Research progress on scar-free healing

Abstract

Introduction

Pathological scars resulting from wound healing can cause functional impairment, cosmetic disfigurement, and psychological distress, highlighting the urgent need for regenerative solutions. Scar-free healing observed in embryonic development and some adult mammalian models have achieved complete tissue repair without fibrosis.

Methods

This article reviews literature on wound healing and scarless healing published over the past 25 years. The databases searched were PubMed/MEDLINE, Embase, Web of Science, CNKI, Wanfang, and ChiCTR (used to supplement research from Chinese scholars). Search terms included ‘wound healing,’ ‘scarless healing,’ ‘scar-free healing,’ ‘embryo,’ ‘immunotherapy’ and ‘tissue engineering therapy,’ among others. Additionally, the reference lists of key articles were examined to identify further relevant studies.

Discussion

In this article, we reviewed the influencing factors of scarless healing, covering both macroscopic and microscopic factors—including fibroblasts, collagen, matrix metalloproteinases, neurological influences, and cytokines—which are key therapeutic targets in clinical practice. It details four critical signalling pathways: (1) the SDF-1/CXCR4 axis, (2) the Wnt signalling pathway, (3) the [Ca²⁺]i and Rho/ROCK pathways, and (4) the Hippo signalling pathway and integrin-linked kinase, along with newly identified genes like YAP and En1. The analysis highlights the promise of combined targeted therapies. Additionally, it reviews advanced treatments and animal models for scarless healing and introduces an innovative immune-targeted strategy to optimize wound healing outcomes.

Conclusion

The formation of scars is influenced by multiple internal and external factors. To achieve scar-free healing, a combination of multiple effective treatment methods is required, such as targeting related signalling pathways or genes. Furthermore, immunotherapy-related approaches hold great potential and require further research to clarify the relevant reaction processes in order to develop new and effective treatment methods.

1. Introduction

The skin is the largest organ and immune defense line in the body, which can prevent water from excessive loss [1,2]. Any compromise of its integrity leads to the formation of wounds, which undergo non-functional healing and produce scars. Chronic wounds are especially prone to scar formation [3,4]. Scarred wounds make the life of patients inconvenient and hinder social reintegration, resulting in numerous negative effects on their physiology and psychology [5,6]. This article reviews literature on wound healing and scarless healing published over the past 25 years. The databases searched include PubMed/MEDLINE, Embase, Web of Science, CNKI, Wanfang and ChiCTR (used to supplement research from Chinese scholars). Search terms included ‘wound healing,’ ‘scarless healing,’ ‘scar-free healing,’ ‘embryo,’ ‘immunotherapy’ and ‘tissue engineering therapy’ among others. Additionally, the reference lists of key articles were examined to identify further relevant studies.

Conventional wound healing consists of three overlapping but distinct processes (as shown in Figure 1): haemostasis and inflammation, proliferation, and remodelling [7]. Once a wound is created, platelets rapidly gather at the wound site to stop bleeding and secrete cytokines that attract neutrophils and macrophages, initiating the inflammatory phase [8]. Within 2–3 days after the injury, fibroblasts from the surrounding tissue migrate to the wound site, proliferate alongside the resident fibroblasts, and begin to produce collagen, extracellular matrix (ECM), and other substances, marking the onset of proliferation stage [9]. Some of the fibroblasts differentiate into myofibroblasts under the action of cytokines such as transforming growth factor-β (TGF-β) [10]. These myofibroblasts stretch and contract the wound to promote healing [11,12]. Finally, the tissue at the wound site undergoes remodeling by matrix metalloproteinases (MMPs) [13]. Excessive and disorganized deposition of collagen and matrix leads to scarring. After healing, the tensile strength of scar tissue can only reach 80% of the original tissue [14]. Additionally, skin appendages, such as glands and hair follicles, do not regenerate [15]. Several in vivo and in vitro studies have demonstrated that the intervention of different cells and cytokines in wound healing can promote scarless outcome. However, a method for achieving scar-free wound healing is lacking in clinical practice. This study reviewed the factors affecting scar-free healing, including various cells, cytokines and signalling pathways, alongside cutting-edge scientific research, biological research models of scar-free healing in nature and means to promote scarless healing. The influencing factors discussed are illustrated in the figure below (Figure 2).

Figure 1.

The stages of wound healing.

Conventional wound healing consists of three overlapping but distinct processes: haemostasis and inflammation, proliferation, and remodelling. A. Once a wound is created, platelets rapidly gather at the wound site to stop bleeding and secrete cytokines that attract neutrophils and macrophages, initiating the inflammatory phase B. Within 2–3 days after the injury, fibroblasts from the surrounding tissue migrate to the wound site, proliferate alongside the resident fibroblasts, and begin to produce collagen, extracellular matrix (ECM), and other substances, marking the onset of proliferation stage. Some of the fibroblasts differentiate into myofibroblasts under the action of cytokines such as transforming growth factor-β (TGF-β). These myofibroblasts stretch and contract the wound to promote healing. C. Finally, the tissue at the wound site undergoes remodelling by matrix metalloproteinases (MMPs). D. Scars form after the wound heals.

Figure 2.

Mechanism diagram.

(a) Once a skin wound occurs, platelets rapidly gather at the site to stop the bleeding. They then release cytokines that attract macrophages, neutrophils, and other immune cells to initiate the inflammatory phase.

(b) Within 2 to 3 days after injury, fibroblasts near the wound migrate to the site, proliferate along with local fibroblasts, and begin producing collagen, extracellular matrix, and other components to initiate the proliferative phase. In response to TGF-β1 and a high-tension environment, fibroblasts can differentiate into myofibroblasts. These myofibroblasts generate tension and contract to promote wound closure, thereby supporting the healing process.

(c) Finally, the tissue at the wound site undergoes remodelling by matrix metalloproteinases (MMPs). When collagen and matrix are deposited excessively and in a disorganized manner, scarring can occur.

(d) The SDF-1/CXCR4 signalling pathway can recruit bone marrow mesenchymal stem cells to migrate to the wound site and promote wound healing. It also promotes the migration of epidermal stem cells and fibroblasts, and enhances the secretion of vascular endothelial growth factor by endothelial cells, thereby accelerating skin epithelialization and dermal structure regeneration.

(e) The SOX family plays a role in the healing of various tissues, including skin, fractures, tendons, and corneas. SOX2 and SOX9 support cell renewal, while SOX7 improves the microvascular environment. SOX2 also contributes to the reprogramming of somatic cells into induced pluripotent stem cells (IPscs) and regulates the migration and proliferation of keratinocytes.

(f) Activation of the Wnt signalling pathway results in fibrotic healing of skin wounds. Wnt-3a interacts with the ligand complex, leading to the accumulation and nuclear translocation of β-catenin. Inside the nucleus, β-catenin binds to TCF/LEF transcriptional enhancers to drive the expression of profibrotic genes.

