Min-Min Zhang and Xiao-Dong Chen,2,?

1Medical School, Nantong University, Nantong, Jiangsu 226001, China, 2Department of Dermatology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China.

Introduction

Keloids are a benign proliferative disease of the skin caused by abnormal healing of physiological wounds.Keloids are similar to hypertrophic scars. However, keloids extend beyond the margin of the original wound and do not spontaneously regress, while hypertrophic scars are confined to the original wound and generally maintain their shape.1Keloids cause pain, pruritus, restricted joint activity and cosmetic problems, and negatively affect quality of life. The bioactivity of keloids is regulated by various factors,such as transforming growth factor(TGF)-β, connective tissue growth factor (CTGF), and hypoxia inducible factor (HIF).2-4These inflammatory factors are involved in keloid fibrosis, collagen production, and the deposition of extracellular matrix. However, the pathogenesis of keloids remain unclarified, and it is still one of the most challenging diseases in clinical practice.

The treatment of keloids includes intralesional corticosteroid injections,laser therapy,cryotherapy,compression therapy, surgery, and combination therapy. The first-line therapy for keloids is intralesional corticosteroid injections. However, no methodology has yet emerged as the“gold standard”of clinical care.1Furthermore,the current keloid therapies have many shortcomings, such as a long treatment course,pain, and a high recurrence rate, which lead to decreases in patient compliance and confidence during treatment.Therefore,the exploration of novel and more effective keloid treatment methods is extremely urgent and important.

Stem cells are self-renewing pluripotent cells that are classified as embryonic stem cells and adult stem cells in accordance with their developmental stage. Stem cells secrete a variety of paracrine factors that areantifibroticand inhibit collagen deposition, and stem cell therapy has achieved good results in fibrosis-related diseases such as renal fibrosis,pulmonary fibrosis,and urethral stricture.4-6

The fate of stem cells is determined by their microenvironment. Under normal conditions, the proliferation of stem cells is tightly controlled. Stem cells with clonality,pluripotency, and self-renewal capacity are present in keloids,and these stem cells contribute to the pathological environment required for the growth of benign tumor-like cells.7This suggests that there may be some triggering factors in keloids that potentially enables the upregulation of embryonic stem cell markers and the induction of cell mesenchymal differentiation to form keloids.

To investigate this possibility, we reviewed the recent application of stem cells in keloids and other fibrotic diseases and summarized the potential therapeutic effects of stem cells on keloids.

Possible mechanisms of stem cell therapy

Anti-proliferative effects

The production of keloids is closely related to the abnormal proliferation of keloid fibroblasts(KFs).Studies have shown that adipose-derived stem cell(ADSC)-conditioned medium(CM)promotesthepresenceof G0/G1phasecellsinKFs,and significantly reduces the proportion of G2/M phase cells,thereby inhibiting the proliferation of KFs.8Compared with normal fibroblasts,KFs significantly reduce apoptosis under serum-free culture conditions.9Thus,the hyperproliferation of KFs in keloids may be related not only to their rate of division but also to their decreased apoptosis. ADSCs also significantly increase the apoptosis of KFs.10All of the following mentionedmechanisms are summarized in(Fig.1).

Antifibrosis

Chemotactic cytokine ligand 2/chemotactic cytokine receptor 2

Monocyte chemoattractant protein-1, also known as chemotactic cytokine ligand 2 (CCL2), is a profibrotic factor that promotes fibroblast proliferation and collagen formationviathe TGF-β pathway. Current research indicates that peripheral CD14+monocytes from patients with keloids induce fibroblast proliferation by releasing CCL2.11Furthermore,CCL2 mediates the amplification of the pro-inflammatory factor interleukin(IL)-17.12Fibrosis is reducedby the inhibition of CCL2 or by the antagonism of its high affinity receptor, chemotactic cytokine receptor 2(CCR2). In bleomycin-induced pulmonary fibrosis models,amniotic fluid-derived stem cells(AFSCs)inhibit pulmonary fibrosis by modulating the level of CCL2 bothin vitroandin vivo. The underlying mechanism by which AFSC reduces CCL2 is thought to be that AFSC secretes matrix metalloproteinase-2 to induce the proteolytic cleavage of CCL2,which induces the formation of the CCR2 antagonist cleavage product. The hydrolysate of CCL2 is a receptor antagonist of CCR2 with a high affinity for CCR2,but does not induce a response when bound. CCR2 is expressed in AFSCs, so the increase in CCL2 concentration has chemotaxis to AFSC.4,6Therefore,AFSC may inhibit fibrosis of keloids through the CCL2/CCR2 pathway.

