Development of a Novel Collagen Wound Model To Simulate the Activity and Distribution of Antimicrobials in Soft Tissue during Diabetic Foot Infection

Abstract

Diabetes has major implications for public health, with diabetic foot ulcers (DFUs) being responsible for significant morbidity and mortality. A key factor in the development of nonhealing ulcers is infection, which often leads to the development of biofilm, gangrene, and amputation. A novel approach to treating DFUs is the local release of antibiotics from calcium sulfate beads. We have developed a novel model system to study and compare the release and efficacy of antibiotics released locally, using collagen as a substrate for biofilm growth and incorporating serum to mimic the biochemical complexity of the wound environment. We found that our soft-tissue model supports the growth of a robust Pseudomonas aeruginosa biofilm, and that this was completely eradicated by the introduction of calcium sulfate beads loaded with tobramycin or gentamicin. The model also enabled us to measure the concentration of these antibiotics at different distances from the beads and in simulated wound fluid bathing the collagen matrix. We additionally found that a multidrug-resistant Staphylococcus aureus biofilm, nonsusceptible to antibiotics, nonetheless showed an almost 1-log drop in viable counts when exposed to calcium sulfate beads combined with antibiotics. Together, these data suggest that locally applied antibiotics combined with calcium sulfate provide surprising efficacy in diabetic foot infections and offer an effective alternative approach to infection management. Our study additionally establishes our new system as a biochemically and histologically relevant model that may be used to study the effectiveness of a range of therapies locally or systemically for infected DFUs.

Susceptibility of monomicrobial or polymicrobial biofilms derived from infected diabetic foot ulcers to topical or systemic antibiotics in vitro

“…The laboratory strains used in this study were the Nottingham Pseudomonas aeruginosa PA01, donated by Miguel Camara and the Staphylococcus aureus ATCC 6538 (LGC standards). Overnight cultures were prepared in tryptic soya broth (Oxoid) and diluted to 10 5 CFU/ml in fibroblast growth media (FGM) (Dulbecco Modified Eagle Medium (Sigma D5546 low glucose) supplemented with 10% fetal bovine serum (FBS)(Sigma), 80 mM HEPES (MP Biomedicals) and 4 mM glutamine (Sigma)) [40], 500 μl of diluted culture were used to inoculate models with S. aureus and P. aeruginosa as previously described [33]. Debrided tissue (DFAN, DFAL, DFAC) was sampled from three different subjects, weighed, and homogenised in a bead beater (Precellys) using glass beads until the tissue had completely homogenised.…”

“…When studying biofilms, investigators often look at lab strains of bacteria in monoculture using readily available substrates in the lab like plastic or agar because these studies can be carefully controlled and the methods and strains are well characterised [32,33], although some polymicrobial models of infection have been developed [34][35][36]. However, clinical isolates often behave differently to lab strains and there is evidence that polymicrobial biofilms have altered tolerance to antibiotics and form more robust biofilms with different architecture because of symbiotic interactions between the different species [35,37].…”

“…We have previously sought to address some of these issues where they apply to wound biofilms by using type 1 collagen, the predominant matrix component in the dermis, as a substrate for biofilm formation [33]. Here we have further developed the model to more accurately reflect the dermis by incorporating hyaluronic acid (HA), extracellular matrix protein extract (ECM) and fibroblasts.…”

Robust and Precise Wounding and Analysis of Engineered Contractile Tissues

“…The 3D models of fibrous tissues can be engineered to recreate various stromal tissues, [27][28][29][30] and these can be adapted for wound models. Wound-like inclusions have been created during fabrication, [31][32][33][34] but this approach does not capture in vivo-like features of wounding, including retraction of the wound edge in tissues under tension. 35 To address these issues, Sakar and colleagues recently developed a 3D bioengineered wounding model, in which fibroblast/collagen tissues contract around anchoring pillars to form prestressed microtissues [36][37][38][39] that were then microsurgically injured using a robotically controlled micromanipulator.…”

Diabetic Foot Ulcers: Current Advances in Antimicrobial Therapies and Emerging Treatments

“…So far, it is not confirmed that the application of various antimicrobials influences the mechanism of action of different biological therapies or the use of different devices. Instead, different models were generated for their collective use as collagen or sulfate beads for the local discharge of antimicrobials, which obtained positive results in the treatment of infection and scarring [149,150]. However, further investigations are required to guarantee that the activity of antimicrobials improves efficiency, in combination with other therapies, against the diverse microorganism populations found in DFU infections.…”