Doxycycline Reduces Scar Thickness and Improves Collagen Architecture

Abstract

Objective: To investigate the effects of local doxycycline administration on skin scarring.

Background: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo.

Methods: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue.

Results: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005).

Conclusions: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

Conflict of interest statement

The authors report no conflicts of interest.

Figures

FIGURE 1.

Doxycycline improves wound thickness without sacrificing tensile strength. A, Scar thickness is significantly reduced in 2 and 20 mg/mL doxycycline treated wounds (n = 10 wounds per group, *P < 0.0001, **P = 0.0023). B, Representative histology of scar treated with vehicle (PBS, left panel) and 2 mg/mL doxycycline (right panel) by hematoxylin and eosin stain. E, Epidermis; D, dermis. C, Relative scar collagen is significantly reduced in 0.1 and 2 mg/mL doxycycline groups as measured by Masson trichrome stain (n = 10 wounds, *P = 0.0357, **P = 0.0317). D, Representative histology of scar treated with vehicle (PBS, left) and 2 mg/mL doxycycline (right) using Masson trichrome stain (top panels), with representative color deconvolution of the above image (blue deconvolution, bottom left panels;red deconvolution, bottom right panels). E, Tensile strength is not significantly different between vehicle and 2 mg/mL doxycycline (Dox) treatment groups (n = 10 wounds, P = 0.4376). F, Representative tensile strength curves for PBS control and 2 mg/mL doxycycline treatment. A, C, Results depicted as mean per animal ± SEM. E, Tensile strength depicted as individual wound strength measurements ± SEM. *,**, denotes statistical significance;NS, not statistically significant.

FIGURE 2.

Reduced skin scar thickness by doxycycline treatment depends on local administration and does not delay wound healing. A, Time to wound closure is not adversely influenced by doxycycline administration in 0.1, 2, and 20mg/mL treatment groups compared with PBS controls (n = 10 wounds, P > 0.24378 at all timepoints). B, Treatment of the right wound with 2 mg/mL doxycycline and left wound with PBS in the same mouse led to comparably reduced scar thickness in the left and right wounds (n = 5 animals, P = 0.6257). C, Oral administration of 2 mg/mL doxycycline via drinking water did not result in reduced scar thickness (n = 10 wounds, P = 0.3006). Control, PBS injection;Dox PO, oral doxycycline. Results depicted as mean ((A), per group perday;(B), per wound;(C), per mouse) ± SEM. NS indicates not statistically significant.

FIGURE 3.

The ability to reduce scar thickness is unique to doxycycline and depends on early administration. A, Improvement in scar thickness is limited to doxycycline (n = 10 wounds, *P < 0.0001). Other tetracycline antibiotics (minocycline, tetracycline) significantly thicken dorsal skin scars compared with control treatment (n = 10 wounds, **P < 0.0001, ***P < 0.0001). B, Delaying doxycycline injection to 12 and 24 hours postoperatively results in increasing scar thickness (n = 4 wounds, *P = 0.0066, **P < 0.0001). A nonsignificant increase in scar thickness but increased variability is seen when doxycycline administration is delayed to 48 hours postoperatively (n = 4 wounds, P = 0.8848, T0 h SEM = 12.37, T48 h SEM = 29.03). C, A decrease in Picro Red fibers (top panel) and increase in Picro Green fibers (bottom panel) is seen in the 2 mg/mL group (n = 10 wounds, *P = 0.0042, **P = 0.0441, ***P = 0.0114). D, Representative Picrosirius staining for wounds treated with PBS (upper left panel) and 2 mg/mL doxycycline (upper right panel), with representative color deconvolution of Picrosirius images (red deconvolution, bottom left panels;green deconvolution, bottom right panels) which allowed for the separate measurement of Picro Red and Picro Green collagen fibers. E, Collagen I immunostaining is significantly reduced in the 2 mg/mL group compared with control (n = 6 wounds, *P = 0.0430). F, Representative anti-collagen I (green) immunostaining in PBS (top panel) and 2 mg/mL doxycycline (bottom panel) groups. *,**,***, denotes statistical significance. Results depicted as mean per animal ± SEM.

FIGURE 4.

Analysis of collagen fiber orientation reveals a favorable phenotype in doxycycline treated skin scars. A, Picro Red fiber number (left panel) is significantly reduced in the 0.1 and 2 mg/mL treatment groups (left panel; n = 10 wounds, *P = 0.0054, **P = 0.0098) and Picro Green fiber number is significantly increased in the 0.1 mg/mL group (right panel;n = 10 wounds, ***P = 0.0292). B, Picro Red fiber length (left panel) is significantly reduced in the 2 mg/mL group (n = 10 wounds, *P = 0.0226). C, Picro Red fiber branching (left panel) is significantly decreased in the 0.1 and 2 mg/mL groups but increased with 20 mg/mL treatment (n = 10 wounds, *P = 0.0159, **P = 0.0053, ***P = 0.0009). Picro Green fibers also have increased branching (right panel; n = 10 wounds, ****P = 0.0444). D, Fiber randomness is increased in Picro Red fibers (left panel; n = 10 wounds, *P = 0.0115, **P = 0.0282, left panel) and Picro Green fibers (right panel;n = 10 wounds, ***P = 0.0289). E, Representation of image deconvolution for analysis, including color deconvolution, conversion to grayscale, image binarization, and skeletonization. *,**,***,***», denotes statistical significance. Results depicted as mean per animal ± SEM.

FIGURE 5.

FACS analysis of inflammatory cell populations within doxycycline treated wounds over time. A, FACS gating strategy for the MAC1+, B220+, and double negative populations. B, Effects of doxycycline on relative populations of macrophage (MAC1+), B cell (B220+), and lymphocyte (double negative, Lymph) cell populations in the wounds at 12 hours (top left panel), 5 (top right), 9 (bottom left), and 14 days (bottom right) postinjury. Macrophages are significantly increased with doxycycline treatment at day 9 postinjury (n = 6 wounds, *P = 0.0407). *, denotes statistical significance. Results depicted as mean per animal ± SEM.

FIGURE 6.

Scarring fibroblasts are significantly reduced in wounds treated with doxycycline. A, Representative histology of En1Cre/;Rosa26mTmG/– skin wounds treated with PBS (left panel) and 2mg/mL doxycycline (right panel). B, Engrailed1 positive fibroblasts (EPFs) in wounds are significantly reduced with doxycycline treatment (n = 6 wounds, *P = 0.0005). *, denotes statistical significance. Results depicted as mean per animal ± SEM.