Beyond Antibiotics: Local Doxycycline Administration Reduces Scarring and Improves Wound Healing by Modulating Scarring Fibroblast Behavior
Heather E. desJardins-Park, AB1, Alessandra L. Moore, MD1,2, Matthew P. Murphy, MB BCh BAO MRCSI1, Dre Irizarry, MD1,3, Bryan Duoto, BS1, Deshka Foster, MD1, Ruth Ellen Jones, MD1,4, Shamik Mascharak, BS1, Leandra Barnes, AB1, Clement Marshall, MD1, Gerlinde Wernig, MD1, Michael T. Longaker, MD, MBA, FACS1.
1Stanford University School of Medicine, Stanford, CA, USA, 2Brigham and Women's Hospital, Boston, MA, USA, 3Beth Israel Deaconess Medical Center, Boston, MA, USA, 4University of Texas Southwestern Medical Center, Dallas, TX, USA.
PURPOSE: Scars can be aesthetically and functionally devastating. Novel scar treatments have the potential to impact millions of patients. Doxycycline possesses known anti-fibrotic properties. However, its role as a potential vulnerary agent has remained unexplored. We hypothesized that topically applied doxycycline would reduce scarring.
METHODS: We employed a murine wounding model wherein full-thickness wounds are stented with silicone rings. This prevents wound contraction and mimics human wound healing kinetics. Antibiotic and PBS (control) solutions were injected locally into the superficial aspect of the wound base following surgery. Wounds were harvested upon complete re-epithelialization (day 15) for tensile strength testing and histologic examination. To quantify scar thickness, a blinded observer analyzed images of H&E-stained wound cross sections in Adobe Photoshop. Three photos were analyzed per wound; for each, dermal thickness was measured at three different scar depths, for a total of nine measurements per wound. Scar thickness was calculated as the mean of these measurements. To quantify collagen branching, picrosirius red-stained images were analyzed using an algorithm in MATLAB to calculate branchpoints per 100 micron2. Bacterial colonization of wounds was assessed via wound swabbing and culture for 24 hours. Cell migration was assessed using an in vitro scratch assay. The population of “scarring” fibroblasts (Engrailed 1-positive fibroblasts, EPFs) in wounds was determined by using the same methods in En1Cre;ROSA26mTmG mice.
RESULTS: Doxycycline treatment significantly reduces scar dermal thickness by 37% compared to PBS (*P<0.001, Figure 1). Picrosirius red staining illustrates that doxycycline-treated wounds have significantly reduced picro-red, scar-like collagen (*P=0.016), and increased picro-green, favorable collagen (*P=0.016, Figure 2). These picro-green fibers also demonstrate significantly more branching (*P=0.032) and are less aligned, like unwounded skin collagen. Notably, ultimate tensile strength is comparable between doxycycline-treated and PBS-treated wounds (respectively: 0.462 MPa, n=10; 0.534 MPa, n=9; P=0.438). Bacterial colonization is not significantly altered by doxycycline treatment (P>0.05 at days 1, 3, 5, 7, and 9). Other tetracycline antibiotics (specifically, minocycline and tetracycline) do not decrease scar thickness. “Scarring” fibroblasts (EPFs) are reduced in doxycycline-treated wounds, demonstrated by a 33.7% decrease in GFP signal in wounds from En1Cre;ROSA26mTmG treated mice (n=6, *P=0.021). Treating “scarring” fibroblasts (EPFs) with doxycycline in vitro significantly reduces migration rate (n=4, *P<0.001).
CONCLUSIONS: Locally administered doxycycline reduces scarring without sacrificing scar strength. These findings may be due to increased picro-green collagen that mimics unwounded skin collagen. Our results suggest that doxycycline's effects are not related to antimicrobial activity. Rather, doxycycline may alter scarring fibroblast behavior during healing. Collectively, our data suggest that doxycycline may represent a novel anti-scarring therapy with the benefits of a well-established safety and dosing profile. We favor rapid transition to studies in human patients to determine whether similar effects are observed.
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