Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications

The formation of structures in poly(lactic acid) has been investigated with respect to producing areas of regular, superficial features with dimensions comparable to those of cells or biological macromolecules. Nanoembossing, a novel method of pattern replication in polymers, has been used for the production of features ranging from tens of micrometers, covering areas up to 1 cm(2), down to hundreds of nanometers. Both micro- and nanostructures are faithfully replicated. Contact-angle measurements suggest that positive microstructuring of the polymer (where features protrude from the polymer surface) produces a more hydrophilic surface than negative microstructuring. The ability to structure the surface of the poly(lactic acid), allied to the polymer's postprocessing transparency and proven biocompatibility, means that thin films produced in this way will be useful for bioengineers studying the interaction of micro- and nanodimensioned features with biological specimen, with regard to tissue engineering, for example.     

Bioactivity improvement of poly(epsilon-caprolactone) membrane with the addition of nanofibrous bioactive glass

Nanofibrous glass with a bioactive composition was added to a degradable polymer poly(ε-caprolactone) (PCL) to produce a nanocomposite in thin membrane form (260 μm). The bioactivity and osteoblastic responses of the nanocomposite membrane were examined and compared with those of a pure PCL membrane. Glass nanofibers with diameters in the range of hundreds of nanometers were added to a PCL solution at 20 wt.%, and the mixture was stirred vigorously and air dried. The obtained nanocomposite membrane showed that many chopped glass nanofibers formed by the mixing step were embedded uniformly into the PCL matrix. The nanocomposite membrane induced the rapid formation of apatite-like minerals on the surface when immersed in a simulated body fluid. Murine-derived osteoblastic cells (MC3T3-E1) grew actively over the nanocomposite membrane with cell viability significantly improved compared with those on the pure PCL membrane. Moreover, the osteoblastic activity, as assessed by the expression of alkaline phosphatase, was significantly higher on the nanocomposite membrane than on the pure PCL membrane. The currently developed nanocomposite of the bioactive glass-added PCL might find applications in the bone regeneration areas such as the guided bone regeneration (GBR) membrane.     

Electrospinning biomedical nanocomposite fibers of hydroxyapatite/poly(lactic acid) for bone regeneration

Development of fibrous matrices of bioceramic-biopolymer nanocomposite offers great potential in the field of bone regeneration and tissue engineering. However, in order to produce electrospun fibers with homogeneous structure, it is essential for the ceramic powder to be fine and to remain stable in suspension. Herein, we developed a novel method whereby the bioceramic hydroxyapatite (HA) was kept in suspension in biopolymer poly(lactic acid) (PLA). The strategy was to introduce a surfactant hydroxysteric acid (HSA) between the hydrophilic HA powder and the hydrophobic chloroform-dissolved PLA. The HA nanopowder was dispersed effectively in HSA and mixed homogeneously with PLA. Continuous and uniform fibers were generated successfully with diameters of approximately 1-2 microm, and featured a well-developed nanocomposite structure of HA nanopowder-dispersed PLA. Initial cellular assays showed excellent cell attachment and proliferation and also enhanced expression of alkaline phosphatase at 7 days of culturing. The HA-PLA nanocomposite fibers may be potentially useful in tissue engineering applications, particularly as three-dimensional substrates for bone growth.     

Immune responses in mice of beta-galactosidase adsorbed or encapsulated in poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres

The immune response induced in mice by beta-galactosidase (beta-gal) adsorbed or encapsulated on poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres was investigated. The encapsulated protein elicited higher antibody response than the protein adsorbed on the microspheres in the case of the PLA microspheres. However, the encapsulated protein elicited weaker antibody response than the adsorbed protein in the case of the PLGA (50:50) microspheres, probably because, in this case, the encapsulation process adversely affected protein immunogenicity. In the case of adsorbed beta-gal, higher antibody response was obtained with the PLA microspheres than with the PLGA (50:50) microspheres. This may be related to the lower rate of beta-gal desorption from the PLA microspheres. Based on the immunoglobulin G1/immunoglobulin G2a ratios and the stimulation indices for interferon-gamma and interleukin-4, beta-gal encapsulated or adsorbed on PLA microspheres induced a Th(1)-biased immune response whereas beta-gal encapsulated or adsorbed on PLGA (50:50) microspheres induced a Th(2)-biased immune response. The results obtained indicate that more potent immune responses are obtained when the protein is encapsulated than adsorbed on the microspheres, providing that the encapsulation process does not adversely affect protein immunogenicity. Also, the type of polymer used to prepare the microspheres, but not the method of protein association with the microspheres, may affect the type of immune response.     

