The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies-therm ally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 mu m. (C) 2005 Elsevier Ltd. All rights reserved.

The visualisation of vitreous using surface modified poly(lactic-co-glycolic acid) microparticles

AIMS To demonstrate the potential use of in vitro poly(lactic-co-glycolic acid) (PLGA) microparticles in comparison with triamcinolone suspension to aid visualisation of vitreous during anterior and posterior vitrectomy. METHODS PLGA microparticles (diameter 10-60 microm) were fabricated using single and/or double emulsion technique(s) and used untreated or following the surface adsorption of a protein (transglutaminase). Particle size, shape, morphology and surface topography were assessed using scanning electron microscopy (SEM) and compared with a standard triamcinolone suspension. The efficacy of these microparticles to enhance visualisation of vitreous against the triamcinolone suspension was assessed using an in vitro set-up exploiting porcine vitreous. RESULTS Unmodified PLGA microparticles failed to adequately adhere to porcine vitreous and were readily washed out by irrigation. In contrast, modified transglutaminase-coated PLGA microparticles demonstrated a significant improvement in adhesiveness and were comparable to a triamcinolone suspension in their ability to enhance the visualisation of vitreous. This adhesive behaviour also demonstrated selectivity by not binding to the corneal endothelium. CONCLUSION The use of transglutaminase-modified biodegradable PLGA microparticles represents a novel method of visualising vitreous and aiding vitrectomy. This method may provide a distinct alternative for the visualisation of vitreous whilst eliminating the pharmacological effects of triamcinolone acetonide suspension.

Polymer-lipid interactions:biomimetic self-assembly behaviour and surface properties of poly(styrene-alt-maleic acid) with diacylphosphatidylcholines

Abstract Various lubricating body fluids at tissue interfaces are composed mainly of combinations of phospholipids and amphipathic apoproteins. The challenge in producing synthetic replacements for them is not replacing the phospholipid, which is readily available in synthetic form, but replacing the apoprotein component, more specifically, its unique biophysical properties rather than its chemistry. The potential of amphiphilic reactive hypercoiling behaviour of poly(styrene-alt-maleic acid) (PSMA) was studied in combination with two diacylphosphatidylcholines (PC) of different chain lengths in aqueous solution. The surface properties of the mixtures were characterized by conventional Langmuir-Wilhelmy balance (surface pressure under compression) and the du Noüy tensiometer (surface tension of the non-compressed mixtures). Surface tension values and 31P NMR demonstrated that self-assembly of polymer-phospholipid mixtures were pH and concentration-dependent. Finally, the particle size and zeta potential measurements of this self-assembly showed that it can form negatively charged nanosized structures that might find use as drug or lipids release systems on interfaces such as the tear film or lung interfacial layers. The structural reorganization was sensitive to the alkyl chain length of the PC.

Poly lactic-co-glycolic acid controlled delivery of Disulfiram to target liver cancer stem-like cells

Disulfiram (DS), an anti-alcoholism drug, shows very strong cytotoxicity in many cancer types. However its clinical application in cancer treatment is limited by the very short half-life in the bloodstream. In this study, we developed a poly lactic-co-glycolic acid (PLGA)-encapsulated DS protecting DS from the degradation in the bloodstream. The newly developed DS-PLGA was characterized. The DS-PLGA has very satisfactory encapsulation efficiency, drug-loading content and controlled release rate in vitro. PLGA encapsulation extended the half-life of DS from shorter than 2 minutes to 7 hours in serum. In combination with copper, DS-PLGA significantly inhibited the liver cancer stem cell population. CI-isobologram showed a remarkable synergistic cytotoxicity between DS-PLGA and 5-FU or Sorafenib. It also demonstrated very promising anticancer efficacy and antimetastatic effect in liver cancer mouse model. Both DS and PLGA are FDA approved products for clinical application. Our study may lead to repositioning of DS into liver cancer treatment.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

Introduction: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). Methods and materials: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium GreenTM-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. Results: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. Conclusion: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

Statistical modelling of the rheological and mucoadhesive properties of aqueous poly(methylvinylether-co-maleic acid) networks: Redefining biomedical applications and the relationship between viscoelasticity and mucoadhesion