(g) Overexpression of YAP in fibroblasts at the wound site can cause excessive fibroblast proliferation, leading to scar formation. YAP contributes to scarring by promoting En1 expression and suppressing Trps1 expression. The Hippo signalling pathway can negatively regulate YAP through a cascade of phosphorylation events, limiting its activity in fibroblasts and helping to reduce scar formation.

(h) TWIST1 is highly expressed in keloid fibroblasts and can activate a cascade of pro-fibrotic genes, including COL1A1, COL3A1, and FN1.

2. Factors influencing scarless formation

2.1. Macroscopic factors

2.1.1. Gestational age and amniotic fluid environment

The shorter the gestational age, the greater the chance of scarless healing. Steven et al. established wound models of foetal rats of various gestational ages and found that the wound recovery of foetal rats with greater gestational age was worse [16]. Most mammals, including rats, rabbits, pigs, sheep, are capable of scar-free healing during the first one-third to one-half period of embryo development [17]. In the latter two-third to one-half of embryonic development, fibrosis becomes apparent during the wound-healing process, ultimately resulting in scar healing [15]. The question of whether scar-free healing in foetal wounds is linked to the amniotic fluid environment has been explored. Experiments revealed that scar-free healing could not be achieved after the transplantation of an adult wound model into foetal sheep and then implantation of the foetal sheep in the amniotic fluid environment of the mother. This indicates that the amniotic fluid environment is not the determining factor for scarless healing [18].

2.1.2. Wound size, depth and tension at the wound site

The healing process of a wound is closely linked to its size, depth and the tension at the wound site [19]. Lovvorn et al. xenografted human aborted foetal skin into mice and conducted experiments using foetal sheep to create a skin incisional model. Their results showed that the incision wound healed without scars, whereas the excision wound showed fibroplasia, suggesting that the smaller the wound, the easier it was to heal without scars [20]. In a study by Morris et al., scars were formed after the healing of larger (8-mm) wounds in foetal lambs, while no scars were observed in 2-mm wounds [21]. Bullard et al. made circular wounds ranging from 2 to 10 mm in diameter on the backs of foetal lambs. Their results demonstrated that larger wounds resulted in more significant scarring [22]. An increase in wound size leads to higher expression of pro-inflammatory and fibrotic genes, triggering inflammatory responses and fibrous hyperplasia, followed by scar formation. The deeper the wound, the less likely it is to heal and the more likely it is to scar. Deeper wounds that extend into the reticular dermis prolong fibroblast proliferation and increase the expression of pro-fibrotic genes, such as TGF-β1 and CTGF, resulting in excessive collagen deposition and scar formation [23]. Additionally, fibroblasts in the reticular dermis (deeper layers) are more likely to differentiate into myofibroblasts under mechanical tension, further promoting fibrosis [23,24]. In contrast, superficial wounds primarily involve the papillary dermis, where fibroblasts have greater migratory capacity and produce more hyaluronic acid. This promotes faster tissue remodelling and reduces fibrotic activity, facilitating scar-free healing. This spatial heterogeneity highlights the importance of targeting depth-specific fibroblast subsets to modulate scar formation. Dunkin et al. conducted a clinical study with 113 human participants, establishing dermal wound models of varying depths on the hip skin. The findings revealed that shallower wounds resulted in less visible scarring after healing. They identified a transition point between scar-forming and non-scar-forming wounds at a threshold depth of approximately 0.56 ± 0.03 millimetres, strongly supporting wound depth as a critical factor in achieving scarless healing [25]. The lower the tension at the wound site, the more conducive it is to scar-free healing. High tension can stimulate the proliferation of fibroblasts, rendering the scar healing characteristics. In addition, collagen is arranged in a basket weave appearance in normal human tissues, but type I and III collagen fibres are aligned in parallel after trauma, resulting in scar formation, possibly due to the high-tension environment at the wound site. The macroscopic factors influencing scar-free healing are summarized in Table 1.

Table 1.

Macroscopic factors influencing scarless formation.

Factor	Mechanism	Outcome	References
Gestational Age	Scarless healing occurs during the first third to half of foetal development; younger gestational age correlates with better wound restoration	Fibrosis emerges gradually in later foetal stages, ultimately leading to scar formation	[15,16]
Amniotic Fluid Environment	Adult wounds transplanted into the amniotic fluid still fail to achieve scarless healing	Amniotic fluid is not a decisive factor in scarless repair	[17,18]
Wound Size	Large wounds (>8 mm) upregulate pro-inflammatory and pro-fibrotic genes, triggering inflammation and fibrosis; 2-mm incisions may achieve scarless healing	Positive correlation between wound size and scar severity	[20–22]
Wound Depth	Deeper wounds exhibit slower fibroblast proliferation and higher TGF-β/CTGF expression	Increased wound depth correlates with slower fibroblast activity and higher scarring probability	[23]
Tension at Wound Site	High tension stimulates fibroblast proliferation and induces parallel alignment of type I/III collagen fibres	Collagen disorganization under high tension promotes scarring; low tension favours scarless healing	[10,14,19,23]

2.2. Microscopic factors

2.2.1. Cells: fibroblasts and myofibroblasts

Foetal fibroblasts are the main effector cells involved in scar-free healing and play a key role in the early embryonic damage repair process [26,27]. These fibroblasts produce ECM and fibrin to repair wounds. Studies have shown that the fibroblasts in foetal wound healing have higher migration capacity, more hyaluronic acid receptors, and different growth factor profiles [28]. Fibroblasts have multiple subpopulations, and different subpopulations have different functions [24]. This heterogeneity has a significant impact on wound healing [29,30]. The differences in the embryonic origin of these fibroblasts are related to their different anatomical locations [31]. Different subtypes of fibroblasts correspond to different expression of homeobox (HOX) genes. Their functions have been explored through the expression profile of HOX [32]. For example, dorsal Engrailed-1(EN1)-positive fibroblasts are responsible for the deposition of most dermal connective tissue during skin wound healing, radiation-induced fibrosis, and cancer interstitial processes [33]. In contrast, Prrx1-positive fibroblasts contribute to ventral dermal fibrosis during wound repair. Fluorescence-activated cell sorting (FACS) and histological analysis of wounds on postoperative day 14 (POD 14) confirmed that the majority of fibroblasts in POD 14 scars were PPFs. Targeted modulation of these fibroblast populations can influence fibrosis progression [34]. Targeted interventions in these fibroblasts can regulate the development of fibrosis [35].

