Problem

Diabetes impairs the body's ability to properly produce or respond to insulin, leading to a variety of health complications. These include a 25% lifetime risk of developing a diabetic foot ulcer (DFU), a non-healing wound that often leads to lower limb amputations. Current treatments are often insufficient in healing and preventing the recurrence of DFUs.

Solution

Develop therapeutic bandages to enhance the treatment of DFUs by targeting their impaired healing mechanism.

Impact

This project laid further groundwork in the investigation of using hydrogels loaded with either MCP-1 or Substance P as a potential treatment for patients suffering from DFUs.

An Impaired Healing Mechanism

There are two types of macrophages primarily involved in the wound healing process—M1 and M2 macrophages:

M1 Macrophages — Pro-inflammatory
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M2 Macrophages — Pro-healing

Patients suffering from diabetic foot ulcers struggle due to an impaired healing mechanism where there is an abundance of M1 macrophages and a lack of M2 macrophages.

Thus, our goal was to:

1. Recruit macrophages to the wound site.

2. Polarize the macrophages to a pro-healing (M2) state.

Bandage Manufacturing Process

click to learn how to make the hydrogels

How to Make:
1. In a sterile environment, create a 2% wt/volume alginate solution using sodium alginate powder and distilled water. Therapeutic factors (MCP-1 or Substance P) are mixed in at this stage.
2. Assemble the middle and bottom sections of the 3-part mold using sterilized binder clips.
3. Pour alginate solution into the circular portion of the mold.
4. Cover the mold with the top piece; use sterilized binder clips to keep mold pieces from shifting.
5. Freeze the alginate solution in the molds overnight.
6. Remove molds from freezer and place into a sterile lyophilization bag.
7. Place lyophilization bag into container and connect to lyophilizer. Run cycle. (This process freeze-dries the hydrogels, allowing micropores to form in the gel.)
8. Remove from lyophilizer and carefully de-mold the gels.
9. Transfer gels to a 24-well plate.
10. Ionically cross-link gels with 100mmol CaCl2 solution. There are covalent ways of cross-linking, but for our studies we used this method because it tends to be more cost-effective and less complex.
11. Store gels for later use.

Freeze-Dried Hydrogels

Therapeutics Tested

Monocyte Chemoattractant Protein-1 (MCP-1) — Beneficial for the recruitment of monocytes and macrophages.

Substance P — Beneficial for the recruitment of macrophages and their polarization to an M2 (pro-healing) state.

Experimental Methods

Therapeutic Release Kinetics Study — The release kinetics of the therapeutic factors were analyzed via enzyme-linked immunosorbent assays (ELISA), and the release profiles of Substance P and MCP-1 from the bandages were optimized to be above the therapeutic levels for both factors at 1.5 μg/ml and 60 ng/ml, respectively, over a 10-day period.

Animal Study — The bandages were tested using a mouse model to assess their capability for wound healing based on two factors: (1) the size of wounds after 5 days and (2) the cell types recruited via fluorescence-activated cell sorting (FACS). The analysis process of the animal study is shown below.

Takeaways

• In the release kinetics study, drug release profiles were in the therapeutic range for both MCP-1 and Substance P gels.
• In the animal study, an increased number of immune cells were recruited by MCP-1 gels compared to pure alginate gels.
• Of the immune cells recruited, more macrophages tended to be recruited.
• Of the macrophages recruited, they were primarily of M2 type(pro-healing). This was expected, as the mice we used in this preliminary study were not diabetic mice and thus did not suffer from the same impaired healing mechanism.
  

Future Steps

• Continue to work on immune cell recruitment.
• Move on to polarization studies to see if M1/pro-inflammatory macrophages can consistently be polarized to a M2/pro-healing state.
  
• Complete animal study with diabetic mice model.
  

Slides