Diversity of Fibroblasts and Their Roles in Wound Healing

Abstract

Wound healing disorders are a societal, clinical, and healthcare burden and understanding and treating them is a major challenge. A particularly important cell type in the wound healing processes is the fibroblast. Fibroblasts are not homogenous; however, there are diverse functional fibroblast subtypes coming from different embryonic origins and residing in dispersed anatomic locations including distinct classes of fibroblasts at various skin depths. In this review, we discuss the implications of fibroblast heterogeneity, with a focus on the fundamental physiological functions of the fibroblast subtypes that govern wound repair and clinical degrees of healing. A better understanding of these diverse functional fibroblast populations will likely lead to novel therapies to enhance wound healing and inhibit excessive scarring.

Wound healing problems present a significant burden, not only to patients but also to the healthcare system. Defects of wound healing include hard-to-heal scenarios leading to chronic nonhealing wounds or excessive scarring and fibrosis. Taking burn wounds and the resulted burn contracture into consideration, wound management is a substantial clinical, social, and economic challenge. Wound care in the United States is estimated to cost between $30 and $100 billion annually (Sen 2019), and the market for global scar treatment is expected to reach $32 billion by 2027 (Sen 2021).

Wound healing can generally be divided into four distinct but overlapping phases: an early hemostasis, and inflammation phases, followed by fibroblast-centric phases that include proliferation with reepithelialization and granulation tissue formation and connective tissue remodeling. Defects in any of these wound healing phases causes under-healing or over-healing problems. Fibroblasts, through their ability to modulate the wound cascade in various ways, are recognized as key modulators in wound healing processes.

With the advent of modern technologies, such as genetic lineage tracing, single-cell multi-omics, and live imaging, fibroblasts are now seen as a heterogeneous mixture of distinct subpopulations of cell types that perform critical and unique functions in tissue development, homeostatic maintenance, and injury repair. Understanding and clinically exploiting the diversity of fibroblastic cells holds tremendous potential for therapeutic enhancement and modulation of wound healing outcomes.

This review focuses on the heterogeneity of fibroblastic cells, primarily within skin where our resolution of fibroblasts and their functions has been extensively studied. In the first part of this review, we catalog the currently known skin fibroblast subtypes including their separate embryonic origins, their tissue-resident locations, and their lineage-specific transcriptomic features. In the second part of our review, we discuss the distinct physiological functions of these fibroblast subtypes and how distinct populations govern the diversity of the skin's response to injury to either scar-over or regenerate.

FIBROBLAST HETEROGENEITY IN SKIN DEVELOPMENT AND WOUND REPAIR

Distinct Origins and Functions of Skin Fibroblasts

The skin is home to an assortment of fibroblastic stromal cells that have unique functions in the skin, and that originate from diverse stem/progenitor sources. Fibroblasts with unique origins/functions are exemplified throughout skin development and during skin injury repair.

Through genetic lineage tracing approaches, various communities of skin fibroblasts have been shown to originate from separate embryonic lineages during development (Fig. 1A). For example, Driskell and colleagues identified two fibroblast lineages during development of the mouse back skin (Driskell et al. 2013) that come from fibroblast progenitors expressing PDGF receptor α (PDGFRα) that is switched on around embryonic day 12.5 (E12.5). From these early stromal progenitors, two distinct cellular lineages with unique differentiation trajectories are seen to emerge from E16.5 and onward. These two lineages are characterized by the expression of either Delta-like noncanonical Notch ligand 1 (Dlk1) or by the expression of leucine-rich repeats and immunoglobulin-like domain protein 1 (Lrig1), respectively. Unique functions of these two populations are revealed at postnatal day 2 (P2), when the Lrig⁺Dlk1^– fibroblasts further express CD26 (also known as dipeptidyl peptidase 4, DPP4) and develop as fibroblasts that colonize the upper “papillary” dermis layer. On the other hand, the Dlk1⁺ fibroblasts diverge into Dlk⁺Sca1^– fibroblasts that colonize the lower “reticular” dermis, and Dlk⁺Sca1⁺ fibroblasts that colonize the deeper layers under the dermis (hypodermis and skin fascia) (Driskell et al. 2013). Lrig1⁺ and Dlk1⁺ fibroblasts further give rise to Neural/glial antigen 2 (Ng2)⁺ fibroblasts, which is a lineage restricted to perivascular locations and that express Ng2⁺ (Goss et al. 2021). In summary, skin is home to a collection of fibroblasts stemming from separate origins that have lineage-specific tissue locations and functions.