Fibroblasts can differentiate into myofibroblasts in response to TGF-β1 and a high-tension environment [36,37], which in turn promote ECM production and the expression of alpha-smooth muscle actin (α-SMA). The traction force of fibroblasts and the coordinated contraction of myofibroblasts are the key factors for wound closure and wound healing [38,39]. Myoblasts are expressed early and disappear rapidly while participating in scarless healing during foetal development. In the later stage of wound healing, myoblasts undergo programmed apoptosis around 14 days after the injury and completely disappear, leaving no scar at the wound site [22]. Studies have shown that prolonged maintenance of myofibroblast-like phenotype can lead to excessive tissue contraction, fibrosis and matrix deposition, ultimately resulting in scar formation [40]. The apoptosis of myofibroblasts does not begin until 15–30 days after the injury in adults [41]. In addition, the expression level of α-SMA is approximately linear with the tension at the wound site, suggesting that the greater the tension, the more abundant the expression of myofibroblasts, thereby promoting scar formation. However, wounds without myofibroblasts do not heal properly and become chronic wounds [14]. Therefore, proper regulation of the metabolism and apoptosis of myofibroblasts has become an effective strategy to control scar occurrence. Studies have shown that myofibroblasts exposed to bone morphogenetic proteins (BMPs) can be reprogrammed into adipocytes. Proper regulation of this plasticity can be a promising approach to managing and reducing scarring.

2.2.2. Extracellular components

• Collagen

Salamander and African spiny mice are typical animal models of scar-free wound healing. During the early stages of repair, these animals primarily use type III collagen, which is later replaced by type I collagen in the later stages. Compared with type I collagen, type III collagen is more easily degraded by different proteases, has a faster metabolic rate and a more regular arrangement, and is the main type of collagen in the early stages of embryo development [42]. In addition, the collagen bundles in the early and middle stages of embryo development are slender, and the precipitation shape is a three-dimensional grid without excessive deposition. After birth, the proportion of type III collagen decreases from 30%–60% to 10%–20%. The collagen bundles are extremely coarse during scar formation, and the deposition format is disordered and parallel. At the same time, the deposition is quite slow but excessive. The proportion and arrangement of type III collagen are closely related to wound healing [43]. In addition, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. Chin et al. found that high levels of DDR1 promoted scarless healing in early foetal life. However, the experimental results showed that the expression of DDR1 decreased with the increase in gestational age [44]. The currently used methods to control collagen metabolism include regulating the activity of enzymes lysyl oxidase, which promote collagen cross-linking [45]. The activity of lysyl oxidase is inhibited, reducing the cross-linking of collagen and thus preventing scar formation. More direct measures, such as an active supplement of type III collagen in the wound area, can also improve healing quality and reduce scar formation [46,47]. Other studies have shown that acid fibroblast growth factor (aFGF) can downregulate the expression of the type I procollagen gene, inhibit collagen synthesis by fibroblasts, reduce excessive deposition of collagen and prevent scar formation [48].

• Hyaluronic acid

Hyaluronic acid is an essential component of the ECM aminoglycan. The content and distribution of hyaluronic acid in embryonic skin differ significantly from those in adult skin, with levels in the adult epidermis and dermis being much lower. Studies have shown that the concentration of hyaluronic acid at the wound site in foetal rabbits is higher and lasts longer than in adult rabbits [49]. Additionally, adding hyaluronic acid can promote the deposition of type III collagen when fibroblasts are cultured in vitro, further supporting its role in enhancing the healing process [17]. The inflammatory response in foetal wounds is lower than in adult wounds, which may be related to the inhibition of platelet aggregation and cytokine release by hyaluronic acid [50]. Studies have shown that more receptors for hyaluronic acid exist on the surface of foetal fibroblasts and hyaluronic acid has a stronger effect on them [20]. A hyaluronan-associated protein–collagen (HA-PC) complex was identified in the scarless healing of foetal sheep. Wounds treated with HA-PC healed faster and had good collagen alignment. This promoted scarless healing by affecting the dynamic changes in collagen microfibrils, aiding in the organization and remodelling of the extracellular matrix during the healing process.

• Expression of enzymes

The expression of enzymes in post-traumatic wound varies between embryos and adults, with matrix metalloproteinases (MMPs) being the most notable. MMPs and their tissue inhibitor of metalloproteinase (TIMP) can affect the structure of ECM. MMPs can degrade ECM, facilitating its remodelling and preventing excessive accumulation in the wound tissue, which is essential for proper tissue repair and scar-free healing [8]. During foetal wound repair, the level of MMPs increases while the level of TIMPs decreases, whereas the opposite trend is observed in adult wounds [51]. This suggests that degradation and remodelling of the wound tissue are more active in foetal wounds. The transition from scar-free healing to scar formation in foetal rats occurred between days 16.5 (E16) to 18.5 (E18) of gestation. Catherine et al. performed full-layer incision on the backs of foetal rats in both E16 and E18 groups. They found that MMP expression was higher in the E16 group than in the E18 group, indicating that MMPs play a crucial role in ECM renewal, promote foetal cell migration, and facilitate scar-free repair [13].

• Transforming growth factor β

TGF-β is closely linked to fibrosis, with TGF-β1, TGF-β2, and TGF-β3 being the primary factors involved in the wound healing of skin tissue. These growth factors are produced by macrophages, keratinocytes, fibroblasts, and platelets [52]. During wound repair, TGF-β1 and TGF-β2 are overexpressed in adults, contributing to fibrosis, while TGF-β3 is predominantly expressed in embryos, playing a role in scar-free healing. Adzick et al. reported TGF-β1 RNA and protein expression were present in injured adult skin, but neither was detected in human foetal skin [53]. When recombinant human TGF-β1 protein was applied to rat wounds, scar fibrosis was aggravated. However, when TGF-β1 and TGF-β2 antibodies were used to neutralize TGF-β1 and TGF-β2 expression around rat wounds, scar fibrosis was alleviated [10]. Subcutaneous injection of exogenous TGF-β3, also known as Avotermin, at the wound site improved scarring outcomes [54]. TGF-β3 promoted fibroblast migration, facilitating their arrangement into a network structure that contributed to scarless healing.

