Integrating carboxytherapy and hypochlorous acid: a novel molecular approach harnessing the Bohr effect for diabetic foot ulcer treatment

Challenges in diabetic foot ulcer treatment: Diabetic foot is a serious complication of diabetes mellitus, primarily caused by prolonged hyperglycemia leading to peripheral neuropathy, vascular insufficiency, and impaired immune response. These factors increase the risk of foot ulcers and infections, often resulting in lower limb amputations. Globally, approximately 15% of diabetic patients develop foot ulcers, with 85% of diabetes-related amputations preceded by such ulcers. Current management strategies focus on glycemic control, proper wound care, infection management, offloading pressure from affected areas, and revascularization procedures for patients with severe arterial disease.1

Molecular mechanisms behind the dual therapy: Carboxytherapy and enhances wound healing: Carboxytherapy is a minimally invasive treatment that involves the subcutaneous or intradermal administration of carbon dioxide (CO₂) for therapeutic and aesthetic purposes. The procedure is based on the physiological effects of CO₂ on microcirculation, tissue oxygenation, and metabolic activity. When CO₂ is introduced into the tissues, it induces localized vasodilation and the Bohr effect, where hemoglobin releases oxygen more readily, leading to enhanced oxygenation and tissue regeneration. Additionally, CO₂ stimulates collagen synthesis and lipolysis, making it a versatile tool in dermatology and regenerative medicine.2 Carboxytherapy is generally considered safe when performed by trained professionals, with minimal side effects such as transient bruising, mild discomfort, and temporary swelling at the injection site. Since CO₂ is a naturally occurring gas in the body, it is rapidly eliminated through the respiratory system, minimizing systemic toxicity. However, contraindications include severe respiratory or cardiovascular conditions, active infections, and pregnancy. While the therapy is well-tolerated, further research is needed to fully understand its long-term effects and optimize treatment protocols. Nonetheless, its non-surgical nature and wide range of applications make it a promising modality in medical and aesthetic fields.3

Hypochlorous acid — a natural warrior against infection & inflammation: Hypochlorous acid (HOCl) is a naturally occurring molecule with potent antimicrobial and anti-inflammatory properties, making it an ideal candidate for enhancing therapeutic strategies in chronic wound management, particularly in diabetic foot ulcers (DFUs). HOCl is produced endogenously by neutrophils during the immune response and is widely used in wound care for its ability to reduce bacterial load and promote tissue healing. When applied topically to diabetic foot ulcers in conjunction with carboxytherapy, HOCl can significantly improve the healing process by modulating several key biological pathways.4

How the combined therapy works synergistically to promote healing? At the molecular level, HOCl exerts its therapeutic effects by interacting with a variety of cellular mechanisms. It has been shown to regulate the nuclear factor kappa B pathway, which plays a critical role in inflammatory responses. By modulating nuclear factor kappa B activity, HOCl can suppress excessive inflammatory signaling, reducing the chronic inflammation that often impedes wound healing in DFUs. Additionally, HOCl has been reported to promote collagen synthesis and fibroblast proliferation, which are essential for wound closure and tissue regeneration. This action is further supported by its ability to influence the extracellular matrix (ECM) remodeling, facilitating the deposition of collagen fibers and enhancing the structural integrity of newly formed tissue.5

Furthermore, HOCl contributes to the modulation of reactive oxygen species (ROS) within the wound microenvironment. While ROS are typically associated with oxidative stress and tissue damage, controlled levels of ROS are crucial for the activation of cell signaling pathways involved in wound healing, such as those mediated by mitogen-activated protein kinases and protein kinase B. HOCl helps maintain a balance between ROS production and scavenging, ensuring that ROS levels remain within an optimal range to promote angiogenesis and tissue repair without causing further damage to surrounding healthy tissue.6

Carboxytherapy, a minimally invasive technique involving the subcutaneous or intradermal administration of CO₂, offers a promising approach for the treatment of DFUs by leveraging the Bohr effect, which enhances oxygen delivery to ischemic tissues. When CO₂ is introduced into the affected area, it induces vasodilation through endothelial nitric oxide synthase activation, leading to increased nitric oxide production, relaxation of vascular smooth muscles, and improved microcirculation.6 Simultaneously, the Bohr effect reduces hemoglobin’s affinity for oxygen in the presence of elevated CO₂ levels, facilitating the release of oxygen into hypoxic tissues, which is crucial for stimulating fibroblast activity, keratinocyte proliferation, and ECM remodeling.7 Additionally, carboxytherapy modulates inflammatory and immune responses by shifting macrophage polarization from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype, reducing excessive levels of tumor necrosis factor-alpha, interleukin-6, and interleukin-1β, and downregulating nuclear factor kappa B signaling, thus creating a balanced environment that favors wound healing.

