Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

Pathologic skin scarring presents a vast economic and medical burden. Unfortunately, the molecular mechanisms underlying scar formation remain to be elucidated. We used a hypertrophic scarring (HTS) mouse model in which Jun is overexpressed globally or specifically in α-smooth muscle or collagen type I–expressing cells to cause excessive extracellular matrix deposition by skin fibroblasts in the skin after wounding. Jun overexpression triggered dermal fibrosis by modulating distinct fibroblast subpopulations within the wound, enhancing reticular fibroblast numbers, and decreasing lipofibroblasts. Analysis of human scars further revealed that JUN is highly expressed across the wide spectrum of scars, including HTS and keloids. CRISPR-Cas9–mediated JUN deletion in human HTS fibroblasts combined with epigenomic and transcriptomic analysis of both human and mouse HTS fibroblasts revealed that JUN initiates fibrosis by regulating CD36. Blocking CD36 with salvianolic acid B or CD36 knockout model counteracted JUN-mediated fibrosis efficacy in both human fibroblasts and mouse wounds. In summary, JUN is a critical regulator of pathological skin scarring, and targeting its downstream effector CD36 may represent a therapeutic strategy against scarring.

Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

Purpose: Skin fibrosis is the end result of injury in human skin. In the US alone >100 million new scars are formed every year, and with no therapy able to prevent or reverse skin fibrosis the medico-economic burden is enormous. Excessive fibrosis, as seen in hypertrophic scarring (HTS), can lead to devastating disfigurement and permanent functional loss. Incomplete understanding of the key pathogenic mechanisms driving pathological skin fibrosis has significantly hindered development of effective treatment strategies. We recently identified JUN, the AP-1 transcription factor, as a key driver of global tissue fibrosis. Here we investigate the role of JUN in skin scarring. Methods: Primary cultures of human dermal fibroblasts (HDF) were derived from HTS and unwounded skin. JUN expression was deleted using CRISPR/Cas9, and the downstream genetic/epigenetic consequences were assessed by RNA-sequencing (RNAseq), the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATACseq), and gene ontology pathway analysis to identify the genes mediating JUN-dependent fibrosis. HDF were treated with salvianolic acid (SAB), a CD36 antagonist, to determine the in vitro effects on HDF proliferation, apoptosis, and production of collagen and TGFβ. The in vivo effects of CD36 antagonism were explored using a novel inducible mouse model of HTS. Dorsal excisional stented wounds were created in JUN (c-JuntetO R26M2rtTA) mice, and either SAB (1mg/ml) or PBS (control) was administred via intraparietal injection every 24h for 14 days. Wounds were compared macroscopically for scar morphology and re-epithelialization rates. On day-14, wounds were harvested and compared for histological fibrosis as well as for the composition fibroblast subpopulations and immune cells by flow cytometry. PCRs were conducted on genes upregulated in JUN fibroblasts to investigate the mechanism by which CD36 antagonism lead to decreased scarring. Results: JUN deletion significantly altered HDF gene expression and chromatin accessibility, affecting genes involved in key fibroproliferative pathways (e.g. PI3K/AKT/mTOR, PPARγ, ECM). Targeted exploration of the 100 candidate genes with closing epigenetic landscapes and reduced gene expression following JUN knock-out (KO) in HTS-HDF implicated CD36 as a surface mediating JUN-dependent fibrosis and proliferative pathways. Immunofluorescence showed close association between JUN and CD36 proteins in HDF. In vitro CD36 antagonism using SAB decreased fibroblast proliferation, increased apoptosis, and decreased production of collagen and TGFβ.. In vivo, SAB treatment did not alter wound closure rate, but significantly prevented the development of HTS; SAB-treated wounds were less raised, less pigmented, had thinner dermal layers, and more ordered collagen fiber networks. At a cellular level, SAB-treated JUN wounds had fewer immune cells and fewer profibrotic reticular fibroblasts. JUN lipofibroblasts exhibited increased expression of genes in the PPARγ pathway (PLIN1, FABP4, FABP5) linking CD36 signaling to adipocyte differentiation. Conclusions: JUN drives pathological skin fibrosis following wounding, and CD36 antagonism with SAB can be used to inhibit JUN-dependent fibrotic pathways within key fibroblast subpopulations and decrease pathological scarring.