Characterization of microrough bioactive glass surface: surface reactions and osteoblast responses in vitro

The current study characterized the in vitro surface reactions of microroughened bioactive glasses and compared osteoblast cell responses between smooth and microrough surfaces. Three different bioactive glass compositions were used and surface microroughening was obtained using a novel chemical etching method. Porous bioactive glass specimens made of sintered microspheres were immersed in simulated body fluid (SBF) or Tris solutions for 1, 6, 24, 48, or 72 h, and the formation of reaction layers was studied by means of a scanning electron microscope/energy dispersive X-ray analysis (SEM/EDXA). Cell culture studies were performed on bioactive glass disks to examine the influence of surface microroughness on the attachment and proliferation of human osteoblast-like cells (MG-63). Cell attachment was evaluated by means of microscopic counting of in situ stained cells. Cell proliferation was analyzed with a nonradioactive cell proliferation assay combined with in situ staining and laser confocal microscopy. The microroughening of the bioactive glass surface increased the rate of the silica gel layer formation during the first hours of the immersion. The formation of calcium phosphate layer was equal between control and microroughened glass surfaces. In cell cultures on bioactive glass, the microrough surface enhanced the attachment of osteoblast-like cells but did not have an effect on the proliferation rate or morphology of the cells as compared with smooth glass surface. In conclusion, microroughening significantly accelerated the early formation of surface reactions on three bioactive glasses and had a positive effect on initial cell attachment.     

Fabrication of novel calcium phosphate/poly(lactic acid) fiber composites

Composites using high-modulus polylactic acid (PLA) fibers coated with calcium phosphate (CaP) were prepared using a cyclic precipitation technique. Scanning electron microscopy revealed that small nuclei of CaP formed after the first soaking cycle, while large quantities of CaP particles were observed after the sixth cycle. The amount of CaP deposited on the PLA yarn increased with deposition time in Ca(2+) and PO(4) (3-) solutions and number of cycles, and decreased with stirring rate during washing cycles. It was observed that around 35 wt % of CaP was deposited on the yarn surface after six cycles of cyclic-soaking. Based on the results, a heterogeneous nucleation and growth mechanism was proposed for the CaP deposition on the surface of hydrolyzed polyester. Composites comprising the coated fibers in a poly(caprolactone) matrix exhibited flexural moduli within the range of that of the cortical bone.     

Composite membranes of poly(lactic acid) with zinc-added bioactive glass as a guiding matrix for osteogenic differentiation of bone marrow mesenchymal stem cells

This study aims to produce a degradable and bone-bioactive membrane for guiding bone regeneration by combining a degradable polymer, poly(lactic acid) (PLA), with a bioactive inorganic zinc-containing bioactive glass (ZnBG). The in?vitro osteogenic development of rat bone marrow mesenchymal stem cells (rBMSCs) upon different membrane substrates (pure PLA control, PLA-BG, and PLA-ZnBG) was investigated in terms of bone cell phenotype syntheses and mineralization. Results showed significantly stimulated production of alkaline phosphatase and osteocalcin at days 14 and 21 in the membranes containing BG and ZnBG, with more in the samples containing ZnBG. The addition of ZnBG in PLA allowed the rBMSCs to express a high level of bone sialoprotein as confirmed by immunostaining. Cellular mineralization of the secreted extracellular matrix showed a significantly higher Ca level on the BG- and ZnBG-added membrane than on the PLA, and the more so in the ZnBG-added one. Based on the in?vitro assessments using rBMSCs, the ZnBG-added PLA is considered to be of potential use in guiding active bone regeneration within the periodontal pocket.     

