(D-124) Effective diabetic wound regeneration via therapeutic exosome-hydrogel composite

Introduction::

Diabetes is a highly prevalent global chronic disease affecting over 37 million people in the U.S. alone. Although it involves various complications, patients commonly experience impaired wound regeneration, which poses a significant risk of infection that may necessitate limb amputation. Therefore, it is critical to develop biomedical strategies for effective diabetic wound regeneration. Recent studies have highlighted that exosomes, nano-vesicles containing various proteins and nucleic acids, secreted from stem cells have physiological effects favorable for tissue regeneration. However, the clinical translation of exosomes is limited by its instability, low bioavailability, and lack of targeting ability. To address these issues, our study proposes that a pyrogallol-conjugated hyaluronic acid hydrogel (HA-PG) can facilitate in vivo delivery of exosomes. We demonstrate that the exosome-encapsulating HA-PG patch strongly adheres to the wounded mouse skin, rapidly undergoes gelation, and releases exosomes to the desired tissues to accelerate wound regeneration, making it a promising therapeutic platform for the treatment of diabetic wounds.

Materials and Methods::

Exosomes were isolated from adipose-derived stem cell-conditioned media through ultracentrifugation at 120,000g followed by resuspension with PBS and quantification by bicinchoninic acid (BCA) assay. HA-PG was synthesized by conjugating 5-hydroxydopamine to hyaluronic acid via the carbodiimide coupling chemistry using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). To fabricate the HA-PG/exo patch, a solution of HA-PG mixed with exosomes was prepared, aliquoted into cylinder-shaped molds, and lyophilized. The HA-PG patch, which was used as the control group, was fabricated via the identical protocol using solely HA-PG solution.

The swelling ratio of the hydrogels was determined by completely immersing each hydrogel in PBS and calculating the ratio of its remaining weight to the initial weight at each time point. The elastic modulus and tissue adhesiveness of the fabricated patch were measured using a rheometer (MCR102). UV-Vis spectroscopy was utilized to uncover the physical and chemical reactions within HA-PG/exo.

The therapeutic efficacy of the HA-PG/exo patch was evaluated with full-thickness skin wound model of diabetic mice. Histological analysis was conducted 14 days post-treatment through Hematoxylin & Eosin, Masson’s trichrome, and immune-fluorescence staining.

Results, Conclusions, and Discussions::

This study revealed that the HA-PG and HA-PG/exo patches possess distinct physical and chemical properties. Rheological analysis uncovered that the elastic modulus of the HA-PG/exo hydrogel was significantly higher than that of the HA-PG hydrogel, while the adhesion force displayed the opposite trend. In addition, the time-dependent UV-Vis absorption spectra of HA-PG/exo and HA-PG confirmed that the absorbance of HA-PG/exo at 300-450 nm, which indicates oxidation of the pyrogallol moieties, increased greater than that of HA-PG. These results verified that the incorporation of exosomes led to changes in physical and chemical properties of the HA-PG/exo hydrogel.

In vitro analyses showed that the HA-PG/exo hydrogel is not only biocompatible but also therapeutic. Specifically, adipose-derived stem cells cultured with HA-PG/exo hydrogel-conditioned media exhibited high viability as well as increased cell proliferation. Furthermore, human umbilical cord-derived endothelial cells incubated with HA-PG/exo hydrogel-conditioned media displayed enhanced tube formation.

Finally, the in vivo therapeutic efficacy of the HA-PG/exo patch was evaluated in the dorsal skin wound of diabetic mice. Treatment of the HA-PG/exo patch promoted mature tissue regeneration as it resulted in reduced scar width, a thicker dermis layer, and more regenerated organelles such as sebaceous glands. Overall, our study demonstrated that the treatment of the HA-PG/exo patch is an effective therapeutic strategy for diabetic wound healing and may be utilized for other biomedical applications.

Acknowledgements (Optional): : This work was supported by the National Research Foundation of Korea (NRF) grant (2021R1A2C3004262) funded by the Korea government, the Ministry of Science and ICT (MSIT), and the Korea Evaluation Institute of Industrial Technology (KEIT) grant (20009125) funded by the Korea government (MSIT).

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