Another critical mechanism is its stimulation of angiogenesis and vascular regeneration through hypoxia-inducible factor-1 alpha activation, which upregulates vascular endothelial growth factor expression, promoting new capillary formation and enhancing nutrient and immune cell delivery to the wound site.8 Furthermore, carboxytherapy accelerates collagen synthesis and ECM remodeling by stimulating fibroblast proliferation and upregulating the transforming growth factor-beta pathway, while mechanical stretching of the ECM due to CO₂ injection activates mechanotransduction signaling via integrins and focal adhesion kinase, further promoting tissue repair.9 In addition to its impact on wound healing, carboxytherapy enhances lipid metabolism and reduces abnormal fat deposition in neuropathic foot ulcers by stimulating cyclic adenosine monophosphate signaling and activating hormone-sensitive lipase, which facilitates lipolysis, decreases tissue stiffness, and improves overall skin elasticity, creating a more favorable wound-healing microenvironment. To maximize its therapeutic potential, a structured protocol for DFU treatment could include early-stage intervention with frequent, low-dose CO₂ injections around the ulcer margin to enhance oxygenation, modulate inflammation, and prevent deep tissue necrosis, potentially in combination with platelet-rich plasma or autologous stem cell therapy for added regenerative effects. In advanced ulcers, higher CO₂ concentrations could be administered via transcutaneous diffusion or controlled intradermal injections to stimulate angiogenesis and fibroblast activity, while hyperbaric CO₂ therapy could serve as an adjunct to conventional wound care for non-responsive cases. For long-term maintenance, periodic carboxytherapy sessions could help sustain vascular health, prevent recurrent ischemic episodes, and strengthen skin integrity, particularly when integrated with lifestyle interventions such as exercise therapy and glycemic control to prolong its benefits. Ultimately, carboxytherapy presents a mechanistically sound and clinically viable strategy for DFU treatment by addressing the core pathophysiological barriers of ischemia, inflammation, impaired angiogenesis, and defective ECM remodeling. Further clinical trials and mechanistic studies are needed to refine its protocols, optimize dosing strategies, and evaluate long-term efficacy, but if successfully integrated into clinical practice, this approach has the potential to significantly reduce amputation rates, enhance wound healing outcomes, and improve the quality of life for diabetic patients suffering from chronic foot ulcers.10

A comprehensive therapeutic protocol for diabetic foot ulcers: The application of carboxytherapy for DFUs, combined with a strategic injection of HOCl within the tissue, follows a meticulous protocol designed to maximize therapeutic benefits while ensuring patient safety. The process begins with a comprehensive patient assessment, including a review of medical history to identify contraindications such as severe cardiovascular disease, active infections, or respiratory disorders. The severity of the ulcer is evaluated using standardized classification systems like the Wagner or University of Texas wound grading scales, and proper glycemic control is ensured to optimize healing outcomes. The ulcerated area is cleaned with an antiseptic solution, typically HOCl, to reduce the risk of infection and inflammation before proceeding with the treatment.

A sterile, medical-grade CO₂ source is used, connected to a precision-controlled carboxytherapy device with adjustable pressure and flow settings. Fine-gauge needles, typically ranging from 30G to 32G, are selected for intradermal or subcutaneous injections to minimize discomfort and tissue trauma. Gas flow rates are typically set between 20 to 80 mL/min, adjusted based on the ulcer size, tissue perfusion, and patient tolerance. The injection procedure involves positioning the patient comfortably and administering CO₂ using a multi-point injection technique, delivering 0.5 to 2 mL per injection site, spaced 1–2 cm apart around the ulcer periphery and ischemic zones. For deeper wounds, subcutaneous injections are delivered at a depth of 3–5 mm to enhance microcirculation and oxygenation. Real-time tissue response is closely monitored, allowing adjustments in gas volume and pressure to prevent excessive distension or discomfort. Topical CO₂ therapy, such as transcutaneous application via CO₂-enriched dressings, may also be employed to enhance diffusion and broader tissue coverage.

In parallel with carboxytherapy, HOCl is injected directly into the ulcerated tissue to improve microbial control and reduce inflammation. This injection is performed after CO₂ is administered to ensure optimal tissue oxygenation and microcirculation, which can enhance the efficacy of HOCl. The injection of HOCl is performed using the same fine-gauge needles, typically 30G, to minimize trauma and ensure accurate delivery to the tissue. The recommended dosage for HOCl is typically 0.1 to 0.3 mL per injection site, injected subcutaneously or intradermally around the ulcer and ischemic zones, spaced 1–2 cm apart. Antimicrobial properties of HOCl help reduce bacterial load in the ulcer, while its anti-inflammatory effects aid in modulating excessive inflammation, thereby promoting faster tissue regeneration and wound closure.

After the procedure, gentle massage is applied to facilitate gas absorption and optimize vascular effects, while HOCl, in particular, stands out as a potent antimicrobial and antiviral agent, effectively eliminating bacterial biofilms and neutralizing viral pathogens within the wound microenvironment. Its broad-spectrum efficacy, coupled with its ability to regulate immune responses and promote extracellular matrix remodeling, positions it as a key component in optimizing chronic wound management.11,12,13

A protective dressing is then applied to maintain a sterile environment and prevent contamination. Patients are advised to avoid excessive pressure on the treated foot for at least 24 hours to allow optimal vascular response.

Monitoring for transient erythema, mild swelling, or discomfort is recommended, with these typically resolving within a few hours. The recommended treatment frequency is two to three sessions per week for moderate ulcers, with the frequency increasing to four sessions per week for severe ischemic ulcers. The total duration of treatment is typically 6 to 12 weeks, depending on the ulcer severity and healing response. Once the ulcer has healed, maintenance therapy is recommended with once-weekly sessions for one to two months to sustain vascular health and prevent recurrence.

Additionally, carboxytherapy can be combined with other regenerative treatments, such as platelet-rich plasma, growth factor therapies, or hyperbaric oxygen therapy, to further enhance the healing potential. This integrated approach, combining the synergistic effects of carboxytherapy and HOCl injections, ensures a targeted, safe, and effective treatment strategy.

Conclusion & recommendations — towards a better therapeutic future: The integration of carboxytherapy and hypochlorous acid offers a promising approach for diabetic foot ulcer treatment by enhancing oxygenation, reducing inflammation, and promoting angiogenesis. Hypochlorous acid, a powerful antimicrobial and antiviral agent, effectively eliminates bacterial biofilms and neutralizes viral pathogens while supporting tissue regeneration. Further clinical studies are needed to optimize protocols and confirm efficacy. If successfully implemented, this approach could reduce amputation rates and revolutionize diabetic wound care, improving patient outcomes worldwide.