1. Context
Scars are distressing memorial signs of a healed wound, which can remain all through the life of a patient. Approaches to optimize the healing process of wounds will minimize the development of hypertrophic scars. Improving strategies in scar treatments are an imitation of what happens in fetal scarless wound healing. The longevity and severity of wound healing phases are different in regular, fibrotic, and scarless wound healing. The inflammation phase is very short and the proliferation and remodeling phases overlap in fetal wound healing. Applying molecular and cellular mechanisms underlying this profile brings about new and effective therapeutic strategies for elevating scars. There are several useful studies targeting signaling pathways, such as TGFβ and Wnt signaling, in scar formation, which none the less could be sufficient individually in scar reduction. Thus, therapeutic plans should be based on targeting relevant concentrations of proteins and their activities. Novel miRNA approaches, as post-transcriptional regulators of multiple proteins and associated pathways, have become an emerging field of interest. The use of fetal cells as the main source of scarless wound healing is beneficial in skin substitute constructions. Cell banks from fetal skin are being developed in short periods of time and with minimal cell culturing requirements and can be easily scaled out. Mesenchymal stem cells are valuable cell sources with their specific characteristics, such as high immuno-modulatory effects in scar environment and unique secreting growth factors, which stimulate keratinocyte migration and increase anti-fibrotic matrix metalloproteinase. Wound healing is commonly quicker and associated with less scarring in the oral mucosa than in the skin, therefore oral mucosal equivalents may contribute to fast and scarless wound healing.
Also, fat grafting is a promising treatment in scars due to mesenchymal stem cells homing in its SVF proportion.
2. Evidence Acquisition
More than 100 million patients in industrialized countries are effected by scars each year, which imposes a heavy financial burden on medical care systems (1). Skin health effects the physiological homeostasis of the human body. Skin is the largest organ of the body, which plays an essential role in protecting the body against mechanical forces, infections, fluid imbalances, and temperature disturbances (2). Wound healing is a defense mechanism against infection and further damage, which is mediated by fibroproliferative response resulted in scar formation (3). The major goals of the treatment of wounds are rapid wound closure in order to restore the protective barrier immediately (4, 5). Scars resulted from pathologic wounds are caused by abnormal fibroproliferative responses and regardless of beauty considerations and deformation that has profound psychological and social effects, they cause devastating disabilities in children with severe burn injuries. They can also have physical effects, such as growth constraints in children and reduce their quality of life (6-8).
Considering the importance of scarless wound healing, therapeutic strategies are expanding, which are categorized to 2 general groups: molecular based therapies and cell based therapies.
The most outstanding molecules involved in molecular based therapies of wounds without scars, were TGF-β3 and IL-10, which failed in phase III clinical trials (9). Among different types of molecular-based therapies, miRNAs are the most promising molecules because one miRNA could target more than 100 genes. Accordingly, miRNA molecules are different from other molecules because of pleiotropic actions through different signaling pathways (10).
Since most molecular-based therapies are not adequate individually and have limitations for scarless wound healing, cell-based therapies have attracted much attentions. There are many different kinds of cell sources for cell-based therapies of wounds in a scarless manner, such as fetal skin cells, mesenchymal stem cells, and oral mucosal cells. Fat grafting may also be considered as a cell-based therapy because its effectiveness is dependent on mesenchymal stem cells in its SVF proportion (1, 11).
Here, this study focused on the most important scarless wound healing strategies, which have been practiced all over the world.
2.1. miRNAs and Scarless Wound Healing
Recent advances have indicated that MicroRNAs play pivotal roles in scarless wound healing (10). miRNAs are defined as a highly conserved family of small noncoding, endogenous single stranded RNA molecules, with an average length of 22 nucleotides that post-transcriptionally repress their target mRNAs in a sequence-specific inhibition manner, predominately by pairing the 6 to 8 nucleotide site in seed sequence of miRNA to 3’UTR of mRNA (12-14).
miRNAs are primarily transcribed by RNA polymerase II to a long initial transcript or pri-miRNA. The pri-miRNA is processed to a shorter hairpin-shaped, double-stranded precursor miRNAs (pre-miRNAs) of approximately 70 nucleotides in length, by Drosha and Pasha/DGCR8. The resulting premiRNAs, with 2 nucleotides overhanging at their 3’ ends, have binding sites for RAN GTPase/exportin-5 that cause translocation of premiRNAs to the cytoplasm. Another RNA-specific nuclease, Dicer, cleaves the pre-miRNAs to 18- to 24-nt double-stranded RNAs. The resulting RNA duplex incorporates with the miRISC, where one of the strands is degraded while the guide strand becomes the mature miRNA. These mature miRNA guides miRISC to the target sequences by binding to complementary regions within the 3’UTR of the target mRNA and their degree of complementarity determines the inhibition of mRNA translation (imperfect matching) or cleavage of mRNA (perfect or near perfect matching) (15-18).
