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Oxygen-Nanobubble biotechnology to enhance physiologic wound healing and tissue regeneration in Vivo

Ntentakis Dimitrios

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Summary

Physiologically, the healing mechanism operates as an orchestrated continuum of molecular responses, organized in 4 stages: 1) hemostasis, 2) acute inflammation, 3) tissue proliferation, and 4) remodeling. There is evidence indicating that, disruptions in the most upstream biological pathways in this continuum of normal healing, are strong prognosticators of downstream wound chronicity and pertinent complications. Clinical and epidemiological interest in wound healing is increasing on a global scale, due to a combination of the following factors: the rising prevalence in systemic vasculopathies predisposing to peripheral ischemia, tissue hypoxia, and suboptimal healing, such as diabetes mellitus, the aging world population, and the questionable capacity of the available management and prevention strategies to confront a potentially generalized surge in complicated wounds. Administration of gaseous oxygen (O2) has consistently demonstrated beneficial outcomes in cutaneous wounds, reflecting the pivotal role of O2 in physiologic healing. A characteristic example is hyperbaric O2 therapy, which has become an established management strategy for diabetic wounds, alongside multiple clinical indications. Yet, an important limitation lies in the short-lived efficacy of O2 when administered in its gaseous form, as a result of passive diffusion through biological tissues in response to local O2 pressure gradients. Thereby, technologies supplying dissolved oxygen (DO) within aqueous solutions, namely DO solutions (DOS), are currently investigated as a novel approach to enhancing wound oxygenation. By enabling O2 to become gradually bioavailable in the liquid medium, a DOS can theoretically supply high oxygen amounts for longer periods compared to gas-based O2 therapies. However, sustaining DO levels without using potentially toxic artificial surfactants and complex chemical additives, remains one of the biggest challenges in the field. The research described herein, aims to document the capacity to manufacture a DOS by exclusively leveraging the physics of the gas-liquid interphase, in the absence of chemical surfactants or additives, and to incorporate the cutting-edge technology of O2 nanobubbles in formulations that are suitable for biomedical applications in wound healing and cutaneous regeneration. First, we conduct a comprehensive analysis of the existing technologies that can be used for supplying DO to healing tissues, with the aim to identify the most promising technological category for further investigation. Via a systematic co-evaluation of engineering principles, oxygenating effects, biomechanics of DO delivery, safety parameters, and healing-related efficacy for all technologies, we highlight the benefits of using a DOS containing no exogenous chemicals, and discuss the potential of O2 nanobubble technology in this approach. Concurrently, we introduce an original classification system for the DO technologies tested in healing applications. Then, we discuss the design, scientific rationale, and implementation of a novel in vivo protocol, aiming to facilitate the clinical translation of biotechnologies developed for healing applications, such as DO-supplying therapies. By simulating the design of early-stage clinical trials, and incorporating standardized methodological components from previous protocols, we enable a holistic examination of engineering fundamentals, quality-control requirements, biological safety, and a preliminary assessment of healing efficacy for new biotechnologies in the field. Of note, the experimental manipulations in the proposed protocol have been selected to maximize simplicity and reproducibility; to account for the fact that, a growing number of relevant research projects are carried out primarily by biochemical and biomedical engineers nowadays. Next, we implement the aforementioned in vivo protocol to study a “plain” DOS containing no physical enhancements to sustain DO delivery, which was manufactured via infusion of high-purity (>99.9%) O2 into sterile normal saline (N/S; 0.9% NaCl) under predetermined conditions, without chemical additives. Our aim is the collection of preliminary data on the in vivo administration of a DOS with the aforementioned characteristics, including the magnitude and sustainability of DO levels in the final formulation, the best routes of administration, optimal dosing parameters, biological safety, and healing outcomes in full-thickness excisional wounds on healthy SKH1 mice. Our findings suggest that the plain DOS can deliver high DO concentrations consistently and sustainably for at least 14 days, while being safe for in vivo administration intravenously, locally, and per os. Also, we show a promising effect of intravenous and per os administration of plain DOS in physiologic wound healing, macroscopically and histopathologically. Finally, we implement the proposed in vivo protocol to evaluate a DOS manufactured by employing the cutting-edge technology of O2 nanobubbles, while also eschewing the use of exogenous chemicals; which we refer to as oxygen nanobubble-enriched water solution (ONBW). Our findings show that the ONBW maintains high DO concentrations throughout a 14-day evaluation period, and is completely safe in vivo for systemic administration, either intravenously or per os. Furthermore, by monitoring full-thickness excisional wounds on healthy SKH1 mice, we extend our observations to show that, independently, two intravenous injections and per os intake of the ONBW can accelerate wound closure macroscopically, and improve epithelial regeneration and collagen deposition histopathologically.

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