Poly(methylvinylether-co-maleic acid) (PMVE/MA) is commonly used as a component of pharmaceutical platforms, principally to enhance interactions with biological substrates (mucoadhesion). However, the limited knowledge on the rheological properties of this polymer and their relationships with mucoadhesion has negated the biomedical use of this polymer as a mono-component platform. This study presents a comprehensive study of the rheological properties of aqueous PMVE/MA platforms and defines their relationships with mucoadhesion using multiple regression analysis. Using dilute solution viscometry the intrinsic viscosities of un-neutralised PMVE/MA and PMVE/MA neutralised using NaOH or TEA were 22.32 ± 0.89 dL g-1, 274.80 ± 1.94 dL g-1 and 416.49 ± 2.21 dL g-1 illustrating greater polymer chain expansion following neutralisation using Triethylamine (TEA). PMVE/MA platforms exhibited shear-thinning properties. Increasing polymer concentration increased the consistencies, zero shear rate (ZSR) viscosities (determined from flow rheometry), storage and loss moduli, dynamic viscosities (defined using oscillatory analysis) and mucoadhesive properties, yet decreased the loss tangents of the neutralised polymer platforms. TEA neutralised systems possessed significantly and substantially greater consistencies, ZSR and dynamic viscosities, storage and loss moduli, mucoadhesion and lower loss tangents than their NaOH counterparts. Multiple regression analysis enabled identification of the dominant role of polymer viscoelasticity on mucoadhesion (r > 0.98). The mucoadhesive properties of PMVE/MA platforms were considerable and were greater than those of other platforms that have successfully been shown to enhance in vivo retention when applied to the oral cavity, indicating a positive role for PMVE/MA mono-component platforms for pharmaceutical and biomedical applications.

Development and characterization of Poly(L-lactic acid) (PLLA) platforms for bone tissue engineering

The development of scaffolds based on biomaterials is a promising strategy for Tissue Engineering and cellular regeneration. This work focuses on Bone Tissue Engineering, the aim is to develop electrically tailored biomaterials with different crystalline and electric features, and study their impacts onto cell biological behavior, so as to predict the materials output in the enhancement of bone tissue regeneration. It is accepted that bone exhibits piezoelectricity, a property that has been proved to be involved in bone growth/repair mechanism regulation. In addition electrical stimulations have been proved to influence bone growth and repair. Piezoelectric materials are therefore widely investigated for a potential use in bone tissue engineering. The main goal is the development of novel strategies to produce and employ piezoelectric biomaterials, with detailed knowledge of mechanisms involved in cell-material interaction. In the current work, poly (L-lactic) acid (PLLA), a synthetic semi-crystalline polymer, exhibiting biodegradibility, biocompatibility and piezoelectricity is studied and proposed as a promoter of enhanced tissue regeneration. PLLA has already been approved for implantation in human body by the Food and Drug Administration (FDA), and at the moment it is being used in several clinical strategies. The present study consists of first preparing films with different degrees of crystallinity and characterizing these PLLA films, in terms of surface and structural properties, and subsequently assessing the behavior of cells in terms of viability, proliferation, morphology and mineralization for each PLLA configuration. PLLA films were prepared using the solvent cast technique and submitted to different thermal treatments in order to obtain different degrees of crystallinity. Those platforms were then electrically poled, positively and negatively, by corona discharge in order to tailor their electrical properties. The cellular assays were conducted by using two different osteoblast cell lines grown directly onto the PLLA films:Human osteoblast Hob, a primary cell culture and Human osteosarcoma MG-63 cell line. This thesis gives also a comprehensive introduction to the area of Bone Tissue Engineering and provides a review of the work done in this field in the past until today, in that same field, including the one related with bone’s piezoelectricity. Then the experimental part deals with the effects of the crystallinity degrees and of the polarization in terms of surface properties and cellular bio assays. Three different degrees of crystallinity, and three different polarization conditions were prepared; which results in 9 different configurations under investigation.

Development of semicrystalline morphology of poly(L-lactic acid) during processing of a vascular scaffold

New and promising treatments for coronary heart disease are enabled by vascular scaffolds made of poly(L-lactic acid) (PLLA), as demonstrated by Abbott Vascular’s bioresorbable vascular scaffold. PLLA is a semicrystalline polymer whose degree of crystallinity and crystalline microstructure depend on the thermal and deformation history during processing. In turn, the semicrystalline morphology determines scaffold strength and biodegradation time. However, spatially-resolved information about the resulting material structure (crystallinity and crystal orientation) is needed to interpret in vivo observations.

The first manufacturing step of the scaffold is tube expansion in a process similar to injection blow molding. Spatial uniformity of the tube microstructure is essential for the consistent production and performance of the final scaffold. For implantation into the artery, solid-state deformation below the glass transition temperature is imposed on a laser-cut subassembly to crimp it into a small diameter. Regions of localized strain during crimping are implicated in deployment behavior.