miRNA-based therapy in the field of dermatology is in its early stage, yet the initial findings are considerable, indicating that there are vast opportunities for developing effective therapies for scarless wound healing. Considering the important roles of miRNAs in skin development and pathology, it was assumed that miRNAs participate broadly in transition of scarless phenotype to a scarred phenotype during development. The comparison of genome-wide profiling of miRNAs showed that miR-29b and miR-29c are differently expressed between mid-gestational (E-16 day) and late-gestational periods (E-19 day). Their potential targets include Smads, βcatenin, and Ras, which were involved in several signaling pathways and ECM remodeling critical to scarless wound healing (19). MiR-192 has been demonstrated to be regulated by TGFβ and might perform its role by maintaining the scarless phenotype (19-21). The altered expression of miR-34 family (which target TGFβ) in mid-gestation fetal keratinocyte, in comparison with late-gestation fetal keratinocyte, may contribute to less scar formation. The MiR-34 family suppresses the expression of genes involved in the TGF-β pathway, such as TGF-β3, TGF-βRI, TGF-βRII, SMAD3, SMAD4, and SAR1A (22, 23).
Additionally, studies have shown that inhibition of miR-145 shows a significant decrease in type I collagen expression, TGF-β1 secretion, and contractility in skin myofibroblasts, which could have roles in scar reduction (24, 25). Furthermore, overexpression of miR-149 could down-regulate the expression of pro-inflammatory cytokines, such as IL-1α, IL-1β, and IL-6 in inflammatory conditions and also indirectly accelerate the expression of TGF-ΒIII and collagen type III in fibroblasts (26).
Another miR, which targets TGFβ-1 is miR-185, which also targets collagen-1 in hypertrophic scar fibroblasts (27). Taken together, all these miRNAs could be promising targets for scarless wound healing.
2.2. Fetal Cell Therapy and Scarless Wound Healing
Since fetal wounds heal without a scar early in gestation, it may hold the key to scarless repair. In fact, the fetus has a more efficient regeneration system to quickly restore tissue integrity and heal the wound in a scarless manner while adults’ wounds are repaired with the formation of scar (28). Among different kinds of cells used for cell-based therapies for wound management of the skin, fetal cells present biological, technical, and therapeutic advantages over others, for instance they are differentiated cells with low immunogenic and high expansion and regeneration characteristics (29).
Their cytokine and growth factor expression profiles are also different from adult cells. Central to this different cytokine expression profile, is the TGF-β family, which has been most widely studied as it is implicated in the transition between scarless healing and repair with scar formation. There are 3 highly homologous isoforms of TGF-β known in humans, including TGF-β1, TGF-β2, and TGF-β3. The relative proportion of TGF-β isoform determines the wound repair outcome (30, 31).
Besides, they are able to secrete more VEFG-A, and fetal fibroblasts and keratinocytes demonstrate no expression of MHC class II with the low expression of MHC class I (HLA-A, -B and -C). They also show expression of indoleamine 2,3 dioxygenase (IDO-1 and - 2), which has immunomodulatory effect on T-cells (32). Their collagens are mainly (30% to 60%) collagen type III, which is composed of smaller and finer fibers than type I that produces higher reticular pattern of fiber deposition in the fetus. They can also proliferate and produce collagen at the same time, and stimulate extra cellular matrix replacement directly or indirectly in tissue repair process for migration to the wound area. Their capability to adapt to biomaterials and strong resistance to oxidative and physical stresses in wound environment, may lead to their durability in the wound milieu (29, 33). Scarless wound healing in fetal skin at early gestation is a result of the unique cytokine or growth factor profile. Considering all these advantages, fetal cells are considered to be a potent candidate for scarless wound healing-based therapies.
Since fetal cells have the advantage of being an organ donation, only very small biopsies are required for developing extensive and stable master cell banks for many tissues. In 2005, Hohlfeld et al. used tissue engineered fetal skin seeded on collagen sheets for 8 children with burns and mean time to healing for the burns was 15.3 days after the first construct application. The results were satisfactory in all 8 children, showing little hypertrophy of new skin (34). In another study, the capacity of fetal cells associated with collagen matrix for treating various wounds was analyzed. Their results indicated that fetal cells accelerated the repair process and reduced scarring in severe burns and wounds of various nature in children (35). Ramelet et al. evaluated the tolerance of several treatments with fetal cell biological bandages and interaction with the chronic wound environment. Their data demonstrated that the fetal skin construct had promising results for both chronic wounds and severe burns (36). In 2013, Zuliani et al. applied fetal fibroblasts and keratinocytes to stimulate wound repair by allogeneic fetal cell-based dressing for acute and chronic wounds. They showed that direct interaction between fetal fibroblast and keratinocytes strongly enhanced wound healing growth factor and had therapeutic benefits over fetal fibroblast cells, individually (32). In 2015, Larijani and Ghahari suggested that applying human fetal skin fibroblast alone or in combination with biocompatible scaffold could be beneficial for treatment of ulcers, such as diabetic wounds (29).
