The interplay of fibroblasts, the extracellular matrix, and inflammation in scar formation

Various forms of fibrosis, comprising tissue thickening and scarring, are involved in 40% of deaths across the world. Since the discovery of scarless functional healing in fetuses prior to a certain stage of development, scientists have attempted to replicate scarless wound healing in adults with little success. While the extracellular matrix (ECM), fibroblasts, and inflammatory mediators have been historically investigated as separate branches of biology, it has become increasingly necessary to consider them as parts of a complex and tightly regulated system that becomes dysregulated in fibrosis. With this new paradigm, revisiting fetal scarless wound healing provides a unique opportunity to better understand how this highly regulated system operates mechanistically. In the following review, we navigate the four stages of wound healing (hemostasis, inflammation, repair, and remodeling) against the backdrop of adult versus fetal wound healing, while also exploring the relationships between the ECM, effector cells, and signaling molecules. We conclude by singling out recent findings that offer promising leads to alter the dynamics between the ECM, fibroblasts, and inflammation to promote scarless healing. One factor that promises to be significant is fibroblast heterogeneity and how certain fibroblast subpopulations might be predisposed to scarless healing. Altogether, reconsidering fetal wound healing by examining the interplay of the various factors contributing to fibrosis provides new research directions that will hopefully help us better understand and address fibroproliferative diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis. Various forms of fibrosis, comprising tissue thickening and scarring, are involved in 40% of deaths across the world. Since the discovery of scarless functional healing in fetuses prior to a certain stage of development, scientists have attempted to replicate scarless wound healing in adults with little success. While the extracellular matrix (ECM), fibroblasts, and inflammatory mediators have been historically investigated as separate branches of biology, it has become increasingly necessary to consider them as parts of a complex and tightly regulated system that becomes dysregulated in fibrosis. With this new paradigm, revisiting fetal scarless wound healing provides a unique opportunity to better understand how this highly regulated system operates mechanistically. In the following review, we navigate the four stages of wound healing (hemostasis, inflammation, repair, and remodeling) against the backdrop of adult versus fetal wound healing, while also exploring the relationships between the ECM, effector cells, and signaling molecules. We conclude by singling out recent findings that offer promising leads to alter the dynamics between the ECM, fibroblasts, and inflammation to promote scarless healing. One factor that promises to be significant is fibroblast heterogeneity and how certain fibroblast subpopulations might be predisposed to scarless healing. Altogether, reconsidering fetal wound healing by examining the interplay of the various factors contributing to fibrosis provides new research directions that will hopefully help us better understand and address fibroproliferative diseases, such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis. Properly resolving wound healing in adults is a complex and tightly regulated process, both spatially and temporally. Sometimes, adult wound healing degenerates toward fibrosis, a process characterized by chronic inflammation, aberrant extracellular matrix (ECM) deposition, and myofibroblast accumulation. The latter develop functional characteristics typical of contractile cells and appear in the context of wound closure and contraction (1Ryan G.B. Cliff W.J. Gabbiani G. Irle C. Montandon D. Statkov P.R. Majno G. Myofibroblasts in human granulation tissue.Hum. Pathol. 