Biodegradable polymer film as a source for formation of human fetal retinal pigment epithelium spheroids.

PURPOSE: To evaluate the attachment of human fetal rctinal pigment epithelial (HFRPE) cells to a biodegradable polymer film with subsequent formation of spheroids in vitro. METHODS: Ten biodegradable polymer films with different compositions were examined for their physical properties and ease of manipulation under a dissecting microscope. The film with the most suitable handling characteristics was chosen, and a purely isolated sheet of HFRPE cells was attached to it. The purity of the cells was assessed by their pigmentation and expression of cytokeratin. Proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdtJ). Cellular structure was analyzed under light and electron microscopes, and the functional capability of the cells was evaluated by rod outer segment (ROS) phagocytosis. RESULTS: The polymer film with composition 50:50 poly (DL-lactide) (PLA)/poly (DL-lactide-co-glycolide) (PLG) with an inherent viscosity of 1.03 dl/g was found to be the most suitable for handling under the microscope. Sheets of HFRPE cells attached to the polymer films within 48 hours and began to form spheroids. All the isolated cells were pigmented and expressed cytokeratin. They possessed a cuboidal morphology, numerous apical microvilli, and no sign of dedifferentiation. HFRPE cells produced extracellular matrix (collagen filaments) on their basal side, filling the cavities of the polymer film. The cells subsequently proliferated, incorporated BrdU, migrated onto the culture plate to form monolayers, and phagocytized ROS. CONCLUSIONS: Biodegradable polymer films can be used as a scaffold for the adhesion of the HFRPE sheet and formation of spheroids. Spheroids represent a source of high density and well-differentiated HFRPE cells that are easy to transfer. Furthermore, the stricture of the membrane makes it suitable for additional applications.

Subgingival controlled release of antimicrobial agents in the treatment of periodontal disease.

Local drug delivery of antimicrobics by sustained release delivery systems can be used to treat periodontal disease. Advantages of these systems may include biodegradation of the system, maintaining high levels of antibiotic in the gingival crevicular fluid (GCF) for a sustained period of time and ease of use with high patient acceptance. This review will identify human in vivo clinical and microbiological studies. Sustained release formulations, application methods, clinical results and microbiological effects are discussed.

The effects of sustained release doxycycline on the anaerobic flora and antibiotic-resistant patterns in subgingival plaque and saliva.

BACKGROUND: The purpose of this study was to determine the effects of periodontal treatment with a sustained-release, biodegradable gel containing 8.5% doxycycline on the anaerobic flora and on antibiotic susceptibility patterns associated with subgingival plaque and saliva. METHODS: Forty-five subjects with adult periodontitis were entered into a parallel design, single-blind study of 6 months' duration. The subjects were randomized to receive either doxycycline treatment (n = 23) or oral hygiene instruction/reinforcement (n = 22). Saliva and subgingival plaque samples were collected prior to and at 7, 21, 91, and 182 days after initiation of treatment. The proportion of the cultivable flora resistant to 10 microg doxycycline/ml was determined relative to total anaerobic counts, and the 3 most predominant colony types resistant to doxycycline were individually enumerated. A representative of each was subcultured, identified to genus and species level, and tested for its susceptibilities to 6 antibiotics. RESULTS: A significant decrease (P <0.01) in total anaerobic counts following doxycycline treatment caused a transient increase in the proportion, but not in the actual counts, of doxycycline-resistant bacteria recovered from both plaque and saliva at 7 and 21 days but not at 91 or 182 days. The same doxycycline-resistant taxa were recovered at all sample periods including baseline. Regardless of treatment, the isolates were similarly distributed and belonged to the same bacterial groups. CONCLUSIONS: Doxycycline treatment significantly reduced the anaerobic population in plaque but did not result in a change in either the number of resistant bacteria present or the acquisition of antibiotic resistance.

Periodontal pocket treatment in beagle dogs using subgingival doxycycline from a biodegradable system. I. Initial clinical responses.

The present study evaluated the clinical response of periodontal pockets in beagle dogs after treatment with a biodegradable delivery system containing 10% doxycycline hyclate (ABDS-D). Eight adult, female beagle dogs had generalized, severe periodontitis with plaque and calculus-laden pockets. In each animal, 3 teeth with multiple pocket sites > or = 4 mm (mean depth = 6.0 mm) associated with attachment loss (mean = 5.4 mm) and which bled on probing (mean score = 2.5) were treated with a single application of either ABDS-D (experimental group) or the delivery system alone without the doxycycline (control group). Residual polymer was removed at day 7. Bioassay of doxycycline in gingival crevicular fluid associated with presence of ABDS-D gave mean levels of bioactivity of approximately 250 micrograms/ml. Levels of bioactive doxycycline were detected for approximately 7 days after ABDS-D removal. Periodontal maintenance consisted of thrice-weekly toothbrushing the treated sites. Clinical responses were evaluated at 2 weeks, and at bi-weekly intervals thereafter for 4 months. Analyses of the data from the control group showed that there was only slight clinical improvement. In contrast, in the experimental group, bleeding on probing and probing depths were significantly reduced from baseline at all post-treatment time points. At 1 month, mean probing depth reduction was 2.4 mm and this was maintained at 4 months (mean reduction = 2.5 mm). These probing depth reductions occurred primarily through gain of clinical attachment which was 2.0 mm at 4 months. Bleeding had been virtually eliminated (mean = 0.2). It was concluded that, for the beagle dogs with severely infected periodontal pockets in this study, treatment with subgingival doxycycline using the delivery system resulted in substantial improvement in periodontal health.

Whole mouth microbiota effects following subgingival delivery of sanguinarium.

An increased incidence of antibiotic-resistant bacteria and yeast overgrowth has been reported following various periodontal treatments. The objective of this study was to detect possible overgrowth of opportunistic bacteria and fungi as well as changes in normal microbiota after application of a biodegradable delivery system containing 5% sanguinarium (ABDS-S) to one quadrant in a split-mouth study. An oral hygiene quadrant served as a control. The ABDS-S treated and control periodontal sites as well as the saliva of 17 subjects were sampled prior to treatment, immediately after ABDS-S removal at 7 days, and again at 30 and 60 days. At Day 7 sanguinarium-resistant bacteria increased in both control and ABDS-S periodontal sites as well as in the saliva. Enteric Gram-negative bacilli in both control and ABDS-S periodontal sites were 2.2 to 3.4 log colony forming units higher at Day 7 compared to baseline. This overgrowth was transient in that levels became undetectable at Days 30 and 60. No such overgrowth was observed for C. albicans or other fungi, or for S. aureus or other staphylococci in any periodontal sites. Levels of Actinomyces increased at Days 30 and 60 in both control and ABDS-S sites as well as saliva. These changes strongly suggest that a 7 day ABDS-S treatment in one quadrant of the mouth led to significant microbiota changes in the treated and control quadrants as well as in the saliva. Future microbial studies involving antimicrobials delivered by local delivery systems must consider the crossover effects of treatment inherent in the split-mouth design.

Antimicrobial action of sanguinarine.

Sanguinarine is a benzophenanthridine alkaloid derived from rhizomes of Sanguinaria canadensis L. (bloodroot). It is a cationic molecule which converts from an iminium ion form at pH less than 6 to an alkanolamine form at pH greater than 7. Sanguinaria extract is composed of sanguinarine and five other closely related alkaloids. The safety profile of both sanguinarine and sanguinaria extract provide a broad margin for their safe use in oral health products. Sanguinarine has broad antimicrobial activity as well as antiinflammatory properties. In vitro studies indicate that the anti-plaque action of sanguinaria is due to its ability to inhibit bacterial adherence to newly formed pellicle, its retention in plaque being 10-100 times its saliva concentration, and due to its antimicrobic properties. The MIC of sanguinarine ranges from 1 to 32 micrograms/mL for most species of plaque bacteria. Long term use of sanguinaria-containing toothpaste and oral rinse products does not predispose users to detrimental shifts in oral flora. Electron microscopic studies of bacteria exposed to sanguinarine demonstrate that bacteria aggregate and become morphologically irregular. Sanguinarine-containing slow release polymer systems are currently being developed for use in periodontitis treatment applications.

Effects of sanguinarium, chlorhexidine and tetracycline on neutrophil viability and functions in vitro.

The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes with minimal toxicity to host cells. Depending on the treatment regimen, antimicrobial agents come into contact with host cells for various intervals of time. Sanguinarium (SANG), chlorhexidine (CHX) and tetracycline (TET) are 3 antimicrobial agents frequently used in the management of periodontal infections. However, their effects on host immune cells during different treatment regimens are not known. Due to their ability to serve as the first line of host defense against microbial infections, we have compared the effects of these antimicrobial agents on human neutrophil functions and viability. The results show that SANG is not lytic to neutrophils from peripheral blood or crevicular fluid, at all concentrations tested. However, exposures of neutrophils to very low concentrations of SANG (0.001%) inhibits neutrophil chemotaxis, oxidative metabolism and degranulation within 5 min. Increasing the exposure time results in a similar inhibition of neutrophil functions, albeit at 50-100 fold lower concentrations of SANG. CHX rapidly disrupts the cell membrane of both crevicular and peripheral blood neutrophils at concentrations above 0.005% within 5 min, and inhibition of all neutrophil functions is due to its lytic properties. While TET is least toxic to neutrophils, a dose dependent inhibition of neutrophil functions is dependent on the calcium concentrations of the cellular environment, and is observed only above 0.04% or higher concentrations in the absence of calcium. The data suggest that a critical cumulative concentration of these drugs is essential for their toxicity and inhibition of neutrophil functions. Therefore, both the length of exposure and the dose of the drug both are critical while considering the effectiveness of SANG, CHX or TET in the treatment of infections. Furthermore, due to differences in their mechanisms of action, the consequences of their effects on neutrophils may have significant bearing on tissue pathology as well as on their therapeutic efficacy.