Figure 1.

Heterogeneity of skin fibroblasts. (A) Fibroblast heterogeneity resulting from developmental lineages. During skin development, a series of lineage markers represent the fibroblastic state. PDGFRα⁺ fibroblast progenitors diverge into Lrig⁺Dlk1⁻ and Lrig-Dlk1⁺ populations, and further develop into CD26⁺ papillary fibroblasts, Dlk1⁺Sca1⁻ reticular fibroblasts, and Dlk1⁺Sca1⁺ hypodermal and fascia fibroblasts. Some Lrig⁺ or Dlk1⁺ fibroblasts give rise to NG2⁺ pericytes (upper panel). The expression history of Engrailed-1 (En1) during embryonic development marks the fibroblastic fate. Skin fibroblasts diverge into two separate lineages, En1-lineage past fibroblasts (EPFs) and En1-lineage-naive fibroblasts (ENFs), which play distinct roles during wound repair and scar formation. In adulthood, En1 can be reactivated upon wounding, converting ENFs to EPFs (lower panel). (B) Fibroblast heterogeneity resulting from anatomic locations. Fibroblasts from mouse dorsal skin diverge into fibrogenic EPFs and nonfibrotic ENFs. Fibroblasts from mouse ventral skin diverge into fibrogenic Prrx1-lineage-positive fibroblasts and nonfibrotic Prrx1-lineage-negative fibroblasts. Fibroblasts from mouse oral mucosa mainly consist of regenerative Wnt1-lineage-positive fibroblasts. These fibroblast lineages are the determinant of the distinct wound repair outcomes at different skin locations. (C) Fibroblast heterogeneity resulting from skin depths. Fibroblasts from different skin depths show different transcriptomic features and harbor different wound healing potentials, with papillary fibroblasts promoting regeneration and reticular and fascia fibroblasts drive for scar formation. (dWAT) Dermal white adipose tissue, (PC) panniculus carnosus.

In parallel to the Watt and Driskell laboratories, our group has uncovered additional examples of unique fibroblast origins/functions during skin repair, using genetic lineage-tracing approaches in reporter mice. We discovered that distinct fibroblast populations of the adult mouse back skin diverge in function depending on transient expression of the transcription factor Engrailed-1 (En1) during early embryogenic development. Genetic lineage tracing in mouse back skin wound models revealed that En1-lineage past (positive) fibroblasts (EPFs) are the cellular subset that is recruited during skin injury repair and that it is just these EPFs that contribute to fibrosis and scar formation. By contrast, En1-lineage naive (negative) fibroblasts (ENFs) do not contribute to fibrosis and scarring (Rinkevich et al. 2015) and appear to be mainly active in early dermal development. Ablation of EPFs at postnatal stages resulted in reduced scars in response to injury. By performing reciprocal transplantations of embryonic fibroblast lineages, we found that the unique scarring outcomes of EPFs (but not ENFs) are intrinsic to them and independent of the local host environment (Rinkevich et al. 2015).

The second example of an origin-function linkage in fibroblast lineages comes from studying the skin's variable repair responses during skin development. In the first two trimesters of fetal life (gestational stage E16.5 in rodents), fetal back skin injuries regenerate intact dermis without scarring, as the wounded dermis restores normal architecture with new collagen and extracellular matrix (ECM). After the third trimester (gestational stage E18.5 in rodents) and throughout adulthood, humans and most mammals patch wounds with scars, which have large tightly packed parallel collagen bundles, erasing the original reticular pattern of fibers in intact skin. The repaired and scarred-over skin never has its normal cellular composition restored, and its ECM lattice organization and function remain structurally and functionally weaker than intact skin. By using genetic lineage-tracing approaches during back skin development and by analyzing the abundance of EPFs and ENFs, our group has identified a shift in fibroblast cell populations and in the composition of the various dermal fibroblast subtypes, which coincides with the phenotypic shift in the skin's response to injury, from scarless repair to scarring. ENF numbers plummet drastically during skin development, from initially comprising 93% of total stromal cells at E12.5 down to 22% at perinatal stages. We showed this decline in ENFs is accompanied by proliferative increase of EPFs from just 3% (at E12.5) to 67% at perinatal stages. This lineage-specific clonal expansion of EPFs is primarily due to proliferative advantage over ENFs, which leads to a dramatic change in the overall composition of fibroblastic lineages in the developing skin (Jiang et al. 2018). The mechanism underlying the clonal advantage of EFPs over ENFs during the last trimester of embryonic development requires further investigation.