PURPOSE: Peripheral nerve injury remains a significant public health issue. Traumatic nerve injuries often necessitate surgical repair with nerve grafts. While autologous nerve grafts are the clinical standard, acellular nerve allograft (ANAs) have been increasingly used. ANAs are prepared from nerve obtained from deceased donors treated with detergents to remove cellular debris and antigenic components. While it has the advantage of being available off-the-shelf, its ability to promote axon regeneration across a long nerve gap is limited. In this study, we evaluate why nerve regeneration across long ANAs is limited.

PURPOSE: Clinical evaluation of dermal fibroses relies on histopathological analysis, which is inherently observer-dependent. Visual analysis is subjective and may preclude detection of subtle phenotypic changes in early-stage or less-severe disease. We present an image processing algorithm which enables objective quantification of multiple parameters of connective tissue architecture. We then classify histologic specimens by their respective dermal fibrotic pathologies, solely using machine learning analysis of their collagen networks. METHODS: Ninety-five human specimens were obtained from the following diagnoses: normal skin, scar, striae distensae (stretch marks), hypertrophic scar, keloid, and scleroderma. Mouse dorsal skin and scar specimens were also obtained. Formalin-fixed, paraffin-embedded histologic specimens were stained with Picrosirius-Red, imaged by polarization microscopy, and analyzed using our image processing algorithm in Matlab 2017a. In brief, this algorithm employs color deconvolution, adaptive filtering, and skeletonization of individual collagen fibers followed by quantification of parameters such as fiber length, branching, and randomness. A neural network was trained on connective tissue parameters (using 70% of images), validated (15% of images), and finally tested (15% of images) on histological images of human specimens. RESULTS: Using our image processing algorithm, 26 connective tissue parameters were identified and quantified. To validate the algorithm, mouse unwounded skin and scar specimens were compared. Using unsupervised hierarchical clustering, these specimens clustered by specimen type (normal skin vs scar) based on four clusters of fiber parameters. The algorithm was then applied to human specimens (unwounded skin, striae distensae, "normal" scars, hypertrophic scars, and keloid). These human specimens were differentiated by five parameter clusters due to the larger degree of variation in connective tissue architecture. The trained neural network classified pathologies with an overall accuracy of 86% (ROC curves > 95% for all specimens), demonstrating high sensitivity and specificity. The neural network also differentiated normal human skin from preclinical scleroderma with a 91% overall accuracy (ROC curves > 95%), demonstrating that our algorithm detected early-stage disease prior to the onset of clinical symptoms. CONCLUSIONS: We present an automated machine learning analysis pipeline for objective characterization of dermal collagen networks. Using a trained neural network, we classify human fibrosis specimens into disease categories based on quantitative analysis of their connective tissue properties alone. The ability to objectively characterize dermal fibroses and to detect preclinical disease has significant implications for clinical diagnosis and management as well as basic research. We intend to expand the use of this technology to fibroses in both skin and other organs, with the goal of establishing a standardized approach for histologic analysis of fibrosis.

Borrelli, Mimi R. MBBS; Garcia, Julia T. MS; Moore, Alessandra L. MD; Patel, Ronak A.; Mascharak, Shamik; Duoto, Bryan MA; Cui, Lu MS; Wan, Derrick C. MD, FACS; Wernig, Gerlinde MD; Longaker, Michael T. MD, MBA, FACS Author Information