Solution-mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSCs) on PLGA by promoting the formation of a calcium-phosphate-rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells was tested. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSCs, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell-derived factors that promoted distant MSC differentiation. Thus PLGA-BG composites demonstrate significant potential as a bone-replacement material.     

Preparation and in vitro characterization of scaffolds of poly(L-lactic acid) containing bioactive glass ceramic nanoparticles

Porous nanocomposite scaffolds of poly(l-lactic acid) (PLLA) containing different quantities of bioactive glass ceramic (BGC) nanoparticles (SiO(2):CaO:P(2)O(5) approximately 55:40:5 (mol)) were prepared by a thermally induced phase-separation method. Dioxane was used as the solvent for PLLA. Introduction of less than 20wt.% of BGC nanoparticles did not remarkably affect the porosity of PLLA foam. However, as the BGC content increased to 30wt.%, the porosity of the composite was observed to decrease rapidly. The compressive modulus of the scaffolds increased from 5.5 to 8.0MPa, while the compressive strength increased from 0.28 to 0.35MPa as the BGC content increased from 0 to 30wt.%. The in vitro bioactivity and biodegradability of nanocomposites were investigated by incubation in simulated body fluid (SBF) and phosphate-buffered saline, respectively. Scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy and X-ray diffraction were employed to monitor the surface variation of neat PLLA and PLLA/BGC porous scaffolds during incubation. PLLA/(20wt.%)BGC composite exhibited the best mineralization property in SBF, while the PLLA/(10wt.%)BGC composite showed the highest water absorption ability.     

The effect of bioactive glass content on synthesis and bioactivity of composite poly (lactic-co-glycolic acid)/bioactive glass substrate for tissue engineering

Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic-co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.     

Biomedical nanocomposites of poly(lactic acid) and calcium phosphate hybridized with modified carbon nanotubes for hard tissue implants

Degradable polymer-based materials are attractive in orthopedics and dentistry as an alternative to metallic implants for use as bone fixatives. Herein, a degradable polymer poly(lactic acid) (PLA) was combined with novel hybrid nanopowder of carbon nanotubes (CNTs)-calcium phosphate (CP) for this application. In particular, CNTs-CP hybrid nanopowders (0.1 and 0.25% CNTs) were prepared from the solution of ionically modified CNTs (mCNTs), which was specifically synthesized to be well-dispersed and thus to effectively adsorb onto the CP nanoparticles. The mCNTs-CP hybrid nanopowders were then mixed with PLA (up to 50%) to produce mCNTs-CP-PLA nanocomposites. The mechanical tensile strength of the nanocomposites was significantly improved by the addition of mCNTs-CP hybrid nanopowders. Moreover, nanocomposites containing low concentration of mCNTs (0.1%) showed significantly stimulated biological responses including cell proliferation and osteoblastic differentiation in terms of gene and protein expressions. Based on this study, the addition of novel mCNT-CP hybrid nanopowders to PLA biopolymer may be considered a new material choice for developing hard tissue implants.     

Nanofibrous matrices of poly(lactic acid) and gelatin polymeric blends for the improvement of cellular responses

Substrates with a nanofibrous morphology are considered as a prospective matrix to populate and support cells in the tissue regeneration area. Although the nanofibers made of synthetic degradable polymers, including poly(lactic acid) (PLA), have been well studied, their poor cell affinity has restricted wider applications. Herein, we produced blending nanofibers made of PLA and gelatin to improve the cellular responses of PLA. For this, both PLA and gelatin were dissolved in an organic solvent, varying the compositions of PLA:gelatin at 1:3 and 1:1 by weight, and the solutions were electrospun into nanofibers. At all compositions, nanofibers could be successfully generated with diameters of approximately hundreds of nanometers. The addition of gelatin into PLA markedly improved the wettability of the nanofibrous substrate. The osteoblastic cells attached and spread well on all the blending nanofibers and pure PLA. In particular, the cellular growth was significantly higher on the gelatin-blended PLA than on the pure PLA nanofiber. On the basis of this study, the PLA/gelatin blending polymeric nanofibers are considered to be useful as a bone cell supporting matrix in the tissue regeneration area.     

