Exposed chronic wounds are often covered with hypoxic tissue and are accompanied by necrosis, ongoing inflammation, and anaerobic infection. Since the oxygen in the atmosphere can only penetrate about 0.3 mm of tissue, and it is difficult for the oxygen circulating in the blood to reach the chronic wound through the damaged blood vessels, a solution for local oxygen delivery is urgently needed.

Recently, Prof. Zhang Zhongtao and Prof. Yao Hongwei from Beijing Friendship Hospital affiliated to Capital Medical University and Cheng Yuhao's team from Nanjing University School of Medicine and School of Life Sciences have studied a nanoparticle that can locally deliver dissolved oxygen to the wound site by adding lyophilized oxygen, thereby changing the traditional gel-type wound dressing (Fig. 1). A related research paper, Nano-oxygenated hydrogels for locally and permeably hypoxia relieving to heal chronic wounds, was published in the journal Biomaterials.

Fig.1 Schematic diagram of the structure of NOX-gel and the treatment of chronic wounds

Hydrogel Material:

Perfluoronaphthalene-coated human serum albumin (FDC@HSA), hyaluronic acid

1. NOX-gel improves cell migration and vascularization

The results of dynamic light scattering (DLS) showed that the hydrodynamic diameter of NOX-gel was 496.3 nm, and in addition, there was no large difference in the oxygen content of NOX-gel before and after lyophilization. The researchers chose CoCl2 to treat the cultured cells, which resulted in high expression of HIF-1α. Tests have shown that NOX gel has a protective effect against hypoxia and down-regulation of HIF-1α. Next, the researchers evaluated the relative viability of the cells, and the results showed that NOX-gel could significantly promote granulation tissue production by releasing oxygen, further reversing the effect of cellular hypoxia on HSF proliferation. Angiogenesis is the key to recovering chronic trauma and providing continuous oxygen. Catheterization experiments using human umbilical vein endothelial cells (HUVECs) were performed to determine the angiogenesis capacity of NOX-gel gel, and the results showed that the NOX-gel group significantly improved the formation of vascular branch points (Fig. 2).

Fig.2 In vitro cytotoxicity of NOX-gel gel and therapeutic effect of wound healing

2. NOX-gel gel promotes wound healing in acute and chronic diabetes

Next, the researchers verified whether NOX-gel can promote cell proliferation and angiogenesis in vivo, and the results showed that in the acute wound model of non-diabetic mice and the chronic wound model of diabetic mice, NOX-gel can deliver oxygen to the wound tissue, further alleviate the hypoxia effect, and accelerate wound closure. In addition, histopathology of HE and Masson staining showed that the granulation tissue thickness of wounds treated with NOX-gel was significantly higher than that of the control group, and that significantly more collagen was produced than wounds treated with other dressings (Figure 3).

Fig.3 NOX-gel gel promotes wound healing in diabetic mice

3. NOX-gel can alleviate the hypoxia phenomenon of diabetic wounds and promote angiogenesis

To investigate whether the rapid healing of wounds by NOX-gel may be due to its delivery of oxygen to the wound tissue, further alleviating hypoxia, immunohistochemistry (IHC) analysis of HIF-1α antibody was performed, which further demonstrated that NOX-gel treatment can successfully prevent hypoxia. In addition, the density of HIF-1α-positive cells in the DM+NOX-gel group was significantly reduced at day 6 and 12 after the gel intervention, confirming that the gel can sustainably improve the hypoxia status of diabetic wounds. In the process of wound healing, the investigators explored the pro-angiogenic ability and anti-inflammatory effect of NOX-gel through CD31 and CD68 staining, respectively, and confirmed that NOX-gel can effectively improve hypoxia, reduce inflammatory response, and promote diabetic wound neoangiogenesis (Fig. 4).

Fig.4 Immunohistochemical analysis of skin tissue sections of diabetic wounds treated with NOX-gel gel

4. NOX-gel enhances flap survival and angiogenesis in diabetic patients

The investigators used a Doppler flow meter to monitor the blood flow of the flap, and the blood flow intensity was displayed in increasing order of blue, green and red. The blood supply of the flap in the DM group and gel group gradually decreased, while the blood supply of the flap gradually recovered in the DM+NOX-gel group. The flap in the NOX-gel group was healthy red and looked like normal skin with an adequate blood supply (Figure 5).

Fig.5 Immunofluorescence results of NOX-gel in promoting postoperative flap survival in diabetic mice 6 days after surgery

TdT-mediated immunofluorescence staining of dUTP-terminal labeling (TUNEL), Caspase-3, HIF-1α and vascular endothelial growth factor (VEGF) showed that TUNEL and Caspase-3 staining were used to monitor apoptosis of transplanted flap tissues. Skin cell apoptosis was the highest in the DM group, and NOX-gel could effectively reverse apoptosis. The mechanism of action of vascular endothelial growth factor in diabetic wound healing has been demonstrated, in particular, VEGF promotes angiogenesis by binding to endothelial cell membranes. To test the hypothesis: oxygen delivery of NOX-gel promotes the survival rate of grafted flaps. HIF-1α and VEGF staining further explain why NOX-gel improves flap survival in diabetic patients. During diabetic wound healing, the expression of HIF-1α in the DM+NOX-gel group was significantly reduced after 6 days of gel intervention, demonstrating that this gel can sustainably deliver oxygen to the flap tissue (Figure 6).

Fig.6 Immunofluorescence results of skin flap sections 6 days after surgery

The researchers proposed a NOX-gel hydrogel with hyaluronic acid, which can effectively improve the hypoxia status of acute or chronic wounds, reduce inflammatory reactions, promote wound neoangiogenesis, and accelerate wound healing. NOX-based oxygen-carrying nanocarriers have good biosafety in vivo. In addition, the preservation and transport of nitrogen oxides is also effective in overcoming the risk of infection. Considering the above advantages, the NOX technology proposed by the researchers can be applied to a variety of biomedical and clinical applications, including but not limited to wound healing, such as the preservation of transplanted tissues and organs.

Article Source:

https://doi.org/10.1016/j.biomaterials.2022.121401