Scar-forming EPFs also newly emerge in response to injury. Mascharak et al. recently showed that En1 (a transcription marker of EPFs) can be reactivated postnatally upon wounding, leading to further conversion of ENFs into EPFs. These newly formed postnatal EPFs further contribute to scar formation (Mascharak et al. 2021a). The reactivation of En1 in ENFs upon wounding appears to be mediated by Yes-associated protein (YAP)-dependent mechanotransduction signaling. During wound healing, genetic knockout of YAP or pharmacologically inhibiting it with the small molecule verteporfin blocks the ENF to EPF conversion and is sufficient for hair follicle regeneration to a certain extent (Jiang and Rinkevich 2021a; Konieczny and Naik 2021; Mascharak et al. 2021a,b). Reexpression of En1 is also found in multiple fibroblast subpopulations from patients with pathological skin fibrotic disease such as scleroderma. En1 was shown to amplify the fibrotic effects of TGF-β, by modulating the activity of SP family transcription factors, and reorganizing the cytoskeleton via Rho-associated kinase (ROCK) activation, which promotes myofibroblast differentiation. Importantly, fibroblast-specific knockout of En1 ameliorated skin fibrosis in mouse models (Györfi et al. 2021), confirming the obligatory role for EPFs and of En1 in scar formation.

Accumulating evidence suggests that targeting EPF lineage cells would provide therapeutic opportunities for promoting wound healing and reducing scarring. However, En1 is not suitable for the purpose of cell isolation or targeting as it is expressed inside the cell. Luckily, CD26, a serine protease, also called dipeptidyl-peptidase 4 (DPP4), has been found to be highly specifically expressed in EPFs in adult murine dorsal skin (Rinkevich et al. 2015). Using CD26 as a selection surface marker provides a 17-fold enrichment of EPFs over ENFs. Conventional fibroblast surface markers such as PDGFR and CD90 (Thy1) do not discriminate between these two functionally different fibroblast lineages. Single-cell RNA sequencing analysis revealed that CD26-expressing fibroblasts are involved in myofibroblast activation during both wound healing and scar formation in mouse (Shook et al. 2018). CD26-expressing fibroblasts are also present in human hypertrophic scars (Patel et al. 2021; Vörstandlechner et al. 2021). CD26 has several known molecular interactions and actions that are of relevance to the fibrotic functions of the EPFs. CD26 has been shown to regulate immune responses, cell-to-cell adhesion (Vörstandlechner et al. 2021), as well as control fibroblast senescence in vivo (Zhao et al. 2022). In addition, CD26 promotes TGF-β1-induced myofibroblast differentiation and ECM production in vitro (Vörstandlechner et al. 2021), providing multiple pathways where CD26 may exert modulatory roles on EPF fibroblasts. The exact enzymatic targets of CD26 in EPFs during wound repair remain to be elucidated, as does the exact chain of command between En1 expression in the nucleus and CD26 expression on the cell surface.

The use of lineage specific embryonic markers to distinguish between various populations of adult fibroblasts may mask a much more complex and transient profile of differentiation in adult fibroblasts. A very recent study from Driskell's group has employed a combination of single-cell isolation with transposase-accessible chromatin sequencing (scATAC-seq) and revealed that chromatin in all fibroblast populations of neonatal murine back skin have broadly similar accessibility profiles, despite their lineage-specific transcriptome profile. This discovery implies that fibroblast lineage markers that appear as membrane proteins, such as Dpp4/Cd26 and Dlk1 that are specifc for papillary and reticular dermal layers, respectively, may define a transient cellular state. This explains why the same marker can be expressed in different fibroblast populations under different conditions (Thompson et al. 2022). In contrast, as a transcription factor, En1 is only active for a short time window (∼48 h) (Rinkevich et al. 2015; Mascharak et al. 2021a). But once it is activated, the characteristics are imprinted in the progenies that do not express En1. Therefore, the history of the En1 expression matters, while the current expression of En1 is not necessary. In this sense, lineage markers that function as transcription factors or transcription coactivators, such as En1, determine the cellular fate of fibroblastic progenies. Such functionally committed populations do already exist at newborn stages that have not yet been segregated anatomically to upper, lower dermal, or fascial compartments. Therefore, the cell-fate lineage markers and cell-state lineage markers can overlap at different skin depths or under different conditions.