• Fibroblast growth factor

The repair process after tissue damage is regulated by various growth factors, among which FGF plays an important role. FGF binds to its receptor, fibroblast growth factor receptor (FGFR), initiating a signalling cascade. Upon binding, FGFR dimerizes and autophosphorylates. These receptors activate downstream signalling pathways through their intracellular domains, initiating multiple signalling pathways, such as phospholipase Cγ (PLCγ), PI3K–Akt and RAS–MAPK pathways [55]. FGFs are categorized into seven subfamilies based on their amino acid sequences [56]. FGF1 and FGF2 were first members identified in the FGF family and are known as acidic FGF (aFGF) and basic FGF (bFGF), respectively [57]. bFGF, which is highly expressed in acute wounds, can promote wound healing and tissue remodelling, playing a critical role in tissue repair [58]. FGF can effectively induce cell dedifferentiation to form pluripotent stem cells, which aids in tissue repair [59]. For example, recombinant FGF2 has been shown to induce the dedifferentiation of mouse astrocytes into neurogenic stem cells [60]. Murota et al. showed that the epithelial–mesenchymal transformation of marginal keratinocytes was enhanced, and wound healing was accelerated in wounds treated with recombinant FGF2 [61]. In addition, FGF2 regulates cell proliferation, migration, and angiogenesis through the NDY1/KDM2B–miR-101–EZH2 pathway [62]. Adding FGF2 in vitro can prolong the life of fibroblasts [63]. However, the evidence for the role of FGF2 in regulating inflammatory response is mixed. Qi and Xin found that FGF2 promoted the production of pro-inflammatory cytokines in primary human aortic smooth muscle cells [64]. It increased the production of interleukin-1β (IL-1β)-dependent pro-inflammatory protein substance P and its receptor NK1R in human articular chondrocytes [65]. However, Tu et al. found that injecting FGF1 or FGF2 reduced the inflammatory response associated with acute pancreatitis in mouse experiments [66]. FGF1 treatment decreased the release of TNF-α and IL-6. Therefore, the effect of FGF on inflammation may be influenced by the dose and duration of recombinant FGF administration as well as the tissue environment. MMPs play a crucial role in the remodelling phase of tissue repair, and FGF can regulate the expression of MMPs. The introduction of the FGF2 gene into HT-1376 cells upregulated the expression of MMPs 2 and 9. Anti-FGF2 antisense oligonucleotides downregulated the expression of MMP2 in KoTCC-1 cells [67,68]. In China and Japan, FGF has been widely used for treating surgical wounds, such as skin grafts, obstetric wounds, burns, and ulcers [69].

2.2.3. Signalling pathways

• Cell signalling pathway of the stromal cell–derived factor-1/CXC chemokine receptor 4 axis

Stromal cell–derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) belong to the CXC chemokine and CXCR-G protein–coupled receptor superfamily, respectively. The cell signalling pathway of the SDF-1/CXCR4 axis plays a crucial role in multiple aspects of growth and development, such as embryonic development and organ homeostasis, as well as inflammation, neurogenesis, haematopoiesis, angiogenesis, cancer metastasis and Human Immunodeficiency Virus (HIV) infection [70,71]. In wound repair, SDF-1 levels are significantly elevated at the wound margins after injury. CXCR4, the receptor for SDF-1, is expressed in epidermal stem cells and proliferating epithelial cells. Blocking the SDF-1/CXCR4 axis results in significantly reduced epidermal stem cell migration to SDF-1 in vitro and delays wound healing in vivo. Conversely, treatment with SDF-1 enhances epidermal stem cell migration and proliferation, accelerating wound healing. It is concluded that the SDF-1/CXCR4 signalling pathway promotes the migration and proliferation of epidermal stem cells to accelerate wound healing [72]. In addition, the SDF-1/CXCR4 signalling pathway recruits bone marrow mesenchymal stem cells to migrate to the wound site, facilitating tissue repair [73,74]. At the same time, this pathway promoted CXCR4-expressing endothelial cells to secrete vascular endothelial growth factor (VEGF), which stimulates angiogenesis, further supporting wound healing by enhancing blood supply to the damaged tissue [75]. Other studies showed that this signalling pathway also accelerated skin epithelialization and dermal structure regeneration by promoting fibroblast migration during the remodelling stage of skin healing [76].

• Wnt signalling pathway

The Wnt signalling pathway is a fundamental signalling pathway involved in growth and development, playing a key role in various biological processes. Wnt ligands can be categorized into two types based on whether their intracellular signalling is mediated by β-catenin. The ‘classical’ Wnt signalling pathway, with Wnt-3A as the prototype, relies on β-catenin to mediate its effects. The ‘non-classical’ pathway, with Wnt-5A as the prototype, operates through β-catenin-independent mechanisms, influencing different cellular responses and developmental processes. Wnt signalling pathway receptors are transmembrane proteins, primarily including frizzled (FZD) protein and lipoprotein receptor–associated protein (LRP). In β-catenin-mediated Wnt signalling, FZD interacts with LRP5 or LRP6 to form a complex. After binding to the complex, the conformation of the dimer changes, resulting in the accumulation and translocation of β-catenin into the cell nucleus. β-catenin binds to TCF/LEF transcriptional enhancers in the nucleus to promote specific gene expression.

The overexpression of the β-catenin gene is the key to skin fibrosis in the Wnt signalling pathway [77,78]. Wnt-3a can induce β-catenin activation, stimulate fibroblast proliferation and migration, collagen gel contraction, myofibroblast differentiation, and enhance the expression of pro-fibrotic genes. In vitro studies showed that the skin fibroblasts from patients with systemic sclerosis had reduced levels of Wnt inhibitor WIF-1, upregulation of Wnt/β-catenin signalling and excessive ECM deposition [79]. Moreover, the Wnt ligand is necessary for TGF-β1 to induce fibroblast differentiation, and the inhibition of β-catenin in fibroblasts can lead to decreased expression of myofibroblast markers [80].

The Wnt/β-catenin signalling pathway can be inhibited through various methods to reduce fibrosis production. Porcupine (PORCN) is an O-acyltransferase responsible for palmitoylating Wnt proteins, which is crucial for their secretion and signalling. PORCN inhibitors, such as LGK974, can effectively inhibit Wnt ligand maturation, which in turn downregulates the Wnt/β-catenin signalling pathway [81] and reduces skin fibrosis [82]. The small-molecule inhibitor XAV939 works by inhibiting ADP ribosyltransferase (tankyrase), which prolongs the half-life of the scaffold protein Axin. Axin forms a β-catenin-degrading complex with proteins like APC, GSK3β, and CK1. By stabilizing Axin, XAV939 promotes the degradation of β-catenin, which in turn reduces fibrosis by inhibiting the Wnt/β-catenin signalling pathway that drives fibrotic processes [83]. The inhibition of β-catenin expression can effectively reduce wound fibrosis. However, β-catenin and e-cadherin maintain the epithelial integrity of cell membranes. Hence, fine-regulating the expression of β-catenin and balancing the negative influence of inhibiting its effect are the challenges in applying the Wnt/β-catenin signalling pathway to promote scarless healing [84].

• Fluctuations in cytoplasmic Ca2+ concentration ([Ca2+]i) and the rho/ROCK signalling pathway

Myofibroblast contraction is the basis of excessive tissue remodelling and the characteristic of scar fibrotic tissue contracture. Tissue remodelling during fibrosis development results in a gradual increase in the hardness of the ECM. The contractile activity of myofibroblasts is further strengthened by external stimulation and the increase in matrix hardness. The myoblasts contract in vivo via two mechanisms, which act independently to cause myosin light chain (MLC) phosphorylation to cause contraction [85–87]. The first mechanism is to stimulate the increase in intracellular Ca2+ concentration in myoblasts with the increase in external tension or matrix hardness. The greater the matrix hardness, the higher the peak of Ca2+ concentration, and the shorter the time required to reach the peak [88]. It is manifested as a series of high-frequency, low-intensity, and low-tension fibroblast contractions initiated by the fluctuations in Ca2+ concentration. Ca2+ membrane channels associated with tissue repair and fibrosis include Piezo1, Piezo2, and members of the transient receptor potential vanilloid channel family (especially TRPV4) [89]. The second mechanism is the conversion of inactive Rho GDP into Rho GTP by guanine nucleotide exchange factors after binding of G protein–coupled receptors to their ligands. Rho family members regulate the activity of ROCK1 and ROCK2 [90]. ROCK1 and ROCK2 can enhance MLC phosphorylation, resulting in strong and long-lasting isometric contractions of fibroblasts. The use of ROCK inhibitor Y27632 can reduce the contraction of granulation tissue at the wound site [91].