The effect of poly (L-lactic acid) nanofiber orientation on osteogenic responses of human osteoblast-like MG63 cells

In this study, poly (L-lactic acid) (PLLA)/trifluoroethanol (TFE) solution was electrospun to fabricate fibrous scaffolds with different fiber orientations. Random and parallel PLLA nanofiber alignments were achieved by using a metal plate and a rolling rod as the receiver, respectively. The parallel PLLA fibrous scaffolds were further hot-stretched to obtain hyperparallel PLLA fibrous scaffolds. The PLLA fibrous scaffolds were characterized by fiber diameter, interfiber distance, fiber array angle, water contact angle, morphology and mechanical strength. The tensile strength of hyperparallel nano-fibers was approximately 5- and 14-times the parallel and random fibers, respectively. Osteoblast-like MG63 cells were cultured on the PLLA scaffolds to study the effects of fiber orientation on cell morphology, proliferation and differentiation. The cells on the randomly-oriented scaffolds showed irregular forms, while the cells exhibited shuttle-like shapes on the parallel scaffolds and had larger aspect ratios along the fiber direction of the hyperparallel scaffolds. Alkaline phosphatase (ALP) activity and collagen I (placeStateCol I) and osteocalcin (OC) deposition exhibited fiber orientation dependence. With an increase in parallelism of the fibers, there was a decrease in ALP activity and placeStateCol I and OC production. These results suggest that exploitation of PLLA fiber orientation may be used to control osteoblast-like cell responses.     

Synthesis of novel cholic acid functionalized branched oligo/poly(epsilon-caprolactone)s for biomedical applications

Novel cholic acid functionalized branched oligo/poly(epsilon-caprolactone)s were synthesized through the ring-opening polymerization of epsilon-caprolactone initiated by cholic acid with hydroxyl groups. The molecular weight of the branched polymers can be adjusted by controlling the feed ratio of the initiator cholic acid to the monomer epsilon-caprolactone. Comparing with linear homopolymer poly(epsilon-caprolactone) (PCL), these branched oligo/poly(epsilon-caprolactone)s show much faster hydrolytic degradation rates, implies that our approach provides a convenient and effective strategy to accelerate degradation of the biodegradable polymers with slow degradation rates such as PCL. The cell culture experiment indicates the incorporation of cholic acid moiety to the polymer chain can improve both cell adherence and proliferation obviously.     

Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.     

Biodegradable honeycomb-patterned film composed of poly(lactic acid) and dioleoylphosphatidylethanolamine

Honeycomb-patterned films have been reported to be useful for scaffolds of cell culture in tissue engineering. In the present study, we investigated a new compound, dioleoylphosphatidylethanolamine (DOPE), a naturally derived phospholipid having unsaturated fatty acid moieties, as a surfactant for fabricating honeycomb-patterned poly(d,l-lactide) (PLA) film. Only DOPE among commercially available phospholipids was useful as a surfactant, and it showed good solubility in PLA/chloroform solution and an excellent property for fabricating honeycomb-patterned film (the concentration of DOPE was from 0.2% to 20% by weight based on the weight of PLA). The pore size of the honeycomb was uniform, and all pores were interconnected with each other. The contact angle of water on the honeycomb-patterned film was affected by the amount of DOPE. Time-of-flight secondary ion mass spectrometer (TOF-SIMS) data suggested that DOPE was concentrated on the surface of the honeycomb-patterned film. To investigate cell proliferation and adhesion on the honeycomb-patterned film, NIH3T3 fibroblast cells were cultured on the film. The NIH3T3 cells adhered well on the honeycomb-patterned PLA film with DOPE (PLA-DOPE) and showed good cell proliferation compared to that on honeycomb-patterned PLA film fabricated with a copolymer (CAP) of dodecylacrylamide and omega-carboxyhexylacrylamide (PLA-CAP). These results suggest that the honeycomb-patterned PLA-DOPE can be applicable as a scaffold for cells with better profiles in comparison with PLA-CAP.     