1974; 5: 55-67Google Scholar). This fibroproliferative response leads to the formation of scar tissue that has increased stiffness and decreased extensibility, and that recovers little, if any, of the original function (2Clark J.A. Cheng J.C.Y. Leung K.S. Mechanical properties of normal skin and hypertrophic scars.Burns. 1996; 22: 443-446Google Scholar, 3Corr D.T. Hart D.A. Biomechanics of scar tissue and uninjured skin.Adv. Wound Care (New Rochelle). 2013; 2: 37-43Google Scholar, 4Richardson W. Clarke S. Quinn T. Holmes J. Physiological implications of myocardial scar structure.Compr. Physiol. 2015; 5: 1877-1909Google Scholar). While adult wound healing typically ends with scar formation, characterized as ex novo connective tissue with myofibroblasts and collagen fibers (5Skalli O. Gabbiani G. The biology of the myofibroblast relationship to wound contraction and fibrocontractive diseases.in: Clark R.A.F. Henson P.M. The Molecular and Cellular Biology of Wound Repair. Springer US, Boston, MA1988: 373-402Google Scholar), it has long been demonstrated that fetal would healing ends in scarless regeneration. The fetal dermis during early gestation can regenerate wounds without a scar because of the ECM being deposited in such a way that resembles the original tissue and restores functional features, such as sebaceous glands and follicles (6Lorenz H.P. Whitby D.J. Longaker M.T. Adzick N.S. Fetal wound healing. The ontogeny of scar formation in the non-human primate.Ann. Surg. 1993; 217: 391-396Google Scholar). In prenatal humans, this capability lasts until week 24 (7Longaker M.T. Whitby D.J. Adzick N.S. Crombleholme T.M. Langer J.C. Duncan B.W. Bradley S.M. Stern R. Ferguson M.W.J. Harrison M.R. Studies in fetal wound healing VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation.J. Pediatr. Surg. 1990; 25: 63-69Google Scholar) and until day 18.5 in rats (8Ihara S. Motobayashi Y. Nagao E. Kistler A. Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage.Development. 1990; 110: 671-680Google Scholar). While this difference in healing outcomes has been known for quite some time, recent discoveries and advances warrant revisiting adult and fetal wound healing conditions. Better understanding of the complex interplay between the ECM, fibroblasts, and inflammation might be leveraged to promote tissue regeneration and avoid, or perhaps even reverse, fibrosis. The wound healing process generally involves four different phases occupying various timescales after injury occurs. In chronological order, these stages are hemostasis, inflammation, repair, and remodeling (Fig. 1). Hemostasis starts following initial injury, as platelet binding to the newly damaged ECM causes them to release their granules (9Li Z. Delaney M.K. O'Brien K.A. Du X. Signaling during platelet adhesion and activation.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2341-2349Google Scholar). These are made up of several molecules, such as clotting factors, cytokines, and ECM proteins. We will explore the differences between adult and fetal wound healing during the hemostasis phase, with particular focus on inflammatory mediators, fibroblast phenotypes, and ECM components in the early provisional matrix. This matrix is made up by proteins in plasma and released from the platelet granules, with fibrin and fibronectin (Fn) representing the lion's share (10Clark R.A. Lanigan J.M. DellaPelle P. Manseau E. Dvorak H.F. Colvin R.B. Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization.J. Invest. Dermatol. 1982; 79: 264-269Google Scholar, 11Magnusson M.K. Mosher D.F. Fibronectin: Structure, assembly, and cardiovascular implications.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1363-1370Google Scholar, 12Barker T.H. Engler A.J. The provisional matrix: Setting the stage for tissue repair outcomes.Matrix Biol. 2017; 60–61: 1-4Google Scholar). The formation of a stable clot and the initiation of leukocyte migration signify the start of the second phase of wound healing, inflammation. Inflammation is initiated by many factors: ATP and nucleic acids released from cells after their injury (13Bours M.J.L. Swennen E.L.R. Di Virgilio F. Cronstein B.N. Dagnelie P.C. Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation.Pharmacol. Ther. 2006; 112: 358-404Google Scholar), proinflammatory cytokines from the platelet granules (9Li Z. Delaney M.K. O'Brien K.A. Du X. Signaling during platelet adhesion and activation.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2341-2349Google Scholar, 14Martínez C.E. Smith P.C. Palma Alvarado V.A. The influence of platelet-derived products on angiogenesis and tissue repair: A concise update.Front. Physiol. 2015; 6: 290Google Scholar), and immune cells recruited by the fibrin matrix (15Güç E. Briquez P.S. Foretay D. Fankhauser M.A. Hubbell J.A. Kilarski W.W. Swartz M.A. Local induction of lymphangiogenesis with engineered fibrin-binding VEGF-C promotes wound healing by increasing immune cell trafficking and matrix remodeling.Biomaterials. 2017; 131: 160-175Google Scholar). Approximately 2 h postinjury, inflammatory signals released by platelets and tissue-resident immune cells, such as macrophages, dendritic cells (DCs), and mast cells, recruit neutrophils to the site of injury (16Henderson R.B. Hobbs J.A.R. Mathies M. Hogg N. Rapid recruitment of inflammatory monocytes is independent of neutrophil migration.Blood. 2003; 102: 328-335Google Scholar). Tissue-resident immune cells are innate immune cells that have matured in the peripheral tissue and remain there for the duration of their lifetime. Tissue-resident immune cells secrete various chemokines, which prompt neutrophil recruitment and chemotaxis, that is, migration toward the wound site (Table 1) (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar). Typically in wound healing, the recruitment of immune cells and subsequent inflammatory cytokine and chemokine expression is spatially and temporally limited. On the other hand, fibrotic disorders across tissue contexts demonstrate chronic activation of tissue-resident and tissue-recruited immune cells (18Mori R. Shaw T.J. Martin P. Molecular mechanisms linking wound inflammation and fibrosis: Knockdown of osteopontin leads to rapid repair and reduced scarring.J. Exp. Med. 2008; 205: 43-51Google Scholar, 19Murray P.J. Wynn T.A. Protective and pathogenic functions of macrophage subsets.Nat. Rev. Immunol. 2011; 11: 723-737Google Scholar). Understanding how the inflammatory phase differs between adult and fetal wound healing will advance two goals: first, identifying inflammatory mediators or cell populations that could be therapeutic targets and second, understanding how inflammation informs fibroblast and ECM biology.Table 1Relevant cytokinesCytokine/chemokineReceptorFunctionInvolved inIL-1⍺/βIL-1RI & IL-1RAPEarly inflammatory marker and is a pyrogen (292Evavold C.L. Ruan J. Tan Y. Xia S. Wu H. Kagan J.C. The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages.Immunity. 2018; 48: 35-44.e6Google Scholar)Fibrosis (293Wilson M.S. Madala S.K. Ramalingam T.R. Gochuico B.R. Rosas I.O. Cheever A.W. Wynn T.A. Bleomycin and IL-1β–mediated pulmonary fibrosis is IL-17A dependent.J. Exp. Med. 2010; 207: 535-552Google Scholar)IL-4IL-4R⍺ & ɣc chain or IL-4R⍺ & IL-13R⍺1B-cell activation, Th2 differentiation, macrophage polarization (145Woytschak J. Keller N. Krieg C. Impellizzieri D. Thompson R.W. Wynn T.A. Zinkernagel A.S. Boyman O. Type 2 interleukin-4 receptor signaling in neutrophils antagonizes their expansion and migration during infection and inflammation.Immunity. 2016; 45: 172-184Google Scholar, 294Stone K.D. Prussin C. Metcalfe D.D. IgE, mast cells, basophils, and eosinophils.J. Allergy Clin. Immunol. 2010; 125: S73-S80Google Scholar)Tissue regeneration, asthma, T-cell immunity (295Spiller K.L. Nassiri S. Witherel C.E. Anfang R.R. Ng J. Nakazawa K.R. Yu T. Vunjak-Novakovic G. Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds.Biomaterials. 2015; 37: 194-207Google Scholar, 296Manson M.L. Säfholm J. James A. Johnsson A.-K. Bergman P. Al-Ameri M. Orre A.-C. Kärrman-Mårdh C. Dahlén S.-E. Adner M. IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways.J. Allergy Clin. Immunol. 2020; 145: 808-817.e2Google Scholar, 297Fort M.M. Cheung J. Yen D. Li J. Zurawski S.M. Lo S. Menon S. Clifford T. Hunte B. Lesley R. Muchamuel T. Hurst S.D. Zurawski G. Leach M.W. Gorman D.M. et al.IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo.Immunity. 2001; 15: 985-995Google Scholar)IL-6IL-6R & gp130Early inflammatory marker and is a pyrogen (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar)Broad involvement with inflammatory pathologies (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar)IL-8CXCR1/2Neutrophil recruitment (298Heukels P. Moor C.C. von der Thüsen J.H. Wijsenbeek M.S. Kool M. Inflammation and immunity in IPF pathogenesis and treatment.Respir. Med. 2019; 147: 79-91Google Scholar)Histological inflammation (298Heukels P. Moor C.C. von der Thüsen J.H. Wijsenbeek M.S. Kool M. Inflammation and immunity in IPF pathogenesis and treatment.Respir. Med. 2019; 147: 79-91Google Scholar)IL-10IL-10R⍺/βAnti-inflammatory processes (116Qayum A.A. Paranjape A. Abebayehu D. Kolawole E.M. Haque T.T. McLeod J.J.A. Spence A.J. Caslin H.L. Taruselli M.T. Chumanevich A.P. Baker B. Oskeritzian C.A. Ryan J.J. IL-10–induced miR-155 targets SOCS1 to enhance IgE-mediated mast cell function.J. Immunol. 2016; 196: 4457-4467Google Scholar)Scarless fetal healing (299Liechty K.W. Kim H.B. Adzick N.S. Crombleholme T.M. Fetal wound repair results in scar formation in interleukin-10–deficient mice in a syngeneic murine model of scarless fetal wound repair.J. Pediatr. Surg. 2000; 35: 866-873Google Scholar)IL-12IL-12Rβ1/2Th1 differentiation (300Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat. Rev. Immunol. 2003; 3: 133-146Google Scholar)T-cell immunity (300Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat. Rev. Immunol. 2003; 3: 133-146Google Scholar)IL-13IL-4R⍺ & IL-13R⍺1 or IL-13R⍺2Th2 differentiation (294Stone K.D. Prussin C. Metcalfe D.D. IgE, mast cells, basophils, and eosinophils.J. Allergy Clin. Immunol. 2010; 125: S73-S80Google Scholar, 301Wynn T.A. Vannella K.M. Macrophages in tissue repair, regeneration, and fibrosis.Immunity. 2016; 44: 450-462Google Scholar)Tissue regeneration, asthma, T-cell immunity (295Spiller K.L. Nassiri S. Witherel C.E. Anfang R.R. Ng J. Nakazawa K.R. Yu T. Vunjak-Novakovic G. Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds.Biomaterials. 2015; 37: 194-207Google Scholar, 296Manson M.L. Säfholm J. James A. Johnsson A.-K. Bergman P. Al-Ameri M. Orre A.-C. Kärrman-Mårdh C. Dahlén S.-E. Adner M. IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways.J. Allergy Clin. Immunol. 2020; 145: 808-817.e2Google Scholar, 297Fort M.M. Cheung J. Yen D. Li J. Zurawski S.M. Lo S. Menon S. Clifford T. Hunte B. Lesley R. Muchamuel T. Hurst S.D. Zurawski G. Leach M.W. Gorman D.M. et al.IL-25 induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo.Immunity. 2001; 15: 985-995Google Scholar, 302Lee A.-J. Ro M. Cho K.-J. Kim J.-H. Lipopolysaccharide/TLR4 stimulates IL-13 production through a MyD88-BLT2–linked cascade in mast cells, potentially contributing to the allergic response.J. Immunol. 2017; 199: 409-417Google Scholar)IL-33ST2 & IL-1RAPAlarmin/DAMP (303Abebayehu D. Spence A.J. Qayum A.A. Taruselli M.T. McLeod J.J.A. Caslin H.L. Chumanevich A.P. Kolawole E.M. Paranjape A. Baker B. Ndaw V.S. Barnstein B.O. Oskeritzian C.A. Sell S.A. Ryan J.J. Lactic acid suppresses IL-33–mediated mast cell inflammatory responses via hypoxia-inducible factor-1α–dependent miR-155 suppression.J. Immunol. 