PURPOSE: Skin fibrosis and scarring can result in devastating disfigurement and permanent functional loss. Currently, there are no treatment modalities able to prevent or reverse this fibrotic process, and scars and their consequences result in an enormous medico-economic burden. Thus, it is of paramount importance to better understand the key pathogenic mechanisms driving this pathological fibrotic process. We recently described a mouse model in which overexpression of JUN, an AP-1 transcription factor, can be induced to produce global tissue fibrosis. Here, we explored the effects of JUN overexpression in skin scarring and fibrosis and the ability of our mouse to model hypertrophic scarring and excessive skin fibrosis in response to wounding. METHODS: Stented excisional dorsal wounds were created in transgenic JUN (c-JuntetO R26-M2rtTA) and Rosa (Rosa26-rtTA) control mice. Doxycycline (2mg/ml) was used to induce JUN overexpression in the wound beds on the day of surgery (POD 0) and every other day until the wounds healed (postoperative day 14, POD 14). On alternate days throughout the healing response, wounds were harvested and analyzed histologically for thickness and collagen deposition, and by fluorescent activated cell sorting (FACS) to compare the relative percentages of fibroblast subpopulations. To study the role of JUN in human scars, dermal fibroblasts were isolated from hypertrophic scars (HTS) and healthy control skin (NS) and transduced to knock out JUN using a CRISPR-Cas 9 method. Proliferation and apoptosis were compared in the knock-out (KO) and non-KO human dermal fibroblasts. RESULTS: The wounds of JUN mice healed at a significantly accelerated rate between POD5 and POD14 (*p<0.05). Compared to the wounds of Rosa control mice, the scars of JUN mice on POD14 were significantly thicker, and although collagen content was not different, it was more disordered on Hematoxylin and Eosin staining, and brighter and more branched upon computational assessment of Picrosirius stained wounds (*p<0.05). JUN overexpression resulted in a significant expansion of reticular fibroblasts at the expense of lipofibroblasts, evident on POD 7 (*p<0.05). Translating these results to human scars; JUN CRISPR-Cas 9 deletion increased apoptosis and decreased proliferation of primary cultures of HTS and NS fibroblasts. CONCLUSION: JUN overexpression increases the fibrotic cutaneous wound healing response by significantly expanding reticular dermal fibroblasts at the expense of the dermal lipofibroblasts. In addition, assessment of HTS and NS fibroblasts isolated from human skin indicate that JUN also mediates the fibrotic response in human disease by inhibiting apoptosis and driving proliferation of the dermal fibroblast subsets. Thus, our novel inducible JUN mouse model can be used to explore the mechanisms driving HTS and other pathological skin fibrosis and facilitate targeted identification of new treatments.

PURPOSE: Skin scarring poses a significant medical burden for tens of millions of patients every year. Recently, Engrailed1-positive fibroblasts (EPFs) were shown to be responsible for the majority of scarring on the dorsal skin after embryonic day (e)18.5 in mice. However, comparatively little is known about the postnatal function of Engrailed1-negative fibroblasts (ENFs), which are present in all layers of the dermis and are putatively non-scarring. We sought to characterize the ENF lineage and assess if ENF-mediated wound healing leads to more ordered repair of skin with regeneration of dermal appendages. METHODS: Experiments were performed in En1Cre;R26mTmG(En1mTmG) and En1Cre;Ai6 (En1Ai6) mice, with Engrailed1-positive cells expressing GFP and Engrailed1-negative cells expressing RFP or no reporter, respectively. Postnatal (p) 30 mice underwent dorsal excisional wounding, and healed wound histology was assessed at 2 or 4 weeks. For mechanotransduction inhibition, the wound bed was injected with 30 uL of 50 uM Verteporfin in PBS. Engrailed1-negative fibroblasts were isolated from whole dorsal p1 skin using fluorescence-activated cell sorting (FACS). In vivo behavior of ENFs was assessed by injecting FACS-sorted ENFs into p30 mouse dorsal skin 2 days prior to wounding. Transcriptomic analysis of ENFs and EPFs was performed using RNA-sequencing of plated cells. RESULTS: FACS-isolated ENFs from p1 mice activate Engrailed1 after 7 days of culture on plastic. However, ENFs do not activate Engrailed1 in vitro after inhibition of stiffness sensing (ROCK inhibitor Y-27632) or culture in soft three-dimensional collagen hydrogels, suggesting a mechanotransduction-mediated mechanism for postnatal Engrailed1 expression. Postnatal ENFs transplanted into dorsal skin also show activation of Engrailed1 after wounding. Transcriptomic analysis by RNA-sequencing reveals that postnatal ENF to EPF transition is accompanied by expression of genes related to fibrosis (e.g., WNT/TGFb) and mechanotransduction signaling, including several target genes of Yes-associated protein (YAP). Accordingly, wounds treated with a single administration of YAP inhibitor Verteporfin yield scars with markedly fewer EPFs, reduced fibrosis, and greater ENF presence at 2 weeks. After 4 weeks, Verteporfin-treated wounds show sustained presence of ENFs, as well as regeneration of dermal appendages. CONCLUSIONS: Postnatal ENFs activate Engrailed1 in vitro by canonical mechanotransduction signaling and take on a fibrotic phenotype; a similar process occurs in the in vivo wound environment. We demonstrate that inhibition of YAP signaling promotes ENF-mediated wound healing with reduced fibrosis and regeneration of secondary elements. Our findings suggest that ENFs may play a critical role in scarring by activating Engrailed1 in response to mechanical cues within the wound bed. Considering that the Engrailed1-negative lineage represents several distinct subpopulations of cells, we aim to assess postnatal Engrailed1 activation in the papillary, reticular, and hypodermal layers to identify a specific ENF population that contributes to scarring.

