A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection

Abstract

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

Three-Dimensional Model of Dorsal Root Ganglion Explant as a Method of Studying Neurotrophic Factors in Regenerative Medicine

“…4809, 19 June 2003) and were approved by the local ethical committee in accordance with internal requirements of the Commission on Bioethics of the Faculty of Medicine of Lomonosov Moscow State University. We used [8][9] week Nestin-GFP male mice that were kindly provided to us by Grigori Enikolopov [22]. Nestin-GFP mice were used to isolate dorsal root ganglia (DRG).…”

“…Although NGF significantly stimulated glial cell migration, its effect on axon growth was insufficient for axon regeneration.Biomedicines 2020, 8, 49 2 of 13 (ECM) that surrounds the cells and creates the microenvironment in vivo [6,8]. The lack of the proper cell-cell and cell-matrix contacts results in change of gene and protein expression profiles [9]. These can adversely affect cell morphology, cell cycle progression, cell survival, and intracellular signaling, potentially resulting in false positive or false negative data acquisition [8,10].In 2D culturing systems, the bioavailability of substances is difficult to evaluate [11].…”

In vitro and ex vivo systems at the forefront of infection modeling and drug discovery

“…However, animal models are more expensive and are not compatible with high-throughput screening of a large number of therapeutic strategies. In addition, anatomical and physiological differences between most animals (with the exception of pigs) and human skin, combined with artificially-induced nonhuman pathology, invariably [243]; C) Schematic diagram of the assembled model to study anaerobic bacteria [244]; and D) BO-Drum skin culture model [57]. result in different healing kinetics and unique complications [57].…”

“…For instance, a skin explant on a surgical gauze was placed in between a sterile agarose plug and an agarose pedestal (Fig. 4C), which was then allowed to equilibrate in 5% CO 2 for 4 h to create a microenvironment with restricted oxygen supply [244]. After inoculation with a Fastidious Anaerobic Agar plug confluent with Dichelobacter nodosus (D. nodosus), the plate was incubated anaerobically.…”

Kinetic Cytokine Secretion Profile of LPS-Induced Inflammation in the Human Skin Organ Culture

“…Though inflammation is a common feature of several skin diseases including psoriasis, atopic dermatitis, seborrheic dermatitis and contact dermatitis [3], the characteristics of the cellular immune response and composition of cytokine profile vary among them [4]. Several in vitro and in vivo models mimic the inflammatory response of the skin [5][6][7][8][9]. Three main in vitro platforms are available: cell cultures (e.g., keratinocyte, Langerhans cells, co-cultures, etc.…”