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Lyophilized Human Amnion Chorion Membrane Mitigates Hypertrophic Scar Formation
Jonathan P. Yasmeh, M.S., Hudson Kussie, B.S., Maia Granoski, B.A., Eamonn McKenna, B.S., Andrew Hostler, B.S., Abdelrahman M. Alsharif, B.S., Maria Gracia Mora-Pinos, M.D., Katharina Fischer, M.D., Kellen Chen, PhD, Geoffrey C. Gurtner, M.D..
University of Arizona College of Medicine - Tucson, Tucson, AZ, USA.

PURPOSE: Following cutaneous injury, chronic inflammation and dysregulated extracellular matrix formation can disrupt the normal wound healing process and result in excessive scar tissue formation known as a hypertrophic scar (HTS). Previous studies have demonstrated that dehydrated human amnion/chorion membrane can effectively hinder the activation of the TGFβ1 signaling pathway, which plays a role in the formation of fibrous tissue and the subsequent development of HTS (Moreno et al. 2021). Here, we investigate whether a tri-layer lyophilized human amnion chorion membrane (LHACM) that includes amnion, intermediate, and chorion layers, can regulate scar formation.
METHODS: Our lab has developed a model that mimics human-like HTS by applying mechanical strain across incisional wounds (Aarabi et al. 2007). We used a modified version of this protocol to create murine HTS. Approximately 2cm long full-thickness incisions were made on the dorsa of C57/BL6 mice and were closed using monofilament nylon 5-0 sutures. On post-incision day 4, sutures were removed, and mechanical loading devices were placed over the wounds and secured using surgical staples. Tension across the wounds were produced by expanding the loading devices by 2mm every other day for 14 days total. Scars were imaged and analyzed on day 14 and tissues were explanted. HTS tissue was analyzed using Picrosirius Red staining combined with ImageJ, Matlab, CurveAlign, and CTfire.
(n=5) did not have LHACM placed and were extended (No treatment, Strain). (n=5) did not have LHACM placed and were not extended (No treatment, No strain). (n=5) mice had LHACM placed subcutaneously prior to incision closure and were extended every other day for the total 14 days (Treatment, Strain). (n=5) had LHACM placed subcutaneously but were not extended (Treatment, No strain).
RESULTS: Mechanical strain caused an average scar width of 1.21cm, which was significantly increased compared to 0.624cm non-strained wounds (p=0.0019). Treatment with subcutaneous LHCAM in mechanically strained and unstrained mice significantly reduced scar widths to 0.697cm (p= 0.0325) and 0.548 (p=0.0011) respectively, similar to non-strained control wounds.
Analysis of extracellular matrix histology using Picrosirius Red showed that even in the presence of mechanical strain, subcutaneous LHACM elicited significantly reduced width (p=<0.0001) and angle (p=0.0016) of collagen fibers as well as fibers that were less straight (p=<0.0001) and aligned (p=0.0056).
CONCLUSION: There is still a great deal that remains unknown regarding the pathophysiology of HTS formation, and available treatment options remain limited. LHACM enhanced the healing of dermal wounds in a murine model of HTS when assessing scar features and tissue histology. Mechanical strain across a wound led to increased gross scar width, and subcutaneous treatment with LHACM significantly reduced scar width as well as improved collagen tissue architecture. These findings can have direct clinical applications to potentially prevent and/or treat hypertrophic scar formation, particularly in wound sites with high tension.
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