Des Jardins-Park, Heather E. AB; Mascharak, Shamik BS; Moore, Alessandra L. MD; Duoto, Bryan A. BS; Longaker, Michael T. MD, MBA, FACS Author Information

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). 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). 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. H.E. desJardins-Park: None. A.L. Moore: None. M.P. Murphy: None. D. Irizarry: None. B. Duoto: None. D. Foster: None. R.E. Jones: None. S. Mascharak: None. L. Barnes: None. C. Marshall: None. G. Wernig: None. M.T. Longaker: None.

Moore, Alessandra L. MD; Murphy, Matthew P. MB, BCh, BAO, MRCSI; Irizarry, Dre M. MD; Des Jardins-Park, Heather E. AB; Duoto, Bryan A.; Mascharak, Shamik; Foster, Deshka S. MD; Jones, Ruth Ellen MD; Wernig, Gerlinde MD; Longaker, Michael T. MD, MBA, FACS Author Information

Moore, Alessandra L. MD; Marshall, Clement D. MD; Des Jardins-Park, Heather E. AB; Duoto, Bryan A. BS; Mascharak, Shamik BS; Barnes, Leandra A. BA; Ransom, Ryan C. BA; Hu, Michael S. MD, MPH, MS; Lorenz, Peter H. MD, FACS; Longaker, Michael T. MD, MBA, FACS Author Information

Moore, Alessandra L. MD; Duoto, Bryan A.; Des Jardins-Park, Heather E. AB; Mascharak, Shamik; Wernig, Gerlinde MD; Longaker, Michael T. MD, MBA, FACS Author Information

PURPOSE: Early fetal wounds heal by regeneration; an important but poorly understood phenomena. Understanding the fetal wound healing mechanism could achieve scarless healing in human patients. In 2015, our group proved that Engrailed-1 (En1) positive fibroblasts (EPFs) are responsible for all scar tissue deposition in adult and postnatal mice. Additionally, these cells appear around the time of phenotypic change from scarless (embryonic day 0–16) to scarring (embryonic day 18+) healing. Given Engrailed-1 positive fibroblasts (EPFs) and Engrailed-1 negative fibroblasts (ENFs) share a common precursor cell, we hypothesized that the EPFs accumulate epigenetic changes over time that result in their phenotypic transition and result in a permanent cellular phenotype. METHODS: Dorsal dermal fibroblasts from En1Cre/-; Rosa26mTmG/- mice were isolated at embryonic day (e)10, e16, e18, post-natal day (p)1, p30, and p30 wounded skin. EPFs and ENFs from these time points were sorted using Fluorescence-Activated Cell Sorting (FACS) and analyzed using the Assay for Transposase-Accessible Chromatin Using Sequencing (ATAC-seq). The data was then compared by time course analysis to generate a list of genes involved in fibrosis and to identify patterns of epigenetic change. E16 EPFs were then isolated by FACS and transplanted into a p1 host, and vice versa, to establish their intrinsic phenotype in vivo. Tissue was harvested 48 hours after transplant and analyzed using immunofluorescence to identify phenotypic differences based on cell type and microenvironment. RESULTS: E10 fibroblasts are of a single lineage and were excluded from analysis. Time course analysis of e16-p30 EPFs and ENFs shows appropriate correlation between samples. Principle Component Analysis shows p30 EPFs and ENFs being the most dissimilar, and EPFs from p30 are most like e16 EPFs. Most epigenetic changes in the EPF lineage occur in embryonic development between e16 and e18, with fewer epigenetic changes occurring postnatally (significant peaks = 173 vs. 336). These epigenetic changes are correlated with open promoter sequences at e18, which then by p1 appear to be closed. In contrast, the ENF lineage accumulates increasing epigenetic changes from e18 and p30 (significant peaks = 88 vs. 545). Lastly, reciprocal transplantation of e16 fibroblasts into a p1 host and vice versa reveal a significant difference in collagen overlap (2.13% versus 24.18%) and morphologic changes suggestive of quiescence versus reactivity. CONCLUSIONS: Our data suggest that fibroblast phenotype is highly cell intrinsic and based on the accumulation of epigenetic change. Epigenetic change correlates with the transition in healing phenotype, and localizes to promoter sequences. By using the CRISPR-Cas9 system in future experiments, we will delineate which genes associated with e18 open promoters are the master regulators of fibrosis. Intervention at these genes may allow for scarless healing in adults. A. Moore: None. U. Litzenburger: None. C. Marshall: None. R.C. Ransom: None. H. desJardins-Parks: None. B. Duoto: None. S. Mascharak: None. L. Barnes: None. E. Brett: None. M. Hu: None. H. Chang: None. H.P. Lorenz: None. M.T. Longaker: None.

PURPOSE: In reconstructive surgery, large areas of tissue loss represent a major surgical obstacle. Where split-thickness skin grafting and flap transfers fail, cell-based treatments represent a promising therapeutic option. Currently, cell therapies are limited to transplants from non-autologous donors, or expanded isolated skin components (e.g., keratinocytes only). However, literature suggests that keratinocytes and fibroblasts act synergistically to restore functional tissue.1 Typical culture conditions poorly mimic in vivo conditions, and skin cells change dramatically after plating.2 Thus, there is a demand for techniques to expand multiple autologous cell types without fundamentally altering cell behavior. Here, we describe methods for the ex vivo culture of skin cells that allow for efficient expansion while maintaining in vivo cell characteristics. METHODS: Adult mouse skin was harvested and sterilized using gradated iodine solutions. Tissue was chopped with sterile scissors followed by digestion with 0.5 mg/mL LiberaseTM DL (Roche). Cells were grown in DMEM/F12 with 10% fetal bovine serum and 1% penicillin-streptomycin, on polystyrene coated with 0.1% gelatin (EmbryoMax) or in 3D collagen hydrogels of varying stiffness. Morphology was assessed via imaging and analysis using Photoshop CS6 (Adobe). Relative cell populations were quantified using fluorescence-activated cell sorting (FACS). Isolation of Engrailed-positive fibroblasts (EPFs), the dermal fibroblast population responsible for wound healing (collagen deposition), was achieved by FACS of cells from En1Cre;R26mTmG mice. RESULTS: Skin cells grown via whole organ culture on gelatin-coated polystyrene had no significant change in resident cell population density over multiple passages (2–4% fibroblasts; of non-fibroblasts, 50–60% blood cells; remainder keratinocytes; P>0.05). Upon isolation from whole organ culture, fibroblasts of a single population (EPFs) demonstrated expansion by over 20-fold in two passages. With traditional culture methods, fibroblasts demonstrate increased cell size over repeated passages; in contrast, these phenotypic shifts in EPFs were rescued by culturing in 3D hydrogels or on gelatin-coated polystyrene. Specifically, EPFs grown on gelatin-coated polystyrene demonstrated no significant change in cell size from passage (P)1 to P3 (average fold change=0.879, n=3 biological replicates, all P>0.05). CONCLUSION: By removing many of the artificial selection pressures that cells experience in culture, we accomplished efficient ex vivo expansion of in vivo-like skin cells. Specifically, by employing whole organ culture rather than culturing cells in isolation, nonselective media, and 3D hydrogels to mimic in vivo mechanical tensions, cells retained their in vivo morphology and population densities. Autologous cell-based therapies hold increasing promise for complex reconstructive surgery, and our results signify a therapeutically relevant advancement that may enable improved cosmesis and functionality of transplanted skin organs. With similar expansion of human skin, a 4mm punch biopsy alone could yield the equivalent of over 250 mm2 of skin for transplantation. In the future, we will verify our technique using epigenetic studies and machine learning-based assessment of cell morphology, and will use our technique to create 3D organoids capable of transplantation. References: 1. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol. 2007;127(5):998–1008. 2. Walmsley GG, Rinkevich Y, Hu MS. Live fibroblast harvest reveals surface marker shift in vitro. Tissue Eng Part C Methods. 2015;21(3):314–21.

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.