• Hippo signalling pathway and integrin-linked kinase

The Hippo signalling pathway inhibits cell growth and plays a role in regulating tissue homeostasis and organ size. It also plays an essential role in stem cell regulation, cell proliferation, cell survival, and tissue healing and regeneration [92]. This signalling pathway consists of a series of conserved kinases, including upstream kinase complexes such as MST1/2, SAV1, LATS1/2, and MOB1 and downstream transcription complexes YAP/TAZ–TEADs [93]. SAV1 and MOB1 are adapter proteins that bind to MST1/2 and LATS1/2, respectively, enhancing their phosphorylation. YAP is the main effector molecule downstream of this pathway and can be directly phosphorylated by LATS1/2. The phosphorylated YAP binds to cytoplasmic proteins, remaining in the cytoplasm where it is subsequently ubiquitinated and degraded. This process inhibits the growth-promoting and anti-apoptotic functions of YAP. The Hippo pathway negatively regulates YAP activity through this cascade of phosphorylation. In contrast, when the Hippo pathway is suppressed, YAP can be transported to the nucleus and binds to transcription factors such as TEADs to promote the expression of target genes [94].

Integrin-linked kinase is a focal adhesion protein that regulates cell proliferation, migration, adhesion, and epithelial–mesenchymal transition [95]. Studies have shown that the overexpression of LIK can lead to the accumulation of YAP in the nucleus and the inactivation of the Hippo signalling pathway. Therefore, the inhibition of LIK can lead to the activation of the Hippo signalling pathway, resulting in the phosphorylation and degradation of YAP and the decline in the transcriptional activity of TEADs [96]. Petrou et al. found that YAP, TAZ, and TEADs were overexpressed in fibroblasts in scar tissue. Also, a significant positive correlation was observed between YAP/TAZ and TEAD expression, indicating that the two could synergistically promote scar formation [97]. Petrou et al. also found that YAP/TAZ and TEAD4 could be inhibited by knocking out YAP or using the YAP inhibitor verteporfin, which significantly inhibited fibroblast proliferation and induced apoptosis, thus reducing scarring.

2.2.4. Expression of wound-healing-related genes

Scar formation after wound healing is highly variable in gene expression. Many upregulated and downregulated antifibrosis genes were detected in the foetal dermal matrix [98]. Hu et al. revealed differences in the expression of 546 genes between fibroblasts and keratinocytes in scar healing and non-scar healing, which were regulated by more than 60 different pathways [99]. In addition, the fibroblasts of different lineages exhibit varying effects on scar formation. This section introduces several target genes and regulatory factors related to scar formation, as discussed in recent literature. Key factors include including the SRY-related HMG-box (SOX) family, TWIST1, YAP-related genes, En1, and Trps1.

• SOX family

SOX is a gene family composed of sex determination region of chromosome (SRY)-related genes, which encodes a series of transcription factors of the SOX family. The SOX family plays a vital role in determining the fate of cells and is involved in the healing process of various tissues, such as skin, fractures, tendons, and corneas. In the SOX family, SOX2 and SOX9 promote cell renewal, whereas SOX7 enhances the microvascular environment. Induced pluripotent stem cells (iPSCs) are known to accelerate wound healing, reduce scarring, and promote regeneration. The induction of somatic cells into iPSCs requires the action of SOX2. In addition, SOX2 can regulate the migration and proliferation of keratinocytes, promote the regrowth of the upper epidermal layers, accelerate wound healing, and reduce scars [100].

• twist1

TWIST1 is an evolutionarily conserved basic helix–loop–helix transcription factor, initially identified for its essential role in embryonic development of Drosophila. In 2022, Zhou et al. conducted a single-cell transcriptome comparative analysis using scRNA-sequencing on 28,064 cells derived from diseased skin tissue and adjacent normal skin tissue of 4 patients with keloids. They analysed each cell type present in the samples [101]. Their unbiased clustering analysis of single-cell data yielded 21 cell clusters involving 11 cell lineages. In these 11 cell lineages, the proportion of fibroblasts and vascular endothelial cells significantly increased in the diseased skin tissue. The team used Monocle 2 to perform a pseudo-time series analysis on all fibroblasts to obtain representative transcription factors that promoted fibrosis. Through constructing a gene regulatory network, they ultimately pinpointed TWIST1 as a candidate target gene involved in the fibrotic process. The intervention at the cellular level using harmine, a specific inhibitor of TWIST1, confirmed that the expression of the TWIST1 gene was significantly inhibited. As a result, the expression of fibrosis-related genes, such as COL1A1, COL3A1 and FN1, as well as TGF-β pathway–related genes, including TGFBR2 and TGFB1, was significantly downregulated after treating fibroblasts of diseased tissue with harmine. Harmine treatment had less effect at the protein level on normal fibroblasts compared with fibroblasts in patients with keloids. These results suggested that TWIST1 could be a promising target for keloid treatment, and harmine may serve as a potential drug for managing keloids.

• yap

YAP is a transcription factor downstream of the Hippo pathway that affects cell growth and response to mechanical stimuli. The specific mechanism is the same as detailed in Section 3.4. In 2021, Mascharak suppressed YAP expression in rat wound models with verteporfin and recorded the recovery at various time points: 0, 14, 30 and 90 days post-operation (POD0/14/30/90). The results showed that wounds recovered significantly faster in the verteporfin-treated group compared to the control group with hairless scars. Also, the wounds had substantial hair growth by day 30 and were indistinguishable from uninjured skin by day 90. These findings suggest that YAP is a key target associated with scarring, and that inhibiting YAP in vivo using verteporfin can promote scarless healing [102].

• Engrailed-1

Fibroblasts expressing Engrailed-1 (En1) are known as En1 lineage–positive fibroblasts (EPFs). This fibroblast lineage plays a dominant role in wound healing and responds to the mechanical cues of the local tissue environment. When En1 expression is activated, it initiates a fibrotic program that results in excessive type I collagen deposition, contributing to fibrosis. Notably, En1-overexpressing fibroblasts are absent in normal skin tissue, suggesting that their presence is a characteristic of pathological wound healing, such as in scar formation. En1 expression needs to be activated by YAP. After skin injury, changes in biomechanical and mechanical signals stimulate the accumulation of YAP in the nucleus. This activation of YAP triggers a series of subsequent reactions that cause fibroblasts to express En1, converting En1 lineage–negative fibroblasts (ENFs) at the wound site into EPFs. Mascharak et al. silenced En1 in fibroblasts with short hairpin RNA (shRNA) and compared it with that in the non-targeted control group. The production and deposition of ECM were significantly reduced, and fibrosis was downregulated by mechanical signalling pathways, such as the Hippo signalling pathway. This suggested that En1 knockdown broadly reduced mechanically induced fibrotic changes. Additionally verteporfin inhibited the fibrotic effect of En1 by inhibiting YAP, which was a key factor activating En1 [34].