TGF-β1

TGF-β is a multifunctional cytokine that contains three different isoforms (TGF-β1, TGF-β2, and TGF-β3) in mammals and activates the membrane receptor serine/threonine kinase complex composed of type II(TβRII)and type I (TβRI) receptors. Upon TGF-β binding, TβRII phosphorylates and activates TβRI,resulting in activation of the TGF-β/Smad signaling pathway. The TGF-β/Smad pathway plays an important role in cell growth,differentiation, apoptosis, and proliferation.2Furthermore, TGF-β1 plays a key role in the progression of fibrosis by inducing matrix productionviaa Smad3-dependent mechanism.13TGF-β1 levels are significantly reduced by bone marrow mesenchymalstem cells(BM-MSCs)ina lungfibrosismodel and skin fibrosis model, resulting in a concentrationdependent antifibrotic effect.14-16TGF-β also significantly increases the expression of collagen I in KFs. The level of collagen I upregulated by TGF-β in KFs is attenuated by amniotic mesenchymal stem cells(MSCs).However,TGF-β and amniotic MSC-CM do not affect the levels of collagen III in keloids, mature scars, or normal skin fibroblasts.2In addition to the Smad family, TGF-β1 also activates the mitogen-activated protein kinase pathway, including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38 kinase. P38 stimulates the transcription and expression of TGF-β2 in KFs under serum stimulation.17Thus, ADSC-CM reduces collagen deposition and inhibits hypertrophic scar formationviathe p38/mitogen-activated protein kinase signaling pathway,18and may play the same role in keloids.

α-Smooth muscle actin

In keloids,the expression of α-smooth muscle actin(α-SMA)is higher than that in mature scars and normal skin.2In a dermalfibrosismodel,α-SMAexpressionisstronglypositive,suggesting that myofibroblasts are active in the formation of skin fibrosis. MSCs promote myofibroblasts to remain dormant and reduce the expression of α-SMA, suggesting potential therapeutic value for keloids.19The expression of α-SMA in keloids is significantly upregulated by TGF-β,while AFSC-CM significantly attenuates α-SMA expression upregulated by TGFβ in KFs.2

Connective tissue growth factor

CTGF is a 36-38-kDa stromal cell protein belonging to the multifunctional cysteine-rich angiogenic inducer 61,CTGF,and nephroblastoma-overexpressed family.CTGF expression is induced by TGF-β, vascular endothelial growth factor(VEGF),angiotensin II,and human growth factor.However,TGF-β is the most important promoter of CTGF expression. Continued overproduction of CTGF was responsible for the maintenance of keloid fibrosis, suggesting that inhibition of CTGF activity may reduce keloid formation.3In a renal fibrosis model, human ADSCs significantly reduce the level of CTGF.20

Anti-inflammatory response

Wound healing in the mammalian fetal period is scar-free,as the healing process involves less inflammatory cells,less inflammatory mediators, and a shorter inflammatory reaction phase than that in the non-fetal period. The oral mucosa has a similar extracellular matrix to the fetal phase, and thus also has minimal scar formation.21In recent years, inflammation has been recognized as the leading cause of keloid formation. Therefore,reducing the concentrations of inflammatory cells and inflammatory mediators, and shortening the process of inflammation are very important for the treatment of keloids.

