Eradication of Bacterial Growth with Topical Antibiotic Treatment in Human Collagen Hydrogel Carrier

PURPOSE: Chronic wounds can be devastating to healthcare systems globally; approximately 6.5 million individuals within the US alone are affected by chronic wounds. (1) The susceptibility of chronic wounds to bacteria makes them prone to long-term infections, yielding complex wound environments that are difficult to treat. Current treatment for infected chronic wounds consists of high-dose oral and intravenous antibiotics; however, this treatment is ineffective in eradicating bacterial biofilms, leads to antibiotic resistance, and can impair physiologic wound repair mechanisms. Our lab has developed a novel human collagen hydrogel (cHG) embedded with antibiotic (cHG-abx) for topical treatment of infected chronic wounds that is able to mitigate the risks of current treatment while providing physiologic ECM proteins needed for wound repair. We hypothesize that topical administration of our novel cHG-abx will effectively inhibit growth of multiple clinically important and common bacteria while maintaining mammalian cell viability. METHOD: C. perfringens and MRSA were treated with 100X minimum inhibitory concentration (MIC) of clindamycin (100 μg/ml) and gentamycin (500 μg/ml), respectively. Human collagen hydrogel preparation: 2.5% cHG was fabricated from a previously established protocol. Prepared cHG was mixed with antibiotic and incubated to induce gelation. Modified Kirby-Bauer: cHG-abx was gelled onto polycarbonate films and allowed to elute in PBS for various timepoints before being placed onto inoculated agar. After 12 hours of treatment, the zone of inhibition was measured. Crystal violet assay: Various eluted cHG-abx were added to bacterial suspensions, incubated, then stained with crystal violet solution. Absorbance was measured at 595 nm and compared to a non-treated well. Mammalian cell cytotoxicity: Wells seeded with human and mouse fibroblasts and ADSCs were treated with cHG-abx for 24, 48, and 72 hours before quantifying viability. RESULTS: No significant mammalian cell death was seen at any time point. Both Kirby-Bauer and crystal violet assays demonstrated significant bacterial inhibition for 48 hours compared to no treatment for C. Perfringens and MRSA. Furthermore, significant differences in bacterial elimination over elution time points indicate sustained release of antibiotics. CONCLUSION: Human collagen hydrogel embedded with antibiotics is capable of sustained low-dose antibiotic release to successfully inhibit growth of various clinically relevant bacterial strains in vitro. Furthermore, the gel shows promise for in vivo application, as no significant mammalian cell death was found. REFERENCES: 1. Sen K, Gordillo G, Longaker M et al. Human Skin Wounds: A Major and Snowballing Threat to Public Health and the Economy. Wound Repair Regen. Off. Publ. Wound Heal. Soc. Eur. Tissue Repair Soc. 17, 763–771 (2009).