Hair follicle–associated fibroblasts including dermal papilla cells and dermal sheath cells are specialized fibroblast populations that are essential for hair follicle development and regeneration after wounding (Shin et al. 2020). Using scRNA-seq, Joost and colleagues systemically analyzed and described the transcriptomic features of dermal papilla fibroblasts and dermal sheath fibroblasts at anagen and telogen phases of hair follicle cycles. They identified a telogen and an anagen dermal papilla population that is distinguished by the expression of Notum and Corin, respectively, whereas dermal sheath fibroblasts are distinguished by high levels of Abi3bp, Tagln, and Grem2 (Joost et al. 2020). Lineage tracing has demonstrated that the dermal papilla and dermal sheath fibroblasts within hair follicles only have a minor contribution to neogenic hair follicles during wound-induced hair follicle neogenesis (WIHN), a phenomenon involving the reemergence of hair follicles at the center of large-size wounds in adult mouse back skin. Instead, a separate extrafollicular lineage of fibroblasts distinguished by Hic1 expression gave rise to 90% of the dermal papilla cells within the newly formed follicles (Abbasi et al. 2020). By combining and reanalyzing three sets of scRNA-seq data, Driskell's group concluded that Hic1-lineage-derived and Crabp1-expressing upper wound fibroblasts that are required for hair follicle neogenesis share a similar gene signature to that of murine papillary fibroblast lineage (Phan et al. 2021). Furthermore, with the addition of the recent scATAC-seq data, the divergent fates of dermal papilla cells and adipocytes from fibroblastic progenitors of neonatal mouse skin is shown to be regulated by distinct chromatin landscapes (Thompson et al. 2022).

Fibroblast Heterogeneity across Anatomic Locations

In parallel to their separate origins, and in accordance with their disparate functions, different fibroblast subtypes colonize different anatomic locations in the skin (Fig. 1B).

As discussed above, in dorsal skin, a period of En1 expression is crucial for distinguishing scarring from regenerative fibroblastic lineages. An analogous fibroblast population has also recently been demonstrated in ventral skin, using genetic lineage tracing. Stromal progenitors that express paired related homeobox 1 (Prrx1) generate the fibroblast lineage in murine chest and abdominal skin, which contributes to ventral skin fibrosis, while the Prrx1-lineage negative fibroblasts are critical for scarless repair there (Currie et al. 2019; Leavitt et al. 2020). Intriguingly, Prrx1⁺ fibroblasts are also the main cellular constituents of blastemas—the undifferentiated mass of cells that produce all cell types needed during limb regeneration in axolotls and frogs (Lin et al. 2021). Axolotls regenerate limbs perfectly, while post-metamorphic frogs only undergo partial regeneration. The authors show that the reason for the different repair outcomes is due to a difference in the dedifferentiation capacity of Prrx1⁺ fibroblasts. In frog blastemas, Prrx1⁺ fibroblasts only undergo partial dedifferentiation and do not fully reexpress the limb bud progenitor program (Lin et al. 2021). It is conceivable that additional fibroblast subtypes differ in as-yet unknown ways that result in different repair outcomes. More insights into the regulatory mechanisms of Prrx1⁺ across these species could give insights into why Prrx1 are pro-regenerative in the salamander blastema while only contributing to scarring in mice.

Oral mucosa is very different from skin from other anatomic locations. Injuries of oral mucosa heal exceptionally quickly and with minimal scarring. Several factors may contribute to this phenotypic difference, such as reduced inflammation and angiogenesis (Pereira and Sequeira 2021), faster reepithelialization (Iglesias-Bartolome et al. 2018), or the presence of saliva and oral microbiota (Glim et al. 2013). Fibroblasts of the oral cavity and cranial skin are derived from neural crest, whereas fibroblasts of the dorsal back skin are derived from paraxial mesoderm. The dominant fibroblast lineage in oral mucosa are Wnt1-lineage-positive fibroblasts (WPFs). This fibroblast subset does not elicit fibrotic responses in response to injury, unlike their En1-lineage-positive counterparts in dorsal skin or Prrx1-lineage-positive counterparts in ventral skin (Rinkevich et al. 2015). A clue to the central differences between the cellular functions of neural crest- and mesoderm-derived fibroblasts is seen in the type and magnitude of collective cellular movements seen during wound healing. Through intravital imaging, we demonstrated that EPFs from dorsal skin migrate directionally and collectively in a supracellular organization, whereas WPFs from oral mucosa move discretely and individually, and lack the intercellular adhesions that support a collective migration of scar-prone fibroblasts into wounds (Jiang et al. 2020a). This lack of collective migration in WPF neural crest fibroblasts leads to significantly less scar formation in wounds, as opposed to EPFs where collective migration leads to accumulations of scar-prone fibroblast in wounds and to more exuberant scars being formed.