2.3. Mesenchymal Stem Cells and Scarless Wound Healing
Adult mesenchymal cells that contribute to the normal healing process of the wound are tissue repair mediators. These cells reduce the inflammation of the wound and reprogram the resident cells and immune cells in order to regenerate the tissue and achieve anti-fibrotic effects. Since in the mammalian fetus healing of the wound is performed without any scar, researchers have made great efforts to model embryonic tissue to devise effective methods for treating wounds. The most prominent of these methods were using Avotermin (TGFβ3) and IL-10 (ilodecakin), which failed in phase III clinical trials. It was indicated that molecular-based methods individually, did not have the potential to treat wounds in a scarless fashion (37, 38).
Recent studies clearly revealed that cell-based therapies are much more powerful than molecular based methods in scarless wound healing.
Adult mesenchymal stem cells express the most signaling molecules involved in the simulation of an environment without ascar, such as TGFβ3, IL-10, and 2,3- dioxygenase indolamin and so on, which have immunomodulatory characteristics involved in wound healing with less scar. Thus, mesenchymal stem cells could be considered as an appropriate candidate for cell therapy to heal a wound without scars (39, 40).
Another characteristic of mesenchymal stem cells, which could be applied in scarless wound healing, is their conditioned media. Exploring the paracrine effects of mesenchymal stem cells on dermal fibroblasts indicated that the proliferation, migration, and the ratio of matrix metalloproteinase to tissue inhibitor of metalloproteinases of fibroblasts increased yet the ratio of TGF-β1/3 was reduced and they transitioned to a phenotype with a lower myofibroblast formation capacity (41, 42).
Seo et al. found that local injection of heterogeneous mixture of 2 million amniotic fluid-derived mesenchymal stem cells and amniotic membrane into the postsurgical scar reduced pain and scarring (43). The use of lipid grafts, which is a source of mesenchymal stem cells, has also led to a reduction in both neuropathic and hypertrophic scars. lipo grafts enriched by mesenchymal stem cells have been used in two studies evaluating their effectiveness on healing chronic ulcers caused by radiation in 10 individuals (44). In another study, 29 cases underwent the injection of lipograft enriched with MSC for removing soft tissue defects. The results of these investigations indicated that lipograft enriched by MSC improves wound healing, increases the effect of fat transplantation, and prevents deep tissue fibrosis and skin scarring (45).
Several studies have been carried out on the efficacy of mesenchymal stem cells in wound healing in multiple animal models. However, due to the fact that laboratory animals like rats and mouse have loose skin, wound healing does not lead to scarring. The model of burn in pigs is the closest model to the creation of scars in humans. Most animal studies have focused on the effectiveness of MSCs in wound healing, and have paid less attention to prevent scarring after wound closure. In 2012, Yun et al. evaluated the effect of adipose-derived stem cells on scar formation and remodeling. They created full-thickness defects on the dorsal skin of pigs and after 50 days, when the defects transformed to scars, they injected 106 adipose-derived stem cells into the scar of the full thickness wound on the back of the porcine models. The results indicated that the scars were reduced, collagen pattern was improved, the number of mast cells was decreased, the expression of TIMP1 and αSMA genes was decreased, and the expression of MMP1 gene was increased (46).
In 2015, Zhang et al. investigated the efficiency of intra-lesional injection of adipose derived mesenchymal stem cell on a hypertrophic scarring in a rabbit ear model. A full-thickness wound was made on the back of the ear of rabbits and 4 × 106 cells were locally injected 14 days later. The conditioned media was also employed as a control. The histological results, ultrasonography, and the level of αSMA gene expression demonstrated the reduction in scar size in relation to internal control 35 days after injection (47). In a recently published research, the secretory exosomes from adipose-derived mesenchymal stem cells caused scarless wound healing by regulating extracellular matrix remodeling. It was found that intravenous injection of exosomes secreted by adipose mesenchymal stem cells caused the level of TGFβ3 to increase in relation to TGFβ1 and prevented fibroblasts from differentiating to myofibroblasts (48).
2.4. Oral Mucosa and Scarless Wound Healing
The first and most obvious difference between skin and mucosa is their different environment; mucosa has a humid environment as a result of salivary and microflora yet findings indicated that wound healing in oral mucosa is likely to involve intrinsic features of mucosal tissue and is not only owing to environmental factors (49).
