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Collagen membranes have been used as bioresorbable barrier membranes in guided tissue/bone regeneration. However, the collagen membranes currently used in clinics lack an active antibacterial function, although infection at surgical sites presents a realistic challenge for guided tissue/bone regeneration. In this study, we successfully prepared novel and advanced collagen composite membranes from collagen and complexes of heparin and chelates of minocycline and Ca2+ ions. These membranes were characterized for chemical structures, morphology, elemental compositions and tensile strength. In vitro release studies were conducted to evaluate the release kinetics of minocycline from these membranes. Agar disk diffusion assays were used to assess their sustained antibacterial capability against model pathogenic bacteria Staphylococcus aureus. The chemical and physical characterization confirmed the successful synthesis of minocycline-loaded collagen composite membranes, namely NCCM-1 and NCCM-2. Both membranes had weaker tensile strength as compared with commercial collagen membranes. They achieved sustained release of minocycline for at least 4 weeks in simulated body fluid (pH 7.4) at 37°C. Moreover, both membranes demonstrated potent sustained antibacterial effects against Staphylococcus aureus. These results suggested that the advanced collagen composite membranes containing minocycline can be exploited as novel guided tissue regeneration membranes or wound dressing by providing additional antibacterial functions.

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Locally applied therapeutic agents have become established in the treatment of periodontal disease. Inhibition of human metalloproteases by metal-chelating antibiotics contributes to the utility of local therapy. Adding inhibitors of bacterial proteases might extend and improve local therapy. The periodontal pathogen Porphyromonas gingivalis (Pg) produces two extracellular cysteine proteases (gingipains Rgp and Kgp) that are virulence factors and contribute to destruction of oral tissues. Our aims were to compare efficacy of protease inhibitors against gingipains and evaluate bactericidal activity of the inhibitors. Protease activity was measured in fluorescent assays with specific Rgp and Kgp substrates. Bacterial viability was measured with BacLight™ (Invitrogen, Inc., Carlsbad, CA) reagents. Pairs of inhibitors of Rgp and Kgp, respectively, were leupeptin and cathepsin B inhibitor II, KYT-1 and KYT-36, and PPACK and Z-FK-ck. The cysteine-protease inhibitor E64 was also tested. Rgp activity was higher than Kgp activity, and activity was higher in Pg 33277 and 49417 cell suspensions than in media. Concentrations required for 50% inhibition of Rgp in cell suspensions were 2 × 10-9, 2 × 10-9, 2 × 10-8, and 5 × 10-5 M for KYT-1, PPACK, leupeptin, and E64, respectively. Concentrations required for 50% Kgp inhibition were 5 × 10-10, 1 × 10-9, and 5 × 10-8 M for Z-FK-ck, KYT-36, and cathepsin B inhibitor II. E64 did not inhibit Kgp. Inhibition of Rgp could be accounted for by competition for binding between the arginine residue of the substrate and the guanidinobutane portion of E64. PPACK was the least selective, with a 10-fold difference in concentrations that inhibited Rgp and Kgp. KYT-1 and Z-FK-ck inhibited both Rgp and Kgp, but inhibitory concentrations differed by 10,000-fold. At up to 1 × 10-4 M, only Z-FK-ck was bactericidal. KYT-1 and KYT-36 were remarkably effective even when used in cell suspensions in which bacterial proteins could bind inhibitors or compete for binding to gingipains. These inhibitors might prove useful as an addition to locally applied therapeutic agents.

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Several recent studies have utilized respondent-driven sampling (RDS) methods to survey hidden populations such as commercial sex-workers, men who have sex with men (MSM) and injection drug users (IDU). Few studies, however, have provided a direct comparison between RDS and other more traditional sampling methods such as venue-based, targeted or time/space sampling. The current study sampled injection drug users in three U.S. cities using RDS and targeted sampling (TS) methods and compared their effectiveness in terms of recruitment efficiency, logistics, and sample demographics. Both methods performed satisfactorily. The targeted method required more staff time per-recruited respondent and had a lower proportion of screened respondents who were eligible than RDS, while RDS respondents were offered higher incentives for participation.

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