Fibroblast Heterogeneity across Skin Layers

Another important tissue profile of fibroblasts is the heterogeneity seen across the different connective tissue matrix layers of the skin (Fig. 1C). Dermal tissues can be divided into upper papillary dermis, lower reticular dermis, and deeper still are the hypodermis and fascia layers.

Surface markers to distinguish the diverging fibroblast populations seen across different skin depths still remain incompletely understood. For example, CD26⁺ fibroblasts, considered as the scar-forming cells of the back skin, are not restricted only to the papillary dermis, as well as EPFs, which are not only present in reticular dermis, but distributed throughout the full thickness of adult mouse back skin (Rinkevich et al. 2015; Shook et al. 2018).

Based on accumulating single-cell RNA-sequencing analyses and immunofluorescence validations, the fibroblasts from different dermal layers display unique subtraits. For example, papillary fibroblasts have been shown to be prone to regenerate wounded skin following a superficial injury, while deeper reticular and fascial fibroblasts are prone to contribute to fibrotic responses. Phan et al. discovered that expression of Lef1, a canonical Wnt transcription factor, is enriched specifically in papillary fibroblasts in neonatal murine skin, a stage where wounded skin may regenerate. However, Lef1 appears to be turned off in adult skin, a stage where wounded skin scars over and does not regenerate. By genetically forcing the reexpression of Lef1 in wound fibroblasts of adult mice, the authors have shown promotion of regeneration including formation of new hair follicles and arrector pili muscle (Phan et al. 2020), mimicking the scarless regenerative phenotypes seen in fetal wounds.

Subclassification of fibroblasts in human skin remains anecdotal and is based on cell surface marker identification. For example, in human breast skin, CD39⁺CD26^– fibroblasts represent the majority of fibroblasts within the papillary skin layers. Whereas deeper reticular fibroblasts in human breast skin can be distinguished based on the expression of the cell-surface marker CD36⁺ (Philippeos et al. 2018).

This subclassification of upper papillary and lower reticular fibroblasts is challenged by a recent study focusing on human hypertrophic scars showing that scar severity is positively correlated with the number of CD39⁺ papillary fibroblasts, but not with CD26⁺, CD36⁺, or FAP⁺ subsets (Huang et al. 2022). CD39⁺ fibroblasts are enriched in hypertrophic scars and seem to facilitate myofibroblast activation and ECM production. Pharmacological inhibition of CD39 reduces mechanical stretch-induced or bleomycin-induced skin fibrosis and scarring (Huang et al. 2022). This discrepancy in classifying human fibroblast populations based on cell-surface markers may be explained by the different anatomic locations of the skin specimens that are examined in these two studies. In addition, in mouse skin, CD39⁺ fibroblasts have been shown to be present throughout all dermal layers, without depth specificity (Huang et al. 2022). This further reminds us to add species differences between model animals and humans when considering translational purposes for wound management or anti-scar therapies.

Further deeper is the fascia layer of the skin. In murine back skin, the fascia is a single layer of loose connective tissue located under the panniculus carnosus (PC) muscle and is further beneath the dermal white adipose tissue (dWAT). Recently, our group discovered that the skin fascia, the deepest dermal tissue layer, plays an important role in wound healing and scarring. Using anatomic fate-mapping techniques, we revealed that, upon deep wounding, the fascia physically moves toward the wound bed. The fascia tissue contains a premade cellular composite that can quickly patch repair wounds. This pre-made kit of cells and ECM includes fascia-resident fibroblasts, macrophages, blood and lymphatic vessels, peripheral nerves, and associated gelatinous ECM that is rich in glycosaminoglycans, proteoglycans, and water, with hyaluronic acid in particular (Correa-Gallegos et al. 2019; Pratt 2021). Fascia movements thus provide the majority of ingredients that are necessary for the early phases of wound repair, prior to the proliferation and activation of myofibroblasts and deposition of new ECM. Physical blockage of fascia movement using an impermeable membrane barrier leads to chronic open wounds that fail to heal (Correa-Gallegos et al. 2019). The key to fascia movements lies in fascia-resident fibroblasts. Several proteins and genes have been shown to be highly expressed by fascia fibroblasts, including Dpp4 (CD26), Ly6a (Sca-1), Plac8, Pi16, GPX3, and MSX1 (Correa-Gallegos et al. 2019; Joost et al. 2020; Boothby et al. 2021; Phan et al. 2021; Usansky et al. 2021; Thompson et al. 2022). Fascial fibroblasts express specific integrins α5β1 and αvβ3, which allows them to behave as the primary physical sensors of the ECM and the surrounding tissue's mechanical forces (Chiquet et al. 2009). The specific set of markers that distinguish fascia fibroblasts from the remaining dermal fibroblast communities and that promote their unique functions, have not yet been fully defined.

