eCM Journal - Manuscript - New quantitative automated model to simulate bacterial dissemination in human tissue during irrigation of contaminated wounds Authors: K Kato, K Akeda, S Miyazaki, J Yamada, C Muehleman, K Miyamoto, YA Asanuma, K Asanuma, T Fujiwara, ME Lenz, T Nakazawa, H An, K Masuda Address: Department of Orthopedic Surgery, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA E-mail: komasuda at ucsd.edu Abstract: While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective methods for direct treatment. Nuclear factor-?B (NF-?B) is a transcription factor that regulates various genes’ expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-?B decoy is an oligodeoxynucleotide containing the NF-?B binding site that entraps NF-?B subunits, resulting in suppression of NF-?B activity. In the present preclinical study, NF-?B decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-?B decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-?B decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-?B decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-?B decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-a, in rabbit degenerated IVDs. NF-?B decoy also reduced the pain response as seen using the “pain sensor” nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-?B decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-?B decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs. Key Words: Intervertebral disc-repair/regeneration, intervertebral disc-degeneration, animal models-general, nuclear factor-?B, decoy, intervertebral disc-histology, intradiscal injection, translational and preclinical research. Publication date: August 3rd 2021 Article download: Pages 63-71 (PDF file) DOI: 10.22203/eCM.v042a05 -True Open Access

2021 Volume No 42 – pages 63-71

Title: New quantitative automated model to simulate bacterial dissemination in human tissue during irrigation of contaminated wounds

Authors: C Brochhausen, F Froschermeier, V Alt, C Pfeifer, A Mayr, I Weiss, M Babel, H Siegmund, M Kerschbaum

Address: Department of Trauma Surgery, University Medical Centre Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg, Germany

E-mail: maximilian.kerschbaum at ukr.de

Abstract: This study presents a simple and cost-effective model using microparticles to simulate the bacterial distribution pattern in soft tissue after low- and high-pressure irrigation. Silica coated iron microparticles [comparable diameter (1 µm) and weight (0.8333 pg) to Staphylococcus aureus] were applied to the surface of twenty fresh human muscle tissue samples in two amputated lower legs. Particle dissemination into deep tissue layers as an undesired side effect was investigated in four measuring fields as positive control (PC) as well as after performing pulsatile high-pressure (HP, 8 measuring fields) and low-pressure flushing (LP, 8 measuring fields). Five biopsies were taken out of each measuring field to get a total number of 100 biopsies. After histological and digital image processing, the specimens were analysed, and all incomplete sections were excluded. A special detection algorithm was parameterised using the open source bioimage analysis software QuPath. The application of this detection algorithm enabled automated counting and detection of the particles with a sensitivity of 95 % compared to manual counts. Statistical analysis revealed significant differences (p < 0.05) in our three different sample groups: HP (M = 1608, S = 302), LP (M = 2176, SD = 609) and PC (M = 4011, SD = 686). While both HP and LP flushing techniques are able to reduce the number of bacteria, a higher effectiveness is shown for HP irrigation. Nevertheless, a challenge for the validity of the study is the use of dead tissue and therefore a possible negative influence of high-pressure irrigation on tissue healing and further dispersion of particles cannot be evaluated.

Key Words: Soft tissue contamination, microparticles, automated particle count, digital whole slide image processing, jet lavage.

Publication date: August 3rd 2021

Article download: Pages 63-71 (PDF file)
DOI: 10.22203/eCM.v042a05