Electrospinning of poly(lactic acid)/polyhedral oligomeric silsesquioxane nanocomposites and their potential in chondrogenic tissue regeneration

The study was conducted to evaluate the cytocompatibility and hydrolytic degradability of the new poly(lactic acid)/polyethylene glycol-polyhedral oligomeric silsesquioxane (peg-POSS/PLLA) nanocomposite as potential material for cartilage regeneration. PLLA scaffolds containing 0 to 5% of peg-POSS were fabricated by electrospinning. Human mesenchymal stem cells (hMSC’s) were cultured in vitro to evaluate the cytocompatibility of the new nanocomposite material. Hydrolytic degradation studies were also carried out to analyze the mass loss rate of the nanocomposites through time. The addition of the peg-POSS to the PLLA did not affect the processability of the nanocomposite by electrospinning. It was also observed that peg-POSS did not show any relevant change in fibers morphology, concluding that it was well dispersed. However, addition of peg-POSS caused noticeable decrease in mean fiber diameter, which made the specific surface area of the scaffold to rise. hMSC’s were able to attach, to proliferate, and to differentiate into chondrocytes in a similar way onto the different types of electrospun peg-POSS/PLLA and pure PLLA scaffolds, showing that the peg-POSS as nano-additive does not exhibit any cytotoxicity. The hydrolytic degradation rate of the material was lower when peg-POSS was added, showing a higher durability of the nanocomposites through time. Results demonstrate that the addition of peg-POSS to the PLLA scaffolds does not affect its cytocompatibility to obtain hyaline cartilage from hMSC’s.     

Preparation and characterization of poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) microspheres for controlled release of paclitaxel

Microspheres of a new kind of copolymer, poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA), are proposed in the present work for clinical administration of an antineoplastic drug paclitaxel with hypothesis that incorporation of a hydrophilic PEG segment within the hydrophobic PLA might facilitate the paclitaxel release. Paclitaxel-loaded PLA-PEG-PLA microspheres of various compositions were prepared by the solvent extraction/evaporation method. Characterization of the microspheres was then followed to examine the particle size and size distribution, the drug encapsulation efficiency, the colloidal stability, the surface chemistry, the surface and internal morphology, the drug physical state and its in vitro release behavior. The effects of polymer types, solvents and drug loading were investigated. It was found that in the microspheres the PEG segment was homogeneously distributed and caused porosity. Significantly faster release from PLA-PEG-PLA microspheres resulted in comparison with the PLGA counterpart. Incorporation of water-soluble solvent acetone in the organic solvent phase further increased the porosity of the PLA-PEG-PLA microspheres and facilitated the drug release. A total of 49.6% sustained release of paclitaxel within 1 month was achieved. Potentially, the presence of PEG on the surface of PLA-PEG-PLA microspheres could improve their biocompatibility. PLA-PEG-PLA microspheres could thus be promising for the clinical administration of highly hydrophobic antineoplastic drugs such as paclitaxel.     

Preparation of poly(lactic acid) composites containing calcium carbonate (vaterite)

A new type of ceramic-polymer biomaterial having excellent apatite-forming ability in simulated body fluid was prepared by hot-pressing a mixture of poly(-L-lactic acid) (PLA) and calcium carbonate (vaterite). After PLA dissolved in methylene chloride was mixed with calcium carbonate consisting of vaterite, the mixture was dried completely and subsequently hot-pressed uniaxially under a pressure of 40 MPa at 180 degrees C. When 30 wt% vaterite was introduced, the modulus of elasticity was effectively improved by 3.5-6 GPa, which was about twice higher than the modulus of PLA. The composite showed no brittle fracture behavior and a comparably high bending strength of approximately 50 MPa. The composite containing 30 wt% vaterite formed a 5-15-microm-thick bonelike apatite layer on its surface after soaking in SBF at 37 degrees C even for 1-3d.