To examine the semicrystalline microstructure development of the scaffold, we employed complementary techniques of scanning electron and polarized light microscopy, wide-angle X-ray scattering, and X-ray microdiffraction. These techniques enabled us to assess the microstructure at the micro and nano length scale. The results show that the expanded tube is very uniform in the azimuthal and axial directions and that radial variations are more pronounced. The crimping step dramatically changes the microstructure of the subassembly by imposing extreme elongation and compression. Spatial information on the degree and direction of chain orientation from X-ray microdiffraction data gives insight into the mechanism by which the PLLA dissipates the stresses during crimping, without fracture. Finally, analysis of the microstructure after deployment shows that it is inherited from the crimping step and contributes to the scaffold’s successful implantation in vivo.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Carbodiimide-mediated synthesis of poly(L-lactide)-based networks

Poly(L-lactide-co-succinic anhydride) networks were synthesised via the carbodiimide-mediated coupling of poly(L-lactide) (PLLA) star polymers. When 4-(dimethylamino)pyridine (DMAP) alone was used as the catalyst gelation did not occur. However, when 4-(dimethylamino)pyridinium p-toluenesulfonate (DPTS), the salt of DMAP and p-toluenesulfonic acid (PTSA), was the catalyst, the networks obtained had gel fractions comparable to those which were reported for networks synthesised by conventional methods. Greater gel fractions and conversion of the prepolymer terminal hydroxyl groups were observed when the hydroxyl-terminated star prepolymers reacted with succinic anhydride in a one-pot procedure than when the hydroxyl-terminated star prepolymers reacted with presynthesised succinic-terminated star prepolymers. The thermal properties of the networks, glass transition temperature (Tg), melting temperature (Tm) and crystallinity (Xc) were all strongly influenced by the average molecular weights between the crosslinks ((M_c). The network with the smallest (M_c )(1400 g/mol) was amorphous and had a Tg of 59 °C while the network with the largest (M_c ) (7800 g/mol) was 15 % crystalline and had a Tg of 56 °C.

Properties of photo-cured poly(D,L-lactide) networks

Poly(D,L-lactide) is a degradable polymer with a long history of use in medical applications. It is strong and stiff and degrades over the course of months into lactic acid, a body-own substance. In the field of tissue engineering it is commonly used to fabricate scaffolds. Stereolithography is a high resolution rapid prototyping technique by which designed 3D objects can be built using photo-initiated radical polymerisations. Poly(D,Llactide) (PDLLA) networks can be obtained by photopolymerisation of oligomers functionalised with unsaturated groups. In this work, PDLLA oligomers of varying architectures (arm lengths, numbers of arms) were synthesised and end-functionalised with methacrylate groups. These macromers were photo-crosslinked in solution to yield PDLLA networks of different architectures. The influence of the network architecture on its physical properties was studied.

catena-Poly[[[diaquacobalt(II)]-mu-(3,5-dinitro-2-oxidobenzoato)-kappa3O1,O2:O1'-[tetraaquacobalt(II)]-mu-(3,5-dinitro-2-oxidobenzoato)-kappa3-O1:O1',O2] dihydrate]

In the structure of polymeric title compound, {[Co2(C7H2N2O7)2(H2O)6] . 2H2O}n from the reaction of 3,5-dinitrosalicylic acid with cobalt(II) acetate, both slightly distorted octahedral Co(II) centres have crystallographic inversion symmetry. The coordination sphere about one Co centre comprises four O donors from two bidentate chelate O(phenolate), O(carboxyl) and bridging dianionic ligands and two water molecules [Co-O range, 2.0249(11)-2.1386(14)A] while that about the second Co centre has four water molecules and two bridging carboxyl O donor atoms [Co-O range, 2.0690(14)-2.1364(11)A]. The coordinated water molecules as well as the water molecules of solvation give water-water and water-carboxyl hydrogen-bonding interactions in the three-dimensional framework structure.

Poly[di-mu-aqua-bis(mu-2-amino-4-nitrobenzoato)dicaesium]

In the structure of title compound [Cs2(C7H5N2O4)2(H2O)2]n the asymmetric unit comprises two independent and different Cs centres, one nine-coordinate, the other seven coordinate, with both having irregular stereochemistry. The CsO9 coordination comprises oxygen donors from three bridging water molecules, one of which is doubly bridging, three from carboxylate groups, and three from nitro groups, of which two are bidentate chelate bridging. The CsO6N coordination comprises the two bridging water molecules, one amine N donor, one carboxyl O donor and four O donors from nitro groups (two from the chelate bridges). The extension of the dimeric unit gives a two-dimensional polymeric structure which is stabilized by both intra- and intermolecular amine N-H...O and water O-H...O hydrogen bonds to carboxyl O acceptors, as well as inter-ring pi-pi interactions [minimum ring centroid separation, 3.4172(15)A].

Poly[aqua([mu]3-2-hydroxy-5-nitrobenzoato-[kappa]3O1:O1':O2)rubidium]

In the structure of title compound [Rb2(C7H4NO2)2(H2O)2]n the centrosymmetric cyclic dimeric repeating unit comprises two irregular RbO4 complex centres bridged by the carboxylate groups of the 5-nitrosalicylate ligands. The coordination about each Rb is completed by a monodentate water molecule and a phenolic O donor which gives a bridging extension [Rb-O range 3.116(7)-3.135(5)A]. The two-dimensional polymeric structure is stabilized by intermolecular water O-H...O(carboxyl) hydrogen bonds and weak inter-ring pi--pi interactions [minimum ring centroid separation, 3.620(4)A].