(I-330) Immunomodulatory Strategies to Improve Burn Wound Healing

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row I - Poster # 330

Presenting Author(s)

KW

Kristopher White

Postdoctoral Scholar
University of California - Irvine
Irvine, California, United States

Co-Author(s)

VP

Victor Pozzo

Research Fellow
Massachusetts General Hospital, United States
AA

Alec Andrews

Senior Research Technologist
Massachussetts General Hospital, United States
PT

Pierre Tawa

Research Fellow
Massachusetts General Hospital, United States
GR

Golda Romano

Research Fellow
Massachusetts General Hospital, United States
AL

Alexandre G. Lellouch

Assistant Investigator
Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Shriners Children's Hospitals, Boston, USA; Groupe Almaviva Santè, Clinique de I’Alma, Paris, France, United States
CC

Curtis L. Centrulo Jr.

Attending Surgeon and Assistant Professor
Massachusetts General Hospital, Harvard Medical School, and Shriners Children's Hospitals, Boston, USA, United States

Primary Investigator(s)

RO

Ronke Olabisi

Associate Professor
University of California, Irvine, United States

Introduction:: The immune system has a critical role in orchestrating wound healing, and strategies to control the immune system in order to effect wound healing have increased. Unlike in acute wound healing, the wound healing cascade in chronic wounds fails to progress beyond the inflammatory phase, and chronic wounds exhibit an imbalance between pro- and anti-inflammatory signals. Mesenchymal Stem Cells (MSCs) have immunomodulatory properties, and we have shown that coencapsulating them with insulin secreting cells (ISCs) activates the PI3K-Akt pathway in the MSCs. The PI3K-Akt pathway plays a key role in regulating immune response and inflammatory factor release. Additionally, our prior work demonstrated improved viability and function of both MSCs and insulin secreting cells (ISCs) when coencapsulated. Furthermore, our dual cell treatment (hydrogel encapsulated MSCs and ISCs) demonstrated a synergistic therapeutic effect of the combined ISCs and MSCs in slow-healing diabetic murine wounds that significantly exceeded healing by MSCs alone. We observed a paracrine wound healing response that rapidly closed diabetic murine wounds (from 40 to 14 days) and minimized scarring. The current study applies this technique to full thickness burn wounds in a swine model and investigates the effect of the therapy on the treated wounds and further probes the effect of insulin on the exogenous MSCs.

Materials and Methods:: Wound healing: Dual cell wound treatment gels (4 cm x 4 cm x 0.4 cm, 8.0 × 106 cells/hydrogel) were formed by encapsulating equal numbers of adult human primary MSCs and ISCs (RIN-m) into polyethylene glycol diacrylate hydrogel sheets via photopolymerization. The hydrogels were applied to 4 cm x 4 cm full thickness thermal burns (N=16 wounds total, n=8 wounds per pig) on the dorsa of mini-pigs (n=2) in accordance with protocols approved by the Harvard MGH IACUC. Hydrogel dressings remained undisturbed for 2 weeks, after which the hydrogels were removed, and wound monitoring continued for 5 weeks. Within-pig control burns were treated with standard of care (cleaning and dressing changes every 2-3 days). Wounds were imaged during dressing changes and analyzed with NIH ImageJ. Biopsies were taken after 5 weeks and stained with H&E for histological analysis. Gene expression: MSCs were also analyzed using single cell RNA sequencing (scRNA-seq) to assess how insulin modulates gene expression. Human MSCs were seeded onto the surface of 6-well plates (10,000 cells/cm2). The cells were then treated with growth media supplemented with 0.1 ng/mL insulin. Control cultures were treated with growth media without insulin. Batches of cells were harvested and fixed after 3 days using a Parse Biosciences cell fixation kit and stored at -80oC until sequencing. The fixed cells were sequenced using a Parse Biosciences Evercode WT kit. Sequencing data was analyzed using Seurat for R and visualized via unmonitored clustering and UMAP reduction. Gene Ontology (GO) enrichment was performed via Metascape.

Results, Conclusions, and Discussions::

Visually, we observed no scab formation in HEMIs treatments and scarring was soft, flat, and non-adherent (Figure 1A). Exudate was clearly visible early in hydrogel-treated wounds. However, this exudate subsided over time and was not determined to be infectious. The exudate volume was also lower after removal of the treatment and replacement of the Tegaderm at week 2. All wounds treated with coencapsulated MSCs and ISCs closed faster than standard-of-care wounds (Figure 1B). Visually, hematoxylin and eosin-stained tissue biopsies show epidermal, dermal, and hypodermal layer formation (Figure 2). The hypodermal layer did not develop in control or MSC-only groups. At 5 weeks post-escharotomy, development and restratification of the epidermis and dermis was observed at the edges of wounds treated with dual cell hydrogels. Our results indicate that co-encapsulation of MSCs and ISCs accelerates wound healing in a porcine burn model, demonstrating increased regenerative capabilities in skin similar in structure to that of human skin.

SCRNA-seq and gene ontology analysis revealed that all differentially expressed genes in MSCs treated with insulin when compared against untreated control MSCs are also known to be involved in wound healing (Figure 3). Sequencing studies are ongoing to further probe how insulin and insulin-secreting cells affect MSC gene expression in juxtacrine and paracrine environments relevant to wound healing.

Taken together, these observations suggest that presence of insulin primes the MSCs to promote skin regeneration through immunomodulatory and wound healing pathways.

Acknowledgements (Optional): : This study was funded in part by an NSF CAREER Award (CBET-2040657) and a Johnson & Johnson Women in Science, Technology, Engineering, Mathematics, Manufacturing, and Design (WiSTEM2D) award.

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