Although healing of both dermal wounds and oral mucosa progresses through the same stages, involving hemostasis, inflammation, proliferation, and remodeling of the collagen matrix yet there are differences in fibroblast, macrophage, and neutrophil activity at various stages of wound healing. Moreover, in oral mucosa, response to the injury are commonly more rapid and with less inflammation and angiogenesis than in the skin, with minimal or no scar formation. Mucosal wounds show a lower inflammatory response with lower neutrophil, macrophage, and T-cell infiltration, as well as decreased expression of pro-inflammatory TGF-β1, yet the level of TGF-β3 expression, which has anti-fibrotic properties in oral mucosa has increased.
Moreover, in oral wounds, remodeling of the wound matrix seems to be quicker than skin wounds (50). It was demonstrated that oral mucosa equivalents (sheet) containing oral keratinocytes and oral fibroblasts may have a role in rapid scarless wound healing. The skin and oral mucosa vary in their histology of each tissue, and keratinocytes from these 2 sites differ in function (51). A previous investigation suggested that differences in response to injury between oral and skin keratinocytes are revealed in gene expression profiles. It was found that 13,710 genes are differently expressed between oral mucosa and the skin. These differently expressed genes might affect keratinocytes and cause skin keratinocytes to show lower intrinsic proliferation and migration capacities than those of oral keratinocytes (52). Also, the cellular response to injury in mucosa and skin are different. There are also differences between fibroblasts in oral mucosa and skin. Dermal fibroblasts proliferated less than mucosal fibroblasts and showed a greater collagen gel contraction because of a greater response to TGFβ1 (53) (Figure 1).
A schematic Representation of Effective Strategies in Reducing Scars
2.5. Fat Grafting and Scarless Wound Healing
Fat is abundant and easily accessible, and for the first time in 1912, it was used for the treatment of facial lipoatrophy by Eugene Holler (54). In 1992, Coleman described a new technique for increasing survival of adipose cells, and nowadays his method is widely used (55). Adipose tissue contains cellular and extracellular matrix components with stem cells in its cellular component, which are capable of differentiating to other cells, such as cartilage, bone, nerve or muscle (56). These cells show less potential than embryonic cells yet compared with bone marrow stem cells, they are easily harvested (55). Lipotransfer seems to improve the structural features of the extracellular matrix and increase its production (57). Histologic assessment after fat transfer revealed new collagen synthesis and dermal hyperplasia with hypervascularity, which was observed locally (55) and in a molecular study, results demonstrated downregulation of fibrotic markers along with upregulation of vasculogenic markers documented using both techniques (58). Jaspers et al. demonstrated the influence of fat grafting on functional scar parameters, such as pliability and overall scar quality in a 3-month follow up and sustainable effectiveness of single-treatment fat transfer after a 12-month assessment (59, 60). Sardesai suggested that lipotransfer appears to improve dermal characteristics, including elasticity, skin thickness, stiffness, and pliability with quantitative and qualitative analysis (61). The exact mechanism of these changes is yet unknown but one hypothesis for the dermal improvement other than direct influence of fat on surrounding tissue, is adhesiolysis. In addition, after fat grafting, perivascular adipose stem cells may result in adipocyte regeneration and revascularization (59). Rapp et al. studied the effect of autologous fat and adipose-derived progenitor stromal cells on swine hypertrophic burn scars and revealed a decrease in scar thickness and reduction in inflammatory profile with altered fibroblast gene expression in RNA sequencing (62).
3. Results
In this review, the known modalities of scarless wound healing, with a focus on cell based therapies, are discussed. Due to the pleiotropic effect that miRNA could exert on scarless wound healing, miRNA-based therapy is considered as the only molecular based strategy that attempts to achieve scarless wound healing in adults, whether through fetal mimicry or attenuating fibrotic response. Fetal cells, mesenchymal stem cells, oral mucosa, and fat grafting are investigated completely. Because the effectiveness of fat grating is associated with its mesenchymal stem cell, this study included it in cell-based therapy. This study attempted to clarify promising clinical targets and evaluated therapeutic strategies currently in practice.
4. Conclusions
Considering all the major experimental and clinical strategies in order to achieve scarless wound healing, it is apparent that most can decrease scar thickness and appearance yet will not generate histological or functional similarity to uninjured skin. Since fetal wound healing is a remarkable process that is primarily different from postnatal healing, it occurs rapidly and without any scar, and offers promising insight for wound healing in a scarless fashion. However, because the factors responsible for the fetal wound healing phenotype were originally ascribed to the intrauterine environment, this has been demonstrated to be neither necessary nor sufficient for scarless healing.The capability of regeneration is now believed to be intrinsic to the tissue itself, and is closely associated with fetal dermal constitution. Consequently, once cell-based therapies will be effective that they were placed in a niche where their normal physiologic condition could be induced with high accuracy. Therefore, using biomimetic materials, which mimic extracellular matrix and produce structural support, is essential.
Current trends in scarless wound healing strategies combine cell-based therapies and regenerative medicine in order to accomplish better results.
