An in vitro study of plasticized poly(lactic-co-glycolic acid) films as possible guided tissue regeneration membranes: material properties and drug release kinetics.

Guided tissue regeneration (GTR), in periodontal therapy, involves the placement of a barrier membrane, to ensure the detached root surface becomes repopulated with periodontal ligament cells capable of regenerating this attachment. GTR procedures exhibit large variability in surgical outcome as a consequence of poor membrane performance. The objective of this study was to evaluate the suitability of plasticized poly(lactic-co-glycolic acid) (PLGA) as a material for GTR membranes. The material was also investigated as a localized controlled release system for the antibiotic, anti-inflammatory agent tetracycline. Films made from PLGA (85:15), plasticized with either 10% w/v methoxypoly(ethyleneglycol) (MePEG) or a diblock copolymer [poly(D,L-lactic acid)-block-methoxypoly(ethyleneglycol)] were loaded with tetracycline base (or hydrochloride salt) and cast by solvent evaporation. Drug release was measured using high performance liquid chromatography (HPLC). The time-course of elasticity changes and swelling were determined using a stress-strain apparatus or gravimetric/dimensional determinations, respectively. Cells extracted from periodontal ligament cell explants were used to evaluate the effect of material and drug loading on cell morphology. Tetracycline·HCl released more rapidly than tetracycline from PLGA films. The addition of either MePEG or diblock caused a concentration dependent increase in release rates for both drugs. Release profiles ranged from a small initial burst phase followed by slow sustained release to almost full drug release after 1 day. After incubation in PBS, the films stiffened and swelled within 30 min. Periodontal ligament cell morphology was not affected by the inclusion of tetracycline. Plasticized PLGA films displayed desired features for possible use as GTR membranes.

A reproducible technique for specific labeling of antigens using preformed fluorescent molecular IgG-F(ab')2 complexes from primary antibodies of the same species.

Immunolabeling two different antigens using the indirect approach with antibodies from the same species is not possible as secondary antibodies can bind to either primary target antibodies. In this study, we describe how preformed complexes of primary and secondary labeled antibodies can be used in such circumstances. In this situation, the first antigen is labeled using the conventional indirect method followed by incubation with the preformed primary-secondary antibody complex against the second antigen. To prevent unbound secondary antibody from binding the indirectly-labeled antibodies, resulting in a false positive, we quenched excess secondary antibody with nonimmune murine serum from the species of the primary antibody. Before the formation of the preformed complex, the optimum dilution of both primary and secondary antibodies was determined. Once these concentrations were established, the concentration of nonimmune murine serum required to quench excess unbound secondary was determined. This step was accomplished by first incubating the sample with an antibody against an antigen known to be localized away from the antigen of interest, followed by the preformed complex. If specific staining was seen, other than that expected from the preformed complex, then the concentration of the serum was deemed insufficient for quenching, and increased accordingly. We demonstrate that this approach is successful in determining the optimum conditions for the preformation of ascites and purified monoclonal primary IgG with fluorescently conjugated F(ab')(2). Double immunolabelling of two focal adhesion antigens and two cytoskeletal proteins, with two murine primary antibodies, are presented as examples of the methodology.

Cryoelectron tomography of isolated desmosomes.

Desmosomes are a complex assembly of protein molecules that form at the cell surface and mediate cell-cell adhesion. Much is known about the composition of desmosomes and there is an established consensus for the location of and interactions between constituent proteins within the assembly. Furthermore, X-ray crystallography has determined atomic structures of isolated domains from several constituent proteins. Nevertheless, there is a lack of understanding about the architecture of the intact assembly and the physical principles behind the adhesive strength of desmosomes therefore remain vague. We have used electron tomography to address this problem. In previous work, we investigated the in situ structure of desmosomes from newborn mouse skin preserved by freeze-substitution and imaged in resin-embedded thin sections. In our present work, we have isolated desmosomes from cow snout and imaged them in the frozen unstained state. Although not definitive, the resulting images provide support for the irregular groupings of cadherin molecules seen previously in mouse skin.

Triton X-100 pretreatment of LR-white thin sections improves immunofluorescence specificity and intensity.

The staining of intracellular antigenic sites in postembedded samples is a challenging problem. Deterioration of antigenicity and limited antibody accessibility to the antigen are commonly encountered on account of processing steps. In this study preservation of the antigen was achieved by fixing the tissues with mild fixatives, performing partial dehydration, and embedding in a low crosslinked hydrophilic acrylic resin, LR-White. Permeabilization of cell membranes with Triton X-100 is well documented but can affect some antigen conformations. We tested the effect of Triton X-100 on the ED1 antigen present in the lysosomal membrane of the macrophage in cell culture. The ED1 antigen in the lysosome was resistant to extraction by Triton X-100. Interestingly pretreating the LR-White sections of macrophage pellets with Triton X-100 improved the staining intensity of ED1. The most intense and clear specific fluorescent staining was observed when sections were pretreated with 0.2% Triton X-100 for 2 min. Longer exposure of sections to 0.2% Triton or 2 min exposure to 2% Triton lead to reduced ED1 labeling. SEM observations indicated that the detergent extracted a component from the cells and not the resin and was determined to be lipid. This novel technique could be applied in many research areas where postembedding fluorescent immunolabeling with higher labeling intensity is desired.

Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study.

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell-peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.

Steps toward a model nanotopography.

A natural lithography technique is employed to create an irregular, submonolayer colloidal topography. Epitenon cells were cultured on these colloidal surfaces, and cell morphology investigations using scanning electron microscropy were conducted. Preliminary experiments brought into question the stability of the colloidal nanotopography, and it was unsure if the surface was presented to cells as a static structure. Investigations using secondary electron and backscattered electron imaging, and also X-ray microanalysis, indicated that the colloidal structure was in fact stable, and cells were capable of direct interactions at the peripheral membrane with the colloids.