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The Safety Of Elution Of Multiple Antibiotics From Collagen-rich Hydrogel For Topical Treatment Of Chronic Wounds
Ayushi Dutt Sharma, BS, Evan H. Jarman, BS, Paige Fox, MD PhD.
Stanford University School of Medicine, Stanford, CA, USA.

PURPOSE:
Chronic wounds pose a significant challenge to modern healthcare systems, affecting 1-2% of the population in developed countries with costs ranging between $28.1 and $96.8 billion annually. Overtime chronic wounds tend to become colonized or infected with multiple different bacteria resulting in patients being treated with several rounds of systemic oral and IV antibiotics. Subsequently, antibiotic resistance develops, and wounds become harder to treat. Comorbidities, such as diabetes and peripheral vascular disease, also make treatment more challenging. Ideally chronic wounds would be treated topically with customized antimicrobial combinations unique to each patient’s wound.In vivo, we have previously shown that a topical collagen-rich hydrogel (cHG) can provide controlled elution of single antibiotics leading to inhibition of bacterial growth while avoiding cytotoxicity towards host cells in a stented wound model. We hypothesize that the elution of multiple antibiotics from collagen-rich hydrogel will also be well-tolerated in mammalian cells. METHODS:
Antibiotic Selection:We performed a literature search to determine the antibiograms bacteria isolated from chronic diabetic wounds; the most common microbes were Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus spp., and beta-hemolytic streptococci. This informed our choice of antibiotics. We tested four antibiotic combinations from six antibiotics. While there are 15 two-antibiotic permutations, these combinations provided the most extensive coverage.Collagen hydrogel formation:2.5% collagen-rich thermoresponsive hydrogel (cHG) was synthesized according to a previously established protocol. After confirmation of composition and gelation, cHG-antibiotic solutions were prepared by determining 100x the minimum inhibitory concentration of each antibiotic and adjusting the final volume of hydrogel to 100 mL per well. The antibiotic concentrations used were 400 ug/mL vancomycin, 400 ug/mL imipenem, 200 ug/mL ciprofloxacin, 500 ug/mL ceftazidime, 1.0 mg/mL gentamicin, and 100 ug/mL clindamycin.Mammalian cell cytotoxicity:Human adipose-derived stem cells (ASCs), mouse ASCs, human fibroblasts (FBs), and mouse FBs were used to study cytotoxicity in both differentiated and undifferentiated mammalian cells. Cells were seeded onto 24-well culture plates at a density of 2 × 104 cells/ml, allowed to attach overnight, and treated with cHG+antibiotic, incubated until 24, 48, and 72-hour time points.A live/dead viability/cytotoxicity assay was used according to the manufacturer’s instructions, and images were analyzed using MATLAB. All experiments were performed in triplicates. RESULTS:
We observed no significant cell death in differentiated and undifferentiated cells in both mouse and human cell lines treated with antibiotic-eluting hydrogel with multiple combinations of antibiotics. All cell lines had >90% survival at all timepoints. (Figures 1-2)
CONCLUSION:
This study demonstrates that the application of a topical drug-eluting hydrogel with a combination of antibiotics in the hydrogel is safe for use in mammalian cells. This could transform the treatment of chronic diabetic wounds, which are often polymicrobial with unique resistance patterns requiring multiple antibiotics.


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