Regenerative Skin Healing Through Targeted Modulation of Engrailed1-Negative Fibroblasts Shamik Mascharak, Heather E desJardins-Park, Alessandra L Moore, MD, Mimi R Borrelli, MBBS, Malini Chinta, BS, Deshka Foster, MD, H Peter Lorenz, MD, Michael T Longaker, MD, MBA, FACS Boston University School of Medicine, Boston, MA; Brigham and Women’s Hospital, Boston, MA; Stanford University Medical Center, Stanford, CA; Stanford University School of Medicine, Stanford, CA INTRODUCTION: Skin scarring poses a significant medical burden for millions of patients every year. Recently, Engrailed1-positive fi- broblasts were shown to be responsible for the majority of scarring on the dorsal skin in mice. However, comparatively little is known about the function of Engrailed1-negative fibroblasts (ENFs), which are present in all layers of the dermis and are putatively non-scarring. METHODS: Experiments were performed in En1 Cre ;R26 mTm- G (En1 mTmG ) and En1 Cre ;Ai6 (En1 Ai6 ) mice, with Engrailed1- positive cells expressing green fluorescent protein and Engrailed1-negative cells expressing red fluorescent protein or no reporter, respectively. Adult mice underwent dorsal excisional wounding, and scar histology was assessed at 4 weeks. For mecha- notransduction inhibition, the wound bed was injected with 50 mM verteporfin (mechanotransduction inhibitor) in phosphate buffered saline. RESULTS: Fluorescence-activated cell sorting-isolated ENFs activate Engrailed1 by 14 days of culture on plastic (green fluorescence in Figure 1A, top row), but not after inhibition of stiffness sensing (ROCK inhibitor Y-27632) or culture in soft collagen hydrogels, suggesting a mechanotransduction mecha- nism for postnatal Engrailed1 expression (Figure 1A, bottom row). ENFs transplanted into dorsal skin activate Engrailed1 after wounding (Figure 1B). Wounds treated with Verteporfin yield scars with dramatically fewer Engrailed1-positive fibroblasts, reduced fibrosis, and regeneration of dermal appendages (Figure 1C top vs bottom row). CONCLUSIONS: Postnatal ENFs activate Engrailed1 in vitro and in vivo by canonical mechanotransduction signaling and take on a fibrotic phenotype. Accordingly, inhibition of mechanotransduc- tion[h1] promotes ENF-mediated wound healing with reduced fibrosis and skin regeneration. Our findings suggest that ENFs contribute to scarring by activating Engrailed1 in response to me- chanical cues within the wound bed. Role of the Skeletal Stem Cell in Achilles Tendon to Bone Interface Healing Ashley L Titan, MD, Ruth Ellen Jones, MD, Ankit Salhotra, Kiana S Robertson, Deshka Foster, MD, Sid Menon, Matthew Murphy, MD, Genesis V Lucero, Charles K Chan, PhD, Michael T Longaker, MD, MBA, FACS Hagey Laboratory for Pediatric Regenerative Medicine, Stanford, CA; Stanford University School of Medicine, Stanford, CA INTRODUCTION: Entheses are complex structures that provide an essential interface between tendons and bones to reduce stress and facilitate movement. Partial injuries to the Achilles tendon- to-bone interface heal in a mechanically inferior manner. The presence and role of the mouse skeletal stem cell (mSSC) in tendon-to-bone healing remains unknown. We hypothesize that activation of mSSCs at the enthesis could promote tendon to bone healing. METHODS: A total of 16 C57BL/6 mice (32 limbs) were used in this study. All study procedures were approved by Stanford’s Administrative Panel on Laboratory Animal Care. To test Achilles healing, we created a partial laceration at the Achilles tendon to bone interface. Sham operation was performed on the contralat- eral limb. Postoperatively, mice were allowed free cage activity and all animals were euthanized at postoperative day 7. Gross evaluation and histology were performed to confirm intactness and investigate healing of the tendon-to-bone interface (n ¼ 12 limbs). Additionally, mSSCs kinetics in the Achilles tendon to bone interface were measured using fluorescence-activated cell sorting (n ¼ 20 limbs). RESULTS: Gross inspection confirmed that the tendon-to-bone interface remained intact at postoperative day 7 (Figure 1A, 1B). Histologic examination and staining with hematoxylin and eosin demonstrated increased cellularity and disorganized collagen fibers at the tendon to bone interface (Figure 1C, 1D). Fluorescence-acti- vated cell sorting analysis revealed a 1.5-fold increased mSSC fre- quency at the tendon to bone interface during healing compared with sham operation (Figure 1E, 1F). Figure. A) ENFs (red) cultured on stiff plastic (top row) activate Engrailedl (green), while those cultured with ROCK inhibitor Y- 27632 (middle row) or in soft collagen hydrogels (bottom row) do not, suggesting a mechanotrarisduction mechanism for postnatal Engrailedl expression. B) Histology of skin injected with ENFs (red) prior to wounding shows activation of Engrailedl (green) in the wound environment. C) Immunofluorescent histology of skin 4 weeks after PBS or Verteporfin treatment. Mechanotransduction Inhibition led to sustained EMF infiltration (red) and regeneration of secondary elements (H&E stain, white arrows). S228 Scientific Forum Abstracts J Am Coll Surg