lacrimal gland device.

Preparation and biodegradation of electrospun PLLA/keratin nonwoven fibrous membrane

As a kind of natural protein, wool keratin was used to improve the cell affinity of poly( l -lactic acid) (PLLA). After small keratin particles were prepared from keratin solution by spray-drying process, they were blended with PLLA solution. PLLA/keratin nonwoven fibrous membrane was produced by electrospinning the blend solutions. The release rate of keratin from the composite membrane was detected by Fourier transform infrared (FTIR) after PLLA/keratin membranes were degraded in PBS up to 4 weeks. The chemical compositions of the PLLA/keratin surface were examined by X-ray photoelectron spectroscope. Although more than half of the keratin was removed from PLLA/keratin membrane during the first few hours of degradation, some keratin particles were still embedded in the PLLA fibers. Osteoblast cells were used to evaluate the cellular behaviors of the composite membrane. After 7 days culturing, more cells were observed on PLLA/keratin membranes than on pure PLLA membranes. MTT assay and alkaline phosphatase (ALP) activity results suggested that keratin could improve the interactions between osteoblast cells and the polymeric membranes.

The behavior of mesenchymal stem cells on micropatterned PLLA membranes

The aim of this study was to evaluate the behaviors of mesenchymal stem cells (MSCs) on Poly(L-Lactic acid) (PLLA) membranes with different surface topographies. The double-sided micropatterns, island-patterned, and sunken-patterned PLLA membranes with diameters of 60 and 100 microm, were fabricated by the soft lithography method. The cell viability of MSCs on the island- and sunken-patterned PLLA membranes were characterized by scanning electron microscopy (SEM), MTT assay, and flow cytometric analysis. Cell adhesion and proliferation capability were superior for the MSCs seeding on the island-patterned PLLA membranes than those on the sunken-patterned PLLA membranes. Especially, we observed the best biocompatibility for MSCs on the island-patterned surface with diameter of 100 microm. In addition, the improvement of cell attachment and augmenting subsequent cellular response are investigated after the island-patterned membranes precoating with collagen and fibronectin. Furthermore, the flow cytometric analysis reveals the MSCs can expand and maintain the phenotype on these PLLA membranes without losing its potential for differentiation. Since scale-up of cell production and optimization of culture conditions are important for stem cell engineering, to control the stem cell proliferation and differentiation is necessary. Therefore, besides topographical properties play a crucial role on the stem cells attachment and proliferative activity, it is suggested that the "relative scale" between cell and pattern also affects the cell adhesion morphologies and cell behaviors. Based on the overall cellular response, this study provides a valuable guidance to prepare appropriate topographic surface for tissue engineering application.

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.

Microscale control over collagen gradient on poly( l-lactide) membrane surface for manipulating chondrocyte distribution

A collagen gradient was constructed to interrogate cell adhesion on a poly( l-lactide) (PLLA) membrane surface. Utilizing a microinfusion pump, gradients of amino groups were generated on the PLLA surface by an aminolysis method. Immobilization of collagen onto the gradient surfaces was performed by glutaraldehyde (GA) coupling to form the collagen gradients. The –NH 2 and immobilized collagen density profiles on the PLLA membranes were quantitatively determined by ninhydrin and hydroproline (Hyp) analysis, respectively. By using fluorescein isothiocyanate (FITC) labeled collagen (FITC-Col), the profile of the as-prepared collagen gradient was directly monitored by a fluorescence microscope. The scanning force microscopy and water contact angle studies revealed that the morphology and wettability of the modified membranes changed progressively as a function of position along the gradient surface. Rabbit auricular chondrocytes were cultured on the collagen gradient membranes to test their cellular response. The attachment and spreading behaviors of the chondrocytes were dependent on the surface collagen density. These results indicate a surface on which the variation of collagen gradient strongly modifies the biological response of chondrocytes.

Chitosan/poly(L‐lactic acid) multilayered membrane for guided tissue regeneration

A chitosan/poly(L-lactic acid) (PLLA) multilayered membrane was prepared for the purpose of guided tissue regeneration (GTR). The membrane was composed of the outer layers of chitosan mesh and the middle layer of nanoporous PLLA membrane. The outer membranes of chitosan fibrous mesh present a highly biocompatible and rough surface for ease of cell adherence. The PLLA membrane is intended to provide mechanical strength to the membrane, thereby preventing epithelial invasion. The PLLA membrane was applied as a local drug delivery carrier for growth factors. Regular pores in PLLA membrane were generated through phase inversion of polymer solution. The membrane retained its integrity during the degradation process in vitro (for up to 8 weeks), which is a requirement for providing enough space in vivo in the GTR procedure. Osteoblasts firmly attached to broad cytoplasmic extensions along with the microfibers of the membrane. The phenotypic expression of cultured osteoblasts on the membrane was measured by a reverse transcriptase-polymerase chain reaction. New bone had formed beneath the chitosan/PLLA membrane. These results suggested that the chitosan/PLLA membranes properly functioned as biocompatible and mechanically stable barriers for GTR.

Surface patterning of poly(L‐lactide) upon melt processing: In vitro culturing of fibroblasts and osteoblasts on surfaces ranging from highly crystalline with spherulitic protrusions to amorphous with nanoscale indentations

Large-scale and reproducible manufacturing of scaffolds for tissue engineering applications will necessitate the adoption of methods relying on the processing of the biodegradable polymers directly from the melt. Such solventless processing will give rise to bulk and surface properties that will differ significantly from those generated upon processing from solution-based methods. Thus, detailed understanding of the microstructures that are developed during melt processing and the resulting surface/cell interactions is needed. Here, surfaces of melt-cast poly(L-lactide) (PLLA) were patterned to furnish membrane samples with a wide range of crystallinity and significant differences in surface topographies, ranging from highly crystalline (60%) with spherulitic protrusions at the surface to amorphous with nanoscale indentations. The PLLA membranes were used to culture in vitro mouse 3T3-Swiss albino fibroblast cells and osteoblast-like MC3T3-E1 cells. The growth rates of 3T3 fibroblasts were significantly lower on highly crystalline PLLA membranes with spherulitic protrusions in comparison to crystalline PLLA without spherulitic protrusions and amorphous surfaces with 5-10-nm-deep indentations. However, the differences in the growth rates of osteoblast-like cells cultured on the PLLA membranes with different surface patterns were only marginally different.