Unlike the anatomy of the rodent fascia in the back skin, human skin fascia is embedded inside the dWAT layer, and may appear as a single layer of viscoelastic loose connective tissue or may appear as multilayered, depending on the skin anatomic location (Jiang and Rinkevich 2021b). Human skin fascia tissue enables a strong attachment of the upper skin to the underlying muscle, and also facilitates the gliding between the dermal and muscle layers (Adstrum et al. 2017; Jiang and Rinkevich 2021b). With these anatomic differences between mouse and human fascia, whether the physiological functions of fascial fibroblasts on wound healing discovered in mice also reflects similar key functions in human wound healing requires further investigation. Boothby and colleagues have looked more closely into human fascia. Using scRNA-seq, they reveal that human skin contains fibroblasts that share a core transcriptional signature with mouse fascia fibroblasts. Furthermore, the number of fibroblasts from such subsets has a positive correlation during inflammation and fibrosis of human skin fascia (Boothby et al. 2021).

FIBROBLAST ROLES IN WOUND REPAIR AND SCAR FORMATION

Phenoconversion and ECM Deposition

The widely accepted myofibroblast theory proposed by Gabbiani 50 years ago states that, upon wounding, the dermal fibroblasts from skin adjacent to the wound site migrate into the wound bed, where they proliferate and subsequently convert to contractile myofibroblasts that express contractile stress fibers, with incorporation of α-smooth muscle actin (α-SMA) (Gabbiani et al. 1971). These myofibroblasts contribute to the formation of the granulation tissue, which is also enriched with newly formed blood vessels through angiogenesis. The granulation tissue, it is stated, is the powerhouse of the wound contraction and new ECM deposition, and thus the healing processes (Fig. 2A; Hinz 2016; Hinz et al. 2019).

Figure 2.

Fibroblast roles in wound repair and scar formation. (A) Myofibroblast/granulation tissue-driven wound contraction and extracellular matrix (ECM) deposition model: fibroblasts from adjacent skin phenoconvert to contractile myofibroblasts, forming granulation tissue and deposit new ECM. TGF-β and c-Jun signaling play key roles in this process. (B) Fascia fibroblast migration-driven wound contraction and ECM mobilization model: fibroblasts from skin fascia migrate collectively from wound peripheral to wound center, creating contraction force and mobilize fascial ECM to repair deep wounds. Temporarily elevated intercellular communication and adhesion among fascia fibroblasts via connexin gap junctions and cadherin adherens junctions are critical for fascia mobilization. (C) Environmental sensing of fascia fibroblasts: fascia fibroblasts sense and adaptively react to environmental changes of mechanical stress via YAP-mediated signaling, and prime and respond to the immune microenvironment by interacting with the fascia-resident immune cells such as T helper 2 (T_H2) cells in paracrine and autocrine fashions during early development. (DDR) Discoidin domain receptor, (PC) panniculus carnosus.

TGF-β/Smad signaling is the most critical molecular switch leading to phenotypic conversion from fibroblasts to myofibroblasts (Hinz 2016; Jiang et al. 2020b). The activation of AP1 transcription factor c-Jun is another indispensable step toward myofibroblast conversion. In murine fibroblasts, c-Jun activates downstream pAkt and CD47, promoting robust cell proliferation and inflammatory cell recruitment (Wernig et al. 2017). Persistent activation of TGF-β and c-Jun signaling, or overexpression of the components in the signal circuits leads to hypertrophic scarring both in mouse and human (Griffin et al. 2021).

Fibroblasts responding to injury may likely undergo numerous consecutive phenoconversion events. For example, the contractile myofibroblast state of skin fibroblasts in normal wound healing is only transient. Granulation tissue is dissolved during the wound remodeling phase, mainly through myofibroblast self-clearance via apoptosis (Hinz 2007). Fully functional myofibroblasts may also revert back to a low contractile/scar-producing activity state that is characteristic of a resting fibroblast in homeostatic tissues (Hinz and Lagares 2020). Alternatively, they can further phenoconvert into new dermal condensate fibroblasts and new adipocytes (Plikus et al. 2017, 2021).