2016; 197: 2909-2917Google Scholar)Inflammation, asthma, Macrophage polarization (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar, 304Vannella K.M. Ramalingam T.R. Borthwick L.A. Barron L. Hart K.M. Thompson R.W. Kindrachuk K.N. Cheever A.W. White S. Budelsky A.L. Comeau M.R. Smith D.E. Wynn T.A. Combinatorial targeting of TSLP, IL-25, and IL-33 in type 2 cytokine–driven inflammation and fibrosis.Sci. Transl. Med. 2016; 8337ra65Google Scholar)TNF⍺TNFR1 or TNFR2Early inflammatory marker and is a pyrogen (305Luheshi G.N. Stefferl A. Turnbull A.V. Dascombe M.J. Brouwer S. Hopkins S.J. Rothwell N.J. Febrile response to tissue inflammation involves both peripheral and brain IL-1 and TNF-alpha in the rat.Am. J. Physiol. 1997; 272: R862-R868Google Scholar)Broad involvement with inflammatory pathologies (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar)IFNɣIFNɣR1/2Macrophage polarization and facilitates antigen presentation (306Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: Therapeutic translation for fibrotic disease.Nat. Med. 2012; 18: 1028-1040Google Scholar, 307Frei K. Siepl C. Groscurth P. Bodmer S. Schwerdel C. Fontana A. Antigen presentation and tumor cytotoxicity by interferon-γ-treated microglial cells.Eur. J. Immunol. 1987; 17: 1271-1278Google Scholar)Antigen-specific immunity and viral immunity (307Frei K. Siepl C. Groscurth P. Bodmer S. Schwerdel C. Fontana A. Antigen presentation and tumor cytotoxicity by interferon-γ-treated microglial cells.Eur. J. Immunol. 1987; 17: 1271-1278Google Scholar, 308Kajita A.i. Morizane S. Takiguchi T. Yamamoto T. Yamada M. Iwatsuki K. Interferon-gamma enhances TLR3 expression and anti-viral activity in keratinocytes.J. Invest. Dermatol. 2015; 135: 2005-2011Google Scholar)MCP-1/CCL2CCR2Immune cell recruitment (306Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: Therapeutic translation for fibrotic disease.Nat. Med. 2012; 18: 1028-1040Google Scholar)Tissue repair, infection clearance, and inflammation (306Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: Therapeutic translation for fibrotic disease.Nat. Med. 2012; 18: 1028-1040Google Scholar)MIP-1⍺/CCL3CCR1/5Immune cell recruitment (309Suthahar N. Meijers W.C. Silljé H.H.W. de Boer R.A. From inflammation to fibrosis—molecular and cellular mechanisms of myocardial tissue remodelling and perspectives on differential treatment opportunities.Curr. Heart Fail. Rep. 2017; 14: 235-250Google Scholar)Tissue repair, infection clearance, and inflammation (309Suthahar N. Meijers W.C. Silljé H.H.W. de Boer R.A. From inflammation to fibrosis—molecular and cellular mechanisms of myocardial tissue remodelling and perspectives on differential treatment opportunities.Curr. Heart Fail. Rep. 2017; 14: 235-250Google Scholar)G-CSFG-CSFRHematopoiesis (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar)Neutrophil maturation and proliferation (17Galli S.J. Borregaard N. Wynn T.A. Phenotypic and functional plasticity of cells of innate immunity: Macrophages, mast cells and neutrophils.Nat. Immunol. 2011; 12: 1035-1044Google Scholar)CXCL1CXCR2Immune cell recruitment (310De Filippo K. Dudeck A. Hasenberg M. Nye E. van Rooijen N. Hartmann K. Gunzer M. Roers A. Hogg N. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.Blood. 2013; 121: 4930-4937Google Scholar)Inflammation and tissue repair (310De Filippo K. Dudeck A. Hasenberg M. Nye E. van Rooijen N. Hartmann K. Gunzer M. Roers A. Hogg N. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.Blood. 2013; 121: 4930-4937Google Scholar)CXCL2CXCR2Immune cell recruitment (310De Filippo K. Dudeck A. Hasenberg M. Nye E. van Rooijen N. Hartmann K. Gunzer M. Roers A. Hogg N. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.Blood. 2013; 121: 4930-4937Google Scholar)Inflammation and tissue repair (310De Filippo K. Dudeck A. Hasenberg M. Nye E. van Rooijen N. Hartmann K. Gunzer M. Roers A. Hogg N. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.Blood. 