(311f) Factor Releasing Regenerative Bandages for Enhanced Healing in Diabetic Wounds

Diabetes mellitus is one of the fastest growing chronic diseases worldwide, with an estimated 285 million adults affected by the disease in 2010.^[1] Approximately 15% of diabetic patients develop diabetic foot ulcers (DFU), which can lead to significant life impairments, including lower extremity amputations.^[2] While a number of methods have been developed for wound healing in recent times, most are not applicable for DFUs since the pathophysiology in diabetic patients differs from healthy individuals. One of the factors unique to the pathology of diabetics is their impaired immune response during the wound healing process, especially the number and types of macrophages present. In healthy patients, macrophages have an integral part in homeostasis and tissue repair, and orchestrate the wound healing process through various mechanisms.^[3] Unfortunately, a decrease in the number of macrophages is observed in diabetic wounds, and those present tend to be pro-inflammatory (M1s) macrophages instead of pro-healing and/or anti-inflammatory macrophages (M2s).^[4] As a result, the wound healing progression from the inflammatory phase to the healing phase is delayed, or prevented altogether, which can lead to non-healing wounds and amputations.

To address this challenge, we hypothesized that increasing the number of macrophages at the wound site and redirecting the macrophages to a more pro-healing state can enhance healing in diabetic wounds. To examine this hypothesis, we have developed a hydrogel bandage system capable of delivering various factors to wounds. The hydrogels are fabricated as alginate cryogels and cross-linked with calcium to produce bandages with large pores (20-400 mm) that can be manufactured on a large scale as off-the-shelf products. The bandages can be loaded with therapeutics (cells or factors) during or after the manufacturing process.

Using this bandage system, polarized macrophages were delivered to diabetic wounds in a murine model of type 2 diabetes (db/db mice) and the wound healing progress was monitored over a 10-day period. Bone marrow derived macrophages were isolated, loaded into bandages, and polarized to either M1 (pro-inflammatory), M2a (pro-healing), or M2c (anti-inflammatory) state. The cell loaded bandages were then applied to wounds on diabetic mice and their effect on healing was analyzed by measuring the size of the wounds over time. Additionally, their cellular components were analyzed by immune-histochemistry (IHC) and immunofluorescence (IF). Wounds treated with macrophage loaded bandages, regardless of their polarization, had smaller wound sizes as compared to the empty bandage controls. Normalized wound size at Day 10 for each group was 23, 18, 22, 19, and 14% of the original wound size for wounds treated with empty bandages, non-polarized (M0), M1, M2a, and M2c macrophages, respectively. Furthermore, wounds treated with M2c loaded bandages were significantly more healed than those treated with M1 loaded macrophages. These results confirmed our hypothesis that the addition of macrophages, especially anti-inflammatory macrophages, to the wound site can enhance healing in diabetic wounds.

To better understand the process and factors that are responsible for the enhanced healing, the conditioned media (CM) of each polarization state was collected, loaded into the bandages, and applied to wounds to determine if the released factors alone could enhance healing. Interestingly, the results from this study were similar to those observed with the cell loaded bandages: wounds treated with CM loaded bandages were significantly smaller in size as compared to the controls. The normalized wound sizes were 23, 10, 14, 11, and 9% of the original wound size for wounds treated with empty, M0, M1, M2a, and M2c CM loaded bandages, respectively. Analysis of the CM demonstrated the presence of various factors for each polarization state, with MCP-1, MIP-1 beta, MIP-2, IL-4, and IL-10 present at higher concentrations in the CM of M2 polarized cells as compared to other factors. MCP-1 and MIP proteins are responsible for the recruitment of monocytes and macrophages and in controlling their function, while IL-4 and IL-10 are responsible for the polarization of macrophages to their M2a and M2c states, respectively.

Using this information, we are currently exploring the use of therapeutic-loaded bandages for the recruitment of macrophages to the wound site and their polarization to the pro-healing state to aid in the healing process. This approach can be advantageous over the delivery of cells in several aspects, including processing time, cost, patient compliance, and feasibility since it eliminates the need for the isolation of cells from patients and further processing to polarize them to the pro-healing state. To this end, we have incorporated various factors into our alginate bandages, modified the bandages to control their release kinetics, and are currently in the process of assessing their effectiveness in wound healing. Specifically, the bandages are loaded with MCP-1 for the recruitment of macrophages, and IL-4 and IL-10 for the polarization of these cells to the M2a and M2c states, respectively. Depending on the factors’ properties such as surface charge, molecular weight, and heparin binding affinities, we have been able to fabricate bandages with different material compositions to establish a prolonged therapeutic release prolife for each factor that can be tuned-in to range from a few hours to days and weeks. While IL-4 and IL-10 display a burst release from alginate bandages, MCP-1 is released in a controlled manner and without a burst release over a 10-day period (cumulative release of 1.2 µg), most likely due to its more positive surface charge. To reduce the burst release of the other factors, laponite, a negatively-charged clay nano-disk, is incorporated into the alginate bandages and results in the elimination of the burst release and a more prolonged release profile for each factor. Depending on the amount of laponite used, the release of IL-4 can be modified from a burst release of 5 µg (~45%) in the first day, to a sustained release of 3 µg over a similar time span. The release of IL-10 can be programmed to range from 4 µg (~35%) in the first day to a sustained release of 0.1 µg during the same period.

Using factor loaded bandages, we have demonstrated that more immune cells, especially macrophages, are recruited to the wound site as compared to traditional bandages. In this study, MCP-1 loaded bandages were placed on wounds in a murine model over a 5-day period and the cell types and numbers present in the wound were analyzed via flow cytometry. Based on this analysis, a significantly higher number of macrophages were recruited to the wound site in samples treated with MCP-1 loaded bandages as compared to traditional bandages (approximately four times more). Our next step is to assess the capability of bandages loaded with IL-4 and IL-10 to polarize the local macrophages to the M2 state and, thereby, enhance wound healing. Such bandage systems can potentially provide significant improvement in the lives of diabetic patients by using their body’s own immune system to directly address their impaired healing mechanism.

References:

[1] L. I. Moura, A. M. Dias, E. Carvalho, H. C. de Sousa, Acta Biomater 2013, 9, 7093.

[2] A. Menke, S. Casagrande, L. Geiss, C. C. Cowie, JAMA 2015, 314, 1021.

[3] T. A. Wynn, A. Chawla, J. W. Pollard, Nature 2013, 496, 445.

[4] S. Nassiri, I. Zakeri, M. S. Weingarten, K. L. Spiller, J Invest Dermatol 2015, 135 (6), 1700.