• Trps1

Mascharak et al. compared the regenerated tissue induced by YAP knockout with scar tissue using single-cell transcriptomics, proteomics, and tissue ultrastructure analysis. Their findings revealed differences in Trps1 gene expression between the two types of tissue [103]. They overexpressed Trps1 during two crucial periods of wound healing: from day 3 (POD3, inflammatory phase) to day 7 (POD7, fibroblast phase), and from day 7 (POD7, epithelialization, and stroma formation phase) to day 17 (POD17, initial scarring phase). The scars of Trps1 OEE (overexpressing Trps1 at POD3/7) and Trps1 OEL (overexpressing Trps1 at POD7/17) wounds were less obvious compared with the scars formed by the control (Trps1 OEC). Additionally, the hair follicles were regenerated, and the matrix structure was similar to that of the undamaged tissue. The regeneration of Trps1 OEE wounds was more significant than that of Trps1 OEL. They also found that the expression of the Trps1 gene was necessary for the knockout of YAP to achieve regeneration. Besides knocking out YAP, Trps1 was also knocked out in POD3/7 and POD7/17, and hairless scars were formed in TRPS1-KDE and TRPS1-KDL wounds compared with regeneration in the control group (TRPS1-KDC). These experimental results clearly demonstrated the correlation between Trps1 and YAP and their significant roles in promoting scar-free healing.

2.2.5. Neurological factors

Tissue regeneration during foetal wound repair consists of two key phases: neuro-dependent and non-neuro-dependent. The first two-thirds of healing require neural induction, whereas the last one-third is non-neuro-dependent. Stelnicki et al. compared the skin incisions of normal foetal lambs with those of denervated foetal lambs and found that the incisions in the innervated lambs healed without scars, whereas those in the denervated lambs healed with scar formation [104].

Wound closure is primarily accomplished through wound edge-to-center contraction, a process driven by the activity of myofibroblasts. Smith et al. found that the myofibroblasts in tissues were closely related to sympathetic nerves [105]. During wound closure, myofibroblasts appeared approximately one day earlier than nerve ingrowth. The matrix components produced by myofibroblasts, such as laminin, played a key role in stimulating nerve axon growth into the wound site. Additionally, the lack of myofibroblasts resulted in poor innervation [106]. Constantinou et al. demonstrated that the expression of nerve growth factor (NGF) increased in the skin wounds of newborn foetuses under traumatic conditions; however, little or no expression of NGF was observed in adults [107]. NGF can regulate the transformation of fibroblasts into myofibroblasts, promote wound recovery, increase the expression of p75mRNA, regulate the apoptosis of myofibroblasts, avoid excessive wound fibrosis, and thus promote scar-free healing. In addition, NGF can also promote nerve growth, promote the release and migration of fibroblasts, stimulate the proliferation of basal keratinocytes, promote the expression of VEGF in capillary endothelial cells, promote cell mitosis, and thus accelerate epithelial repair.

3. A Biological model for the study of scarless healing

3.1. Early gestation foetus, oral mucosa and female endometrium

Trauma in early gestation foetus can heal without scars [108]. Scar-free healing has been observed when early gestation foetal skin is transplanted subcutaneously into immunodeficient nude mice, followed by the creation of incisions. Similarly, scar-free healing can also occur after transplanting age-matched human skin and making incisions [109,110]. Compared with human skin, oral mucosa wound healing, especially in gums, is basically free of scar formation. This is related to the difference in the expression of TGF-β at the wound. TGF-β1 can activate myoblasts and promote extracellular collagen secretion, whereas TGF-β3 inhibits scar repair. The low expression of TGF-β1 and the high expression of TGF-β3 in oral mucosal wound repair, in contrast to the scar formation seen in adult skin, promote the scarless healing in the oral mucosa. A high concentration of TGF-β3 can also regulate B cells and induce the expression of protease and integrin, thus inhibiting tissue inflammatory response [111,112]. A rapid and mild inflammatory response can facilitate scarless healing [113]. Additionally, the salivary mucin in saliva forms a hydrophilic viscoelastic gel, which can act as a barrier to protect the underlying mucosa from mechanical damage, isolate pathogens and other harmful substances, protect the wound, and promote wound healing [18]. Saliva also contains basic fibroblast growth factor (bFGF), which can inhibit TGF-β1, accelerate extracellular collagen degradation, promote ECM remodelling, and play an anti-scarring role [114,115].

The endometrium is a unique mucous membrane in the human body, consisting of basal and functional layers. It heals without scarring after various injuries and during each menstrual cycle, achieving complete regeneration [110]. This regenerative ability may be attributed to the tissue-specific characteristics of stromal cells (SCs) or their microenvironment, as the transformation of SCs into myofibroblasts plays a key role in the development of scars. Eremichev et al. found the formation of soluble factors during endometrial healing, which inhibited the transformation of SCs into myofibroblasts [116]. Christine Galant et al. discovered that the functional-layer residual tissue shed during the menstrual cycle is rich in gene products, such as collagen and its processing enzymes, that are related to ECM biosynthesis. This supports the hypothesis that the functional-layer residual tissue is involved in endometrial regeneration during the later stages of the menstrual cycle [117]. Additionally, oestrogen can also regulate the Wnt signalling pathway to promote endometrial regeneration [118]. Oestrogen can induce the rapid expression of ligands of Wnt4 and Wnt5a [119], which are essential molecules in the Wnt signalling pathway. Its signalling pathway antagonist SFRP2 inhibits DNA synthesis and cell growth of endometrial epithelial cells by inhibiting oestrogen-induced Wnt pathway–related molecules. The interstitial and epithelial interactions play an essential role in early endometrial repair. During early repair, the oestrogen level in circulating blood is extremely low and the number of receptors is also small. Despite this, temporal cells can still convert oestrogen signals into growth-stimulating signals, which they then transmit to promote epithelial proliferation. In addition, the normal wound repair matrix also plays a role in endometrial repair through factors such as MMPs, TGF-β, VEGF, and inflammatory cells. After an endometrial injury, residual endometrial dry/progenitor cells or bone marrow–derived stem cells from circulating blood may be stimulated to migrate to the site of injury, aiding in regenerative repair.

