Theresa A. Freeman

Replacing implanted medical hardware due to infection often requires one or more revision surgeries. Each surgery triggers a tissue injury response and disrupts the established bacterial biofilm. However, the complex tissue response to reinjury and biofilm disturbance is not well understood. Our results show that with an existing infection, immunological niches such as the bone marrow, lymph nodes, and circulating blood further upregulate pro-inflammatory programs in response to revision. Rather than reducing bacterial burden, this heightened inflammation provokes virulence factor expression and tissue damage, including bone osteolysis and muscle fibrosis. While muscle fibrosis appears transient and begins resolving by 14 days post-revision, osteolysis continues to progress. This study defines the timing and pathophysiology of coordinated multi-tissue responses to revision during infection. Understanding how host–pathogen interactions influence tissue recovery after revision can help identify risks and guide interventions that minimize damage and maximize bacterial clearance.

The FDA‐approved Renuvion cold plasma device is currently used for dermal skin tightening procedures and subdermal tightening after liposuction. Anecdotally, patients report improved tissue healing outcomes following treatment. The most likely explanation for this is plasma‐generated reactive species which are inflammatory but also activate cellular signaling pathways, stimulate antioxidant responses, and activate immune cells. In this study, we aimed to determine the immediate and long‐term molecular effects of a single plasma treatment on surgically injured muscle and the soft tissue envelope. We used RNA sequencing, histology, and immunohistochemistry to determine changes to the tissue following treatment. Neutrophils and mast cells rapidly mobilize 6 h after treatment in conjunction with an upregulated cellular antioxidant response. Additionally, genes identified by RNAseq indicate upregulated pro‐regenerative muscle‐tissue‐protective gene transcripts and downregulated apoptotic pathway transcripts in the muscle tissue 6 h after treatment. The histology and RNAseq results from 4‐ and 14‐days post plasma treatment indicate that these early inflammatory and antioxidant events drive muscle regeneration to skew toward myogenic differentiation over adipogenesis. Thus, we conclude that a single plasma treatment results in an immediate inflammatory and antioxidant response that enhances long‐term muscle fiber repair through reduced adipogenesis.

Cold atmospheric plasma devices generate reactive oxygen and nitrogen species that can be anti-microbial but also promote cell migration, differentiation, and tissue wound healing. This report investigates the healing of surgical incisions created using cold plasma generated by the J-Plasma scalpel (Precise Open handpiece, Apyx Medical, Inc.) compared to a steel scalpel in in vivo porcine and rat models. The J-Plasma scalpel is currently FDA approved for the delivery of helium plasma to cut, coagulate, and ablate soft tissue during surgical procedures. To our knowledge, this device has not been studied in creating surgical incisions but only during deeper dissection and hemostasis. External macroscopic and histologic grading by blinded reviewers revealed no significant difference in wound healing appearance or physiology in incisions created using the plasma scalpel as compared with a steel blade scalpel. Incisions created with the plasma scalpel also had superior hemostasis and a reduction in tissue and blood carryover. Scanning electron microscopy (SEM) and histology showed collagen fibril fusion occurred as the plasma scalpel incised through the tissue, contributing to a sealing effect. In addition, when bacteria were injected into the dermis before incision, the plasma scalpel disrupted the bacterial membrane as visualized in SEM images. External macroscopic and histologic grading by blinded reviewers revealed no significant difference in wound healing appearance or physiology. Based on these results, we propose additional studies to clinically evaluate the use of cold plasma in applications requiring hemostasis or when an increased likelihood of subdermal pathogen leakage could cause surgical site infection (i.e., sites with increased hair follicles).