Inhibition of inflammatory cell infiltration

The development of keloids is closely related to the longlasting inflammatory response.Mouse ADSCs(mADSCs)reduce the infiltration of neutrophils,macrophages,and T lymphocytes in the inflammatory phase of lung injury,suggesting that similar effects may be exerted in keloids.16

Inhibition of pro-inflammatory factors

IL-12 is a cytokine with a wide range of biological activities that is mainly produced by activated inflammatory cells.The expression of IL-12 is significantly increased in keloid macrophages.22The expressions ofTNF-α andIL-12mRNA in macrophages cocultured with mADSCs are significantly downregulated in a dose-dependent manner,indicating that mADSCs may control the inflammatory response in keloids by inhibiting the expression of IL-12.16IL-6 is a pleiotropic cytokine that is involved in the inflammatory pathway,hematopoiesis and immune regulation. Keloids are associated withIL-6gene polymorphisms in Japanese and Chinese populations.23-24Furthermore,BM-MSCs reduce the level of IL-6 in a liver fibrosis model,suggesting that they may play the same role in keloids.25-27

Anti-vascular factors

The mechanical biology of the dermis and blood vessels may play an important role in the pathogenesis of scars. An increase in wound tension may result in increased inflammation and vascular permeability as a result of the expansion of the gap between the endothelial cells of the blood vessel wall.This increase in vascular permeability allows inflammatory cells to migrate into the interstitial space.28Keloids have a significantly greater degree of angiogenesis than normal skin.Therefore, inhibition of angiogenesis may inhibit the development of keloids by slowing down the inflammatory response.CD31+and CD34+vasculature in keloid tissue is significantly inhibited by ADSC-CM.8

Inhibition of collagen production

TGF-β: is described earlier in “Antifibrosis” section.

Plasminogen activator inhibitor-1 (PAI-1) gene: PAI-1 plays an important role in the progression of tissue fibrosis. ADSC-CM attenuates the accumulation of collagen in keloids by inhibiting the expression of thePAI-1gene.8

Collagen I:The increase in extracellular matrix deposition is characterized by overexpression of collagen I and collagen III, and ADSC-CM significantly downregulates the expression of thecollagen Igene,but has no significant effect on collagen III expression.8Similarly, BM-MSCs reduce the level of collagen in a skin fibrosis model.15

Hypoxia inducible factor(HIF)-1α:After hypoxia,HIF-1α,CTGF and VEGF are significantly upregulated in KFs and normal fibroblasts, thereby increasing collagen deposition.13Human AFSCs inhibit the expression of HIF-1a and reduce collagen deposition.4

Tissue inhibitor of metalloproteinase(TIMP)-1:TIMP-1 is a glycoprotein of the TIMP family that may lead to the deposition of collagen I in keloids.29The level of expression of TIMP-1 in keloids is significantly reduced by ADSC-CM.8

Heat shock protein 47kDa (HSP47): HSP47 is an endoplasmic reticulum-resident and collagen-specific chaperone that recognizes the collagen hydrophobic amino acid sequence (Gly-Pro-Hyp) and aids in the secretion of correctly folded collagen.Elevated collagen in keloids is associated with HSP47 expression.30BM-MSCs reduce the transcription level of HSP47 in a bleomycininduced skin fibrosis model.15

Inhibition of cell invasion

The enhanced invasiveness of dermal fibroblasts is a key factor in the development of keloids, while ADSC-CM significantly inhibits the invasion of KFs.8

Antibacterial activity

The formation of keloids is thought to be related to local bacterial infections. Human MSCs mediate antibacterial activity againstEscherichia coliandPseudomonas aeruginosaby secreting the antimicrobial peptide LL-37.31

Stem cell treatment

Abnormally proliferating stem cells in keloids may be a source of inflammatory factors.7Thus,the introduction of normally functioning stem cells or their secreted factors may have a therapeutic effect on keloids.

Stem cell therapy is currently a hot topic, and stem cell transplantation has achieved a certain amount of success.Methods of stem cell transplantation include intravenous injection and local injection.The injection of stem cell-CM and topical stem cell-CM also has an effect on some fibrotic diseases.