2013; 121: 4930-4937Google Scholar)Abbreviation: MCP-1, monocyte chemoattractant protein-1. Open table in a new tab Abbreviation: MCP-1, monocyte chemoattractant protein-1. The third phase of wound healing is repair, which is characterized by the early provisional matrix being used to produce a more mature late provisional matrix based on coordination between inflammatory mediators, fibroblasts, and ECM components that now include Fns, proteoglycans, and collagens (12Barker T.H. Engler A.J. The provisional matrix: Setting the stage for tissue repair outcomes.Matrix Biol. 2017; 60–61: 1-4Google Scholar, 20Sottile J. Hocking D.C. Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions.Mol. Biol. Cell. 2002; 13: 3546-3559Google Scholar). During primary repair in dermis, the tissue most studied by the field, skin cells known as keratinocytes move from the basal lamina through the provisional matrix. Upon reaching the wound location, keratinocytes undergo a proliferative burst that is stopped only by contact inhibition once the wound is closed. Secondary repair in skin occurs when re-epithelialization is not sufficient (21Gurtner G.C. Werner S. Barrandon Y. Longaker M.T. Wound repair and regeneration.Nature. 2008; 453: 314-321Google Scholar, 22Reinke J.M. Sorg H. Wound repair and regeneration.Eur. Surg. Res. 2012; 49: 35-43Google Scholar). In several tissues including dermis, fibroblasts migrate into the wound. First, they secrete Fn, but after transforming growth factor beta (TGF-β, a pleiotropic cytokine with several isoforms) signaling from macrophages (23Assoian R.K. Fleurdelys B.E. Stevenson H.C. Miller P.J. Madtes D.K. Raines E.W. Ross R. Sporn M.B. Expression and secretion of type beta transforming growth factor by activated human macrophages.Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6020-6024Google Scholar), they enter an activated state named myofibroblasts, characterized by the expression of alpha-smooth muscle actin (αSMA) (24Tomasek J.J. McRae J. Owens G.K. Haaksma C.J. Regulation of α-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-β1 control element.Am. J. Pathol. 2005; 166: 1343-1351Google Scholar) and collagen secretion. Myofibroblasts secrete more ECM (mainly collagen and Fn) and contract the wound bed. However, unchecked myofibroblasts can lead to fibrosis. Afterward, keratinocytes migrate over the newly produced ECM and release vascular endothelial growth factor (VEGF) (25Detmar M. Yeo K.-T. Nagy J.A. van de Water L. Brown L.F. Berse B. Elicker B.M. Ledbetter S. Dvorak H.F. Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells.J. Invest. Dermatol. 1995; 105: 44-50Google Scholar). The ensuing angiogenesis and neovascularization, needed to support myofibroblastic presence, leads to the formation of granulation tissue. This tissue derives its name from a high density of myofibroblasts, macrophages, capillaries, and loosely organized collagen bundles (22Reinke J.M. Sorg H. Wound repair and regeneration.Eur. Surg. Res. 2012; 49: 35-43Google Scholar). While a single marker for pathological myofibroblasts has not been discovered yet, a combination of αSMA, Fn-extra domain type III A (EDA), and lack of thymocyte differentiation antigen 1 (Thy-1, a glycosylphosphatidylinositol [GPI]-anchored protein that modulates certain integrins engagement) could be employed as markers. Such a strategy would enable precise targeting of the effector cells in pathological fibrotic contexts other than skin wounds, where topical therapeutic delivery may be sufficient. We will explore how the repair phase differs between the fibrosis that occurs during adult healing and the scarless fetal wound healing scenario. We will focus on the ECM components, the integrins that they ligate, and fibroblast phenotypes that dictate tissue repair. With the exception of small dermal wounds, remodeling is the last step of wound healing. This phase, which may last weeks or months, is characterized by the crosslinking of the ECM secreted by myofibroblasts, as well as other modifications that might alter tissue remodeling and function. We will identify the differences during tissue remodeling in adult and fetal wound healing by focusing on differences in cytokine signaling to