Collective Migration and ECM Mobilization

Despite the above, the myofibroblasts and granulation tissue theory does not completely explain skin contraction during wound healing, especially at the early phase of the wound healing response. Indeed, several observations challenge the granulation tissue theory. A series of earlier experiments showed that repeated removal of granulation tissue did not impair wound closure or skin contraction (Grillo et al. 1958; Watts et al. 1958; Gross et al. 1995), indicating that granulation tissue is not needed for wound contraction to occur.

Indeed, it was demonstrated that simply excising the wound edge, rather than the granulation tissue itself, immediately changed wound shape and releases wound tension (Watts 1960; Gross et al. 1995), which implies that wound contraction comes from the wound edges rather than wound bed. An alternative model for wound contraction has been raised by Harris and colleagues. In this separate hypothesis, the initial wound contraction is generated during the migration of fibroblasts from wound edge toward wound center. The traction force generated by the migrating fibroblasts is much greater than the force they need for their local motion. Harris postulates that this additional force generation by fibroblasts is used for pulling the wound edge to enable tissue contraction and wound closure (Harris et al. 1981). While the myofibroblast theory has been widely accepted, largely due to the clear observation of α-SMA staining, Harris's fibroblast migration-driven wound contraction model proposed at a similar time has been neglected over these past decades, likely due to a simple unanswered question—how can fibroblast migration at wound edges generate such strong pulling force? Recently, studies on skin fascia using fate mapping and in vivo time-lapse imaging techniques have given important new insights that explain Harris's model of fibroblast traction forces as a primary means for wound closure and that start to put the jigsaw puzzle of wound healing in place.

As discussed above, patch repair of deep wounds requires the mobilization of skin fascia. The fascia that is present underneath the wound moves from the wound periphery toward its center. This movement of the fascia is dependent on its resident fibroblasts, and indeed genetic depletion of En1-lineage-positive fibroblasts in fascia is sufficient to drastically reduce fascia movements and inhibits wound closure (Jiang et al. 2020a). The mobilization of fascia tissue is closely associated with migration of fascia fibroblasts. Fascia fibroblast migration from the wound edge toward its center occurs collectively as a group of cells, and this collectivity is triggered by the wound response. It is this directed supracellular migration that can generate robust contraction forces (Fig. 2B).

Collective migration has been described during morphogenesis, wound healing, and cancer metastasis (Friedl and Gilmour 2009; Shellard et al. 2018; Shellard and Mayor 2019). In the fascia, collective cell migration is achieved by reinforced intercellular communications and adhesion among fascia fibroblasts, via the temporary up-regulation of gap junctions through Connexin43 (Wan et al. 2021), and adherens junctions comprised of Cadherin-2 (N-cadherin) (Hinz et al. 2004; Jiang et al. 2020a) and Cadherin-11 (OB-cadherin) (Lodyga et al. 2019; Beckmann et al. 2021).

This collective migration of fascia fibroblasts and the subsequent mobilization of fascia tissue into wounds quickly generate a structurally imperfect but mechanically stable patch that seals the wound. Collective migration leads to anisotropic alignment of fascia fibroblasts, which redistributes mechanical stress preventing force transmission to the nucleus to protect from cell damage (Nava et al. 2020). The consequence is the anisotropic alignment of ECM fibers, made from fascia fibroblasts dragging on fascia ECM, which results in scar formation (Correa-Gallegos et al. 2019; Jiang et al. 2020a; Correa-Gallegos and Rinkevich 2021). Depletion of fascia fibroblasts by genetic targeting significantly reduces scar severity after wound repair providing new opportunities for clinically modulating wound healing (Jiang et al. 2020a).

The myofibroblast/granulation tissue model (Fig. 2A) and the fascia fibroblast migration model (Fig. 2B) may not necessarily contradict each other. Rather, they may reflect two separate phases of wound healing and contraction. For example, early wound ECM may be primarily derived by fascia mobilization, whereas newly deposited ECM from myofibroblasts may supplement later phases of scar tissue remodeling. The ratios of fascia fibroblasts to adjacent dermal fibroblasts in wounds, and the ratios of mobilized fascia ECM to that of newly deposited ECM in wounds are not quantified so far. The long-term contribution of fascia fibroblasts in wounds and scars requires further investigation.

Environmental Immuno- and Mechanosignaling

Another important function of fibroblasts during injury repair is to sense and adaptively respond to environmental changes (Fig. 2C). Fibroblasts both regulate mechanical and immunological cues during wound healing, as well as being affected by these two cues. Both inflammatory and mechanical cues directly influence fibroblast-to-myofibroblast phenotypic conversion (Hinz et al. 2019; Merkt et al. 2021; Sawant et al. 2021), thereby directly influencing wound healing. In turn, fibroblasts are also one of the major orchestrators that modulate immune responses and matrix stiffness during wound healing (Correa-Gallegos et al. 2021; Plikus et al. 2021; Schuster et al. 2021; Seo et al. 2021).

Fascia is the primary tissue where fibroblasts sense mechanical stress (Stecco et al. 2015). Fascia fibroblasts are equipped with a full set of molecular tools that enable mechanoperception of the environment and transduction signaling within the cells needed to modulate the fibroblasts. These mechanical stress modulators include Wnt (Plikus et al. 2021), sonic hedgehog (Shh) (Lim et al. 2018), integrins, and discoidin domain receptors (DDRs) (Hinz et al. 2019). Fibroblasts bind to ECM fibers through integrins and DDRs, which form focal adhesion, transmitting the ECM stiffness and mechanical force as regulatory signals via the activation of focal adhesion kinase (FAK) and force-dependent nuclear translocation of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), promoting the expression of profibrotic genes (Fig. 2C). Pharmacological inhibition or genetic inactivation of YAP or FAK in fibroblasts reduces scar formation and promotes a more regenerative healing phenotype as indicated by formation of new hair follicles within the wounded skin (Nardone et al. 2017; Chen et al. 2021; Koester et al. 2021; Mascharak et al. 2021a,b).

Roy and colleagues (2020) went one step further by demonstrating that modulation of mechanical environment with a defined 3D collagen matrix functionally rejuvenates the aged fibroblasts for enhanced matrix contraction, deposition, and remodeling. In fact, there are specific mechanical cues that promote perfect skin regeneration in nature, with African spiny mice (Acomys) as the best example studied so far. Acomys’ skin is 20 times softer than in laboratory mice (Mus musculus) (Seifert et al. 2012). Recent study with atomic force microscopy found that the wound bed in Acomys is also significantly softer than in M. musculus, which creates a permissive mechanical environment that is essential for regeneration (Harn et al. 2021). Further transcriptomic analysis revealed that Twist1 and Zeb2 are the key transcription factors that respond to matrix stiffness and that allow new hair follicles to reform. Therefore, manipulating mechanotransduction in fibroblasts, especially in fascia fibroblasts would be an attractive therapeutic option for pro–wound healing and anti-scarring purposes.

Finally, fascia has a more global function in wound response as a tissue reservoir for immune cells, including macrophages, neutrophils, and T cells. Indeed, fascia fibroblasts license peripheral immune responses. For instance, mouse fascia fibroblasts, defined as Lin⁻CD34⁺PDPN⁺ Sca1⁺CD26⁺CD9⁻, preferentially express the T helper 2 (T_H2) cell cytokine receptors IL-4R and IL-13R. The interaction of T_H2 cells with fascia fibroblasts at the neonatal stage is required for prime fascia toward development and maturation (Boothby et al. 2021). In return, fascia fibroblasts produce T_H2 alarmin IL-33 promoting selective accumulation of T_H2 cells, and FGF18 that may promote proliferation and activation of fascia fibroblasts in an autocrine fashion function (Fig. 2C). The interplay between fascia fibroblasts and T_H2 cells is critical for induction of local inflammation and fascia contraction for wound closure.

CONCLUDING REMARKS

The heterogeneity of skin fibroblasts can be seen through the resolution of developmental lineages, as well as through anatomic locations and skin depth. Mixtures of the variations from these factors create a very heterogeneous population with diverse functions during skin wound repair.

Specialized fascia fibroblast promotes wound contraction and healing processes through collective migration and mobilization of fascia's ECM into wounds, further adding to myofibroblast phenoconversion and to ECM deposition, which further modulates the mechano- and immune microenvironment to enhance wound healing.

Continued development and application of techniques, such as spatial single-cell transcriptomics that integrate single-cell transcriptomes with spatial information from histological sections (Vickovic et al. 2019), will continue to deepen our view of different lineages and subpopulations of fibroblasts during skin development, wound repair, and under pathological skin conditions. Once we can better understand fibroblast functional diversities, through targeting different subsets of fibroblasts, we may even be able to manipulate the outcome of wound healing, and finally achieve the goal of rapid and scarless skin regeneration.