3.2. Other typical scar-free healing models

Besides the aforementioned models of scar-free healing, many animal models in nature can achieve scar-free healing of wounds. For example, zebrafish, which share similar wound-healing stages with mammals, demonstrate distinct advantages as a model for exploring scar-free healing. Each stage in zebrafish healing overlaps less and extends over a longer growth period, making them an excellent model for in-depth research on the healing process that occurs without scar formation. Rebecca used it as a model and found that the ready-made FGF was not necessary for wound re-epithelialization, but for epidermal remodelling and granulation tissue formation [120]. Other amphibians, such as Xenopus frogs and salamanders, also have scarless healing properties and can regenerate skin appendages. This ability does not disappear with age [121]. Besides embryos in mammals, antler regeneration, dolphins, and African spiny mice can also achieve scarless regeneration. Antler regeneration is one of the most typical examples of tissue regeneration. Mesenchymal cells from the periosteum of the pedicle form the base of the regenerated blast, and from these cells, new antlers are formed, including skin, nerves, bone and blood vessels [122]. The skin of the African spiny mouse is easily torn as a defense mechanism against predators. However, the wound quickly closes, and the tissue regenerates, including hair follicles and other related structures, returning the tissue to a state nearly identical to its original form [123].

4. New ideas for scar-free healing

4.1. Stem cell therapy

Besides the relevant influencing factors and treatment methods mentioned earlier, stem cell therapy is also an effective means to promote scar-free healing. The primary purpose of using stem cells is to improve the quality of wound healing, accelerate wound closure, and reduce adhesion and contracture [124]. It can not only reduce the formation of scars but also promote the recovery of skin accessory organs and achieve functional healing [125]. This makes it an effective treatment for chronic wounds [126]. Stem cells are of various types and perform different functions. Studies showed that human embryonic stem cells (hESCs) differentiated into basal keratinocytes and reconstituted the epithelium when placed in an induction medium containing bone morphogenetic protein 4 (BMP4) and ascorbic acid. When these cells were subsequently transplanted into nude mice, wound healing was accelerated [127]. Compared with adult stem cells, hESCs may be the most suitable cells for skin tissue regeneration due to their ability to self-renew and provide an unlimited supply of differentiated keratinocytes or keratinocyte progenitor cells for treating skin damage [128]. However, their clinical use is subject to ethical controversy and legal limitations. Besides hESCs, iPSCs are also effective in wound repair. This circumvents the ethical issues associated with hESCs and the risk of immune rejection [129]. Bilousova et al. demonstrated that iPSCs could differentiate into skin-related cells such as epidermis, hair follicles, and sebaceous glands in vitro [130]. The exosomes of mesenchymal stem cells (MSCs) derived from human iPSCs promoted wound healing in rat skin by promoting collagen synthesis and angiogenesis [131]. However, iPSCs still face several challenges in clinical application, including the risk of cancer development associated with retroviral vector use, issues with genetic stability, and the high costs of treatment [132]. At present, adult stem cells are mostly used in the clinical application of stem cell therapy to promote wound healing. More attention is paid to hMSCs and adipose-derived stromal cells (ASCs) [133]. hMSCs can secrete soluble factors to induce proliferation, migration, and chemotaxis of dermal fibroblasts [134]. In addition, hMSCs can improve skin wound healing when combined with bFGF [135]. A clinical study showed that the combination of bone marrow autologous MSCs and autologous skin fibroblast grafts in treating diabetic ulcers steadily reduced the wound size and increased the thickness of dermal vessels and dermis [136]. hMSCs can also downregulate the inflammatory response at the wound site, such as reducing inflammatory cell infiltration and lowering the levels of IL-1, IL-6, and TNF- [137]. ASCs are more readily available and have no ethical restrictions compared with other stem cells [138]. ASCs can promote collagen synthesis and differentiate into endothelial cells, where they secrete VEGF to stimulate the formation of new blood vessels, thereby enhancing wound healing [139]. All these stem cells affect wound healing and regeneration to varying degrees and in different aspects. Despite their limitations, the appropriate type of stem cells and regulating them effectively are crucial strategies for treating skin wounds.

4.2. Tissue engineering therapy

Tissue engineering therapy is a recently developed approach for wound repair. It combines biological materials with scaffolds to replace damaged skin tissue. This method accelerates wound healing, reduces wound contracture and scarring, and has remarkable efficacy and significant application prospects [140]. Scaffolds have various forms, including porous structures, fibres, microspheres, hydrogels, composite materials and cell-free structures. They are composed of biological materials that support cell growth and differentiation, similar to the native cellular microenvironment [141]. Their main function is to provide physical support, promote cell migration and proliferation, and thus accelerate wound healing [142,143]. Appropriate cell sources, such as hair follicle stem cells, melanocyte stem cells, adipose-derived stem cells, fibroblasts, and keratinocytes, can be selected based on specific therapeutic goals. These cells are inoculated on scaffolds and cultured for a designated period of time before transplantation to assist wound healing. Currently, tissue engineering products used in clinical application can be broadly classified into three categories: allografts (skin tissue cultured from donors of the same species), cultured epithelial autografts and synthetic skin substitutes. Allogeneic skin biomaterials are usually produced using live neonatal foreskin fibroblasts combined with a mesh structure, that can effectively cover deep lesions, venous ulcers, diabetic foot ulcers, and other chronic foot ulcers [144]. Clinically available allogeneic skin constructs (e.g. Apligraf^®, Dermagraft^®) primarily serve as biologically active dressings rather than permanent skin replacements [145,146]. Multicentre trials have shown that these products offer temporary protection for deep wounds by maintaining moisture, providing a barrier against microbes, and delivering short-term release of growth factors. Biodegradable synthetic substrates, including polyglycolic acid and polylactic acid; along with polymeric films like polyurethane or silicone; hydrogels typically containing synthetic components such as polyethylene oxide or polyvinyl alcohol [147]. However, the necessary microenvironment for exogenous stem cells is often absent. Cai et al. introduced an enzyme-crosslinked hyaluronic acid hydrogel designed to support the growth of exogenous bone marrow mesenchymal stem cells and enhance acute wound healing. This hydrogel is created by grafting dopamine onto hyaluronic acid, followed by enzyme crosslinking using horseradish peroxidase and hydrogen peroxide, which creates a loose, porous structure. In vivo experiments demonstrated that this hydrogel significantly accelerated the healing of acute full-thickness skin wounds, promoting the formation of appendages such as hair follicles and minimal scarring [148]. Additionally, synthetic nanofiber scaffolders made from material like polyε-caprolactone, all provide a three-dimensional (3D) structure that supports cell attachment and growth. With the advancement of 3D printing technology, it has become a promising tool for skin tissue replacement. Peng et al. used polylactide-co-caprolactone (PLCL) and collagen gel to print functional units of skin. Wounds treated with these PLCL-based constructs healed faster and showed the ability to regenerate skin appendages, such as hair follicles [149]. However, despite these advancements, tissue engineering techniques for wound repair still have limitations, such as the regulation of vascularization, immune rejection, and difficult regeneration of skin appendages, which need to be further examined and improved [150].

4.3. Inflammation and immune response regulation therapy

Another big difference between scar-free healing of foetal wounds and scar healing of adult wounds is the degree of inflammation and immune response. The inflammatory response is an integral part of the wound-healing process. However, an excessive inflammatory response can lead to uncontrollable scarring. The process of foetal scarless wound healing is characterized by minimal inflammation, whereas pathological scars usually involve an unregulated inflammatory response [42]. Ferguson et al. investigated the cellular and molecular differences between scar-free healing in foetal wounds and scar healing in adult wounds [151]. In embryonic wounds, the inflammatory response consists of a smaller number of less differentiated inflammatory cells. These cells remain at the site of the wound for a much shorter period than in adult wounds [108]. Walraven et al. reported a lower number of macrophages, dendritic cells, T cells, mast cells, and Langerhans cells and a higher proportion of M2-type anti-inflammatory macrophages in foetal tissue [152]. Brosnan et al. compared the inflammatory features of foetal wounds in E16 mice that healed without scarring with those in adult scar wounds. The results showed that inflammatory cells were recruited to the wound later and in fewer numbers in foetal wounds, and their persistence was much lower than that in adults [153]. In foetal wounds, macrophages and neutrophils were less abundant and showed lower phagocytic activity [154]. In addition, fewer mast cells and cytoplasmic particles were observed during foetal wound healing. Experiments using mouse models showed that mice with more mast cell expression also had more wound scarring [155]. The peritoneal injection of polydeoxyribonucleotides reduced mast cell degranulation and the release of inflammatory cytokines, thereby reducing the number of inflammatory cells and scar size [156]. The expression of inflammatory mediators cxygenase-2 (COX2) and prostaglandin E2 (PGE2) was lower during scar-free healing compared with scar wounds, and the application of PGE2 to foetal wounds increased the expression of TGF-1 and scar formation [157]. These studies suggested that manipulating the number or activation status of inflammatory cells at the wound site might be one way to control scarring during adult wound healing. For example, systemic administration of neuropeptide-alpha-melanocyte-stimulating hormone (alpha-MSH) has been shown to promote regenerative healing, resulting in reduced scarring at the macroscopic level and collagen fibre organization more closely resembling that of uninjured skin [158]. Although this effect requires the administration of alpha-MSH prior to injury, the prophylactic administration of antifibrotic drugs still holds clinical value, especially in situations such as scar repair surgery.

Compared with scar healing, scarless healing wounds present an “immunosuppressive” condition. From the perspective of animal models, although salamanders possess both T and B cells, acute rejection does not occur during tissue transplantation. Compared with conventional mouse models, African spinous mice had fewer circulating neutrophils, more mast cells than neutrophils, and no F4/80-positive macrophages [159]. In addition, Zhang et al. found that serum-soluble human leukocyte antigen E (sHLA-E) demonstrated a sensitivity of 83.69% and a specificity of 92.16% in identifying keloids. Furthermore, an incomplete reduction of serum sHLA-E level was associated with a higher recurrence rate [160]. This finding underscores the potential role of the immune system in the diagnosis of keloid disorders. Evidence suggests that excessive inflammation and immune response are detrimental to wound repair. So, does this mean that adult skin wounds can be reduced with immunosuppressive treatment at the wound site or throughout the body? Where does the balance lie? Although further scientific research and clinical evidence are needed, immunosuppressive therapy remains in its early stages and holds significant potential. (The scarless healing treatment strategies discussed in this fourth section are summarized in Table 2.)

Table 2.

Summary of treatment methods for promoting scar-free healing.

Therapeutic Strategy	Mechanism	Key Cells/Molecules	Advantages	Limitations	Clinical Applications
Stem Cell Therapy	Promotes tissue regeneration through differentiation/paracrine effects, modulates inflammation, reduces fibrosis	hMSCs, iPSCs, ASCs, hESCs	Multipotent differentiation potential Promotes angiogenesis Anti-inflammatory effects Reconstructs skin appendages	Ethical concerns (hESCs) Oncogenic risk (iPSCs) High cost	Bone marrow MSCs combined with autologous dermal fibroblasts for diabetic ulcers (Vojtassák et al., 2006)
Tissue Engineering	Provides 3D structural support via biomaterial scaffolds combined with cells/growth factors, reduces contraction	Synthetic materials (PLGA, PLCL), Natural materials (collagen, HA), 3D bioprinting	Customizable architectures Deep defect repair Epidermal-dermal regeneration	Vascularization challenges Immune rejection Limited appendage regeneration	PLCL-collagen gel bioprinted skin units for hair follicle regeneration (Peng et al., 2022)
Immunomodulation Therapy	Suppresses excessive inflammation (M1 macrophages), inhibits pro-fibrotic cytokines (TGF-β1), enhances M2 phenotype	α-MSH, Anti-TGF-β antibodies, NGF	Targets core scarring mechanisms Prophylactic potential	Requires early intervention Systemic immunosuppression risks	Systemic α-MSH administration reduces scar formation in rats (de Souza et al., 2015)
Signaling Pathway Targeting	Blocks pro-fibrotic pathways via small molecule inhibitors	LGK974 (PORCN inhibitor), XAV939 (β-catenin degradation), Verteporfin (YAP inhibitor)	Precise pathway modulation Inhibits fibroblast activation	Balance required between normal healing/fibrosis Long-term safety concerns	LGK974 treatment in systemic sclerosis models (Chen et al., 2017)

5. Conclusions

Since the discovery of scarless healing after fetal skin trauma in 1971 by Burrington and defining the concept of scarless healing, many related studies have been conducted. Despite advancements, currently no clinical method exists that is completely suitable for humans and guaranteed to achieve 100% regeneration. Thus, our efforts can only promote scar reduction indefinitely. In addition, the difference in adult individuals significantly impacts scar formation, and hence we should judge the overall situation of individual patients during the initial diagnosis period. Effective and accurate diagnosis is the cornerstone of subsequent recovery, especially the initial diagnosis and treatment of keloids. The early development of a personalized treatment plan is crucial for optimal recovery. We should take effective measures immediately after the injury to minimize tension at the wound site as much as possible by using tension reduction sutures and scar tape. Additionally, targeted intervention involving specific cells, cytokines, or signalling pathways, or the regulation of related genes, can be applied to modulate the wound healing process. However, promoting scarless healing will require a multifaceted approach, as no single strategy alone can achieve complete regeneration. Achieving scarless healing demands extensive scientific research and innovative thinking. In addition, more biological models of scar-free healing need to be explored to provide a scientific basis for achieving scar-free healing.

Funding Statement

This work was supported by the Traditional Chinese Medicine Science and Technology Plan of Hebei Province Administration of TCM. (Grant Number: 2025319).

CRediT author statement

Wenhui Yang: Conceptualization,Writing - Original Draf; LiuLiu: Project administration; Xingjian Cheng: Visualization, Supervision (All contributions must meet Taylor & Francis authorship criteria)

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data availability is not applicable to this article as no new data were created or analyzed in this study.