fibroblasts and enzymatic changes to the ECM. Given the overall complexity of wound healing, solving fibrotic scarring by recapitulating fetal scarless healing appears to be out of reach with a traditional single target and silver bullet drug. For example, in the indication of idiopathic pulmonary fibrosis (IPF), even the two Food and Drug Administration–approved therapies demonstrate only moderate extension of quality-adjusted life years, with nintedanib and pirfenidone adding 1.41 and 0.73 quality-adjusted life years over standard care, respectively (26Clay E. Cristeau O. Chafaie R. Pinta A. Mazaleyrat B. Cottin V. Cost-effectiveness of pirfenidone compared to all available strategies for the treatment of idiopathic pulmonary fibrosis in France.J. Mark Access Health Policy. 2019; 7: 1626171Google Scholar). We believe that studies with a more holistic approach with respect to the pathways involved will ensure the field's success in the search for scarless healing. As such, the focus on elucidating the mechanism of fibrosis across organ systems should shift toward the combinatorial effects of the major factors involved: fibroblasts, the ECM, and inflammation, respectively, the actors, stage, and setting of the saga of wound healing. These types of studies could be inspired by the fetal wound healing process, given that a fetus can regenerate wounds without scar tissue into the second trimester of human gestation. This review compares and contrasts fibrotic wound healing in adults to the ideal standard of fetal scarless healing, attempting to tease out pathways and molecules that, when dysregulated, become biomarkers or promoters of fibrosis. In doing so, we follow the chronological event of the traditional healing response, with emphasis on proteins contributing to hemostasis and the provisional matrix, the mediators of inflammation, and the phenotype of fibroblasts, the effector cells. We conclude by proposing new pathways and areas to investigate in the future. Hemostasis has two parts: primary hemostasis and secondary hemostasis, both of which aim to stop the loss of blood from the circulation (27Gale A.J. Current understanding of hemostasis.Toxicol. Pathol. 2011; 39: 273-280Google Scholar). In primary hemostasis, platelets aggregate around the site of injury to form a primary clot. In secondary hemostasis, insoluble fibrin is generated by the coagulation cascade and used to strengthen the primary clot. Understanding the mechanisms and molecules that facilitate these early parts of wound healing is critical to devise therapeutic approaches. Damage to resident cells and the surrounding ECM caused by the wound initiates the coagulation cascade as well as other pathways that reach well beyond the effort to close the wound. Damaged cells often lyse and release ATP into the extracellular space, initiating inflammation and cell-mediated immunity via P2 purinergic signaling (13Bours M.J.L. Swennen E.L.R. Di Virgilio F. Cronstein B.N. Dagnelie P.C. Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation.Pharmacol. Ther. 2006; 112: 358-404Google Scholar). At the same time, platelets from circulating plasma bind to the now exposed and damaged endothelial ECM, causing their activation and eventual primary clot formation. Platelets, considered the first responders to sites of vascular injury (9Li Z. Delaney M.K. O'Brien K.A. Du X. Signaling during platelet adhesion and activation.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 2341-2349Google Scholar), lack a nucleus; yet possess three organelles: alpha granules, dense granules, and lysosomes. Alpha granules are the key organelles for protein storage, including growth factors, coagulation factors, extracellular adhesion molecules, cytokines, proteoglycans, and chemokines (14Martínez C.E. Smith P.C. Palma Alvarado V.A. The influence of platelet-derived products on angiogenesis and tissue repair: A concise update.Front. Physiol. 2015; 6: 290Google Scholar). Upon adhesion t