Sterilization of skin prior to surgery is challenged by the reservoir of bacteria that resides in hair follicles. Atmospheric pressure plasma jets (APPJs) have been proposed as a method to treat and deactivate these bacteria as atmospheric plasmas are able to penetrate into structures and crevices with dimensions similar to those found in hair follicles. In this paper, we discuss results from a computational investigation of an APPJ sustained in helium flowing into ambient air, and incident onto a layered dielectric similar to human skin in which there are idealized hair follicles. We found that, depending on the location of the follicle, the bulk ionization wave (IW) incident onto the skin, or the surface IW on the skin, are able to launch IWs into the follicle. The uniformity of treatment of the follicle depends on the location of the first entry of the plasma into the follicle on the top of the skin. Typically, only one side of the follicle is treated on for a given plasma pulse, with uniform treatment resulting from rastering the plasma jet across the follicle over many pulses. Plasma treatment of the follicle is sensitive to the angle of the follicle with respect to the skin, width of the follicle pocket, conductivity of the dermis and thickness of the underlying subcutaneous fat layer, the latter due to the change in capacitance of the tissue.

Plasma medicine, specifically non-thermal plasma (NT-plasma) technology, has clinical potential in medicine and biology, which has yet to be realized. Current concepts being tested range from wound healing, cancer suppression and dental applications. To date clinical trials testing NT-plasma have mainly focused on its bacteriostatic and bacteriocidal properties in patients possessing chronic and acute wounds, or diverse skin and itching diseases. Further development of NT-plasma as a clinical tool depends on gaining a better understanding of the mechanisms underlying NT-plasma interactions with living organisms. Several strategies have been employed including; characterizing NT-plasmas, determining cellular response to specific NT-plasmas and identification of NT-plasma modifications to the environment surrounding cells and tissues. The successful accomplishment of these goals and to prove plasma medicine has true clinical potential requires a multiple disciplinary approach where physicists, chemists, biologists, and electrical, mechanical and biomedical engineers work together with clinicians to meet the needs of the medical community. The best-characterized mechanism underlying NT-plasma activity on both eukaryotic and prokaryotic cells is the generation of ROS and RNS at the cell/environment interface which elicits an immediate intracellular oxidative response. This thesis tests the hypothesis that NT-plasma promotes redox dependent changes to enhance developmental and cellular signaling. Initially, NT-plasma parameters including electrode size, plasma dose, filament formation and energy density were compared to determine both cellular response and treatment effectiveness. Once established, we were able to establish the mechanisms by which NT-plasma generated intracellular ROS and extra- and intracellular calcium flux promote murine limb autopod development and mesenchymal cell differentiation. Results of this study indicate that if NT-plasma is applied under the appropriate conditions it induces chondrocyte differentiation, osteoblast differentiation and promotes mouse limb autopod development. Effective NT-plasma dose was governed by both the biological model to which it was applied and to electrode geometry and size. Specifically, energy density was not an effective method for predicting cellular response nor to determine the resulting filament pattern created by the electrode. The cellular response at frequencies above 50 Hz filamentation pattern were instead strongly dependent on electrode size. Having established these conditions, we determined the biological responses to NT-plasma were dependent on increases in intracellular ROS production and the resulting activation of oxidative-stress responsive proteins and gene expression. Furthermore, both cellular differentiation and the enhancement of limb autopod elongation by NT-plasma were dependent on increases in intracellular calcium, through the activation of voltage gated calcium channels on the plasma membrane. In conclusion, this thesis identifies the potential of plasma medicine technology to induce and recapitulate normal cellular signaling and its potential in regenerative medical applications for skeletal tissue engineering, and if directly applied to improve healing after skeletal injury.

Bacterial infection and antibiotic resistance are major threats to human health and very few solutions are available to combat this eventuality. A growing number of studies indicate that cold (non-thermal) plasma treatment can be used to prevent or eliminate infection from bacteria, bacterial biofilms, fungi and viruses. Mechanistically, a cold plasma discharge is composed of high-energy electrons that generate short-lived reactive oxygen and nitrogen species which further react to form more stable compounds (NO₂, H₂O₂, NH₂Cl and others) depending on the gas mixture and plasma parameters. Cold plasma devices are being developed for medical applications including infection, cancer, plastic surgery applications and more. Thus, in this review we explore the potential utility of cold plasma as a non-antibiotic approach for treating post-surgical orthopedic infections.