Intravenous injection

Intravenous injection is a very simple method of stem cell transplantation.Many studies have shown that injury has a chemotactic effect on stem cells,which provides a basis for the intravenous injection of stem cells.32-33For example,BM-MSCs migrate to the site of liver injury.19

MSCs have special immunological properties, and cord blood stem cells can escape the surveillance of the immune system, even if they are incompatible with the major histocompatibility complex.Thus,the intravenous infusion of cord blood stem cells does not result in serious adverse events.34However,despite the chemotaxis, systemic administration of cord blood stem cells results in a massive loss of stem cells during transplantation. Therefore, researches are needed to improve the effectiveness of stem cell transplantation.

ADSCs are easier to obtain than other types of stem cells. During the extraction of ADSCs,N-acetylcysteine is added to the swelling fluid used in liposuction, asNacetylcysteine protects ADSCs from oxidative stress and increases the survival rate of ADSCs.N-acetylcysteine also reduces thedifferentiationofADSCsintomatureadipocytes and improves the survival rate of transplanted ADSCs.35At present,liposuction and fat transplantation techniques are relatively mature and involve few ethical problems.

During the culturing of stem cells,the induction of MSCs under hypoxic conditions (the creation of hypoxiapreconditioned MSCs) results in increased inhibition of extracellular matrix production by fibroblast cells compared with normally cultured MSCs. However, it also promotes the expression of HIF and VEGF and promotes cell proliferation. As keloid development is a type of hyperplastic disease and the expression of HIF and VEGF is upregulated in keloids,hypoxia-preconditioned MSCs may not be suitable for keloid treatment.36The homing potential of MSCs is improved by melatonin pretreatment, and adenovirus-mediated overexpression of decorin in BMMSCs effectively prevents fibrotic changes in animal models of cirrhosis.37-38Other drugs can also synergize with stem cells to increase the effectiveness of stem cell treatment.39

Local injection

Local injection of stem cells results in poor stem cell survival rates. In an attempt to overcome this problem,recent research has focused on stem cell scaffolds; the effectiveness of stem cell transplantation is greatly improved by the use of scaffolds, including umbilical cord blood-derived MSC-seeded fibronectin-immobilized polycaprolactone nanofibers,decellularized scaffolds,and three-dimensional hydrogels.40

Stem cell derivatives

Stem cell research has recently focused on exosomes,which are cell secretions containing proteins and micro-RNAs that alter cellular activity in target cells.41The therapeutic effect of stem cells is thought to be mainly achieved through paracrine effects. The exosome contains the paracrine factors of stem cells,and some studies have even suggested that exosomes have the same characteristics as their stem cells.42Thus, exosomes may replace stem cells as a more accessible and safer treatment option.

Existing problems and prospects

Although most of the factors secreted by stem cells have a therapeutic effect on keloids, there are still some complications. A small number of stem cell types, such as Wharton’s jelly-derived MSCs, enhance the expression of the profibrotic marker PAI-1d while downregulating the expression of the antifibroticTGFβ-3gene and promoting the proliferation of KFs.43Furthermore, the transplantation of some stem cells, such as human AFSCs, promotes VEGF expression and may be counterproductive to the treatment of keloids.4At present, most studies on keloids are performed in vitro,and the lack of established animal models makes it difficult to convert research findings to clinical applications. In addition, the application of stem cells is ethically controversial, which makes it difficult to advance to clinical treatment. Despite these difficulties,stem cells are becoming increasingly widely used in the treatment of skin diseases. For example, hypertrophic scars and acne scars are improved by fat grafting, the intravenous injection of MSCs is being performed in various animal model studies of systemic scleroderma,and various bioscaffolds carrying stem cells are being used in the study of skin wound healing.44-46Due to the compact nature of keloid tissue, stem cell scaffolds and exosome sustained release devices may have good clinical prospects.

Conclusion

Stem cells can inhibit the proliferation of KFs and have antifibrosis,anti-inflammatory response and other functions that can inhibit the development of keloids.Furthermore,the technologyofstemcelltransplantationisbecomingmoreand more mature. It appears that stem cells have invaