Poster N14 - Development of a vascularized skin organoid to study diabetic wound healing

Introduction: Diabetic foot ulcers (DFUs) pose severe clinical and economic burdens on society. DFUs affect more than 400 million people worldwide, greatly contributing to both morbidity and mortality rates of diabetic patients as a consequence of repeated hospitalizations (1). DFUs account for more than 73,000 nontraumatic lower-limb amputations in the United States alone, with healthcare costs ranging from $9 to $13 billion, without considering the cost of diabetes care and related infections (2). Adding to the burden of daily diabetes care, dermal wounds among type 2 diabetics, such as DFUs, often become infected with bacteria leading to wounds that fail to heal properly (3). In addition, as a result of hyperglycemia, affected patients also show a significantly impaired healing rate and microvascular complications due in part to the narrowing of their blood vessels and stiffening of their arteries (4). Overall, DFUs increase already high treatment costs due to their longer care time and high likelihood of sepsis and/or death (5). Currently, most investigational or clinically available treatments rely on the release of drugs, proteins, microRNA, DNA, or cells to engineer skin tissue equivalents (6). These approaches require long waiting periods for novel tissue to develop with little to no improvement or positive outcomes in affected patients.

Materials and

Methods: In order to address these issues, the Ameer Laboratory has developed a regenerative wound dressing that consists of the thermoresponsive macromolecule poly (polyethylene glycol citrate-co-N-isopropylacylamide) (PPCN) with a covalently conjugated laminin-derived peptide A5G81 that is dermo-conductive, dermo-inductive, and antioxidant. PPCN-A5G81 has been demonstrated to accelerate dermal wound closure in diabetic mice by stimulating keratinocyte and fibroblast proliferation and migration and reducing oxidative stress6. Our objective is to establish a more complex skin organoid model by including vascular cells to help us understand potential mechanisms of action for the accelerated wound healing observed when treating diabetic wounds in mice and swine with PPCN-A5G81 and ultimately develop better therapies for patients with DFUs. To establish our model, we cultured skin organoids with microvascular endothelial cells (human umbilical vein endothelial cells, HUVECs) and incorporated the cells into the collagen-based dermal compartment hydrogel to create a microvascular network. A challenge of increasing cellular complexity in co-culture models is satisfying the metabolic needs of all cell types in one media. Therefore, prior to combining the HUVECs into the skin organoids, we conditioned the cells by growing them in specially made media. To optimize the vascularized skin organoid model, we generated different types of organoids including 1) collagen hydrogel-embedded HUVECs “sandwiched” between two collagen-fibroblast hydrogels with keratinocytes seeded on the upper compartment, 2) HUVECs mixed with fibroblasts in the same collagen hydrogel with keratinocytes on top, or 3) HUVECs only in the collagen with keratinocytes on top.

Results, Conclusions, and Discussions: Our preliminary results show keratinocyte stratification and both the presence of the endothelial cells and tubulogenesis forming vascular networks in the “sandwiched” model (fig. 1). Both the HUVEC and keratinocyte model and the keratinocyte, fibroblasts, and HUVEC model exhibited tubule formation but significantly less than the “sandwiched model” due to their lack of structure they resulted in very small and fragile models. Our ongoing studies will determine functionality of the organoid tissue and the individual cell types including the function of the tubules as well as healing rate of each model post-injury. Our goal is to use this fully human skin organoid to model diabetic wounds using patient-derived cells for testing the efficacy and mechanism of action of bioengineered materials such as PPCN-A5G81 for superior wound-healing treatments.

Acknowledgements (Optional): This research is supported by the CARE Center, the NIH grants T32-EB031527 and 5P30AR075049-04, and the National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK131302-02).

Bibliography
(1) Jodheea-Jutton, A.; Hindocha, S.; Bhaw-Luximon, A. Health Economics of Diabetic Foot Ulcer and Recent Trends to Accelerate Treatment. The Foot 2022, 101909. https://doi.org/10.1016/j.foot.2022.101909.
(2) Xiao, J.; Zhu, Y.; Huddleston, S.; Li, P.; Xiao, B.; Farha, O. K.; Ameer, G. A. Copper Metal–Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes. ACS Nano 2018, 12 (2), 1023–1032. https://doi.org/10.1021/acsnano.7b01850.
(3) Kolluru, G. K.; Bir, S. C.; Kevil, C. G. Endothelial Dysfunction and Diabetes: Effects on Angiogenesis, Vascular Remodeling, and Wound Healing. International Journal of Vascular Medicine 2012, 2012, 1–30. https://doi.org/10.1155/2012/918267.
(4) Deshpande, A. D.; Harris-Hayes, M.; Schootman, M. Epidemiology of Diabetes and Diabetes-Related Complications. Physical Therapy 2008, 88 (11), 1254–1264. https://doi.org/10.2522/ptj.20080020.
(5) Bowler, P. G.; Duerden, B. I.; Armstrong, D. G. Wound Microbiology and Associated Approaches to Wound Management. Clinical Microbiology Reviews 2001, 14 (2), 244–269. https://doi.org/10.1128/cmr.14.2.244-269.2001.
(6) Zhu, Y.; Cankova, Z.; Iwanaszko, M.; Lichtor, S.; Mrksich, M.; Ameer, G. A. Potent Laminin-Inspired Antioxidant Regenerative Dressing Accelerates Wound Healing in Diabetes. Proceedings of the National Academy of Sciences 2018, 115 (26), 6816–6821. https://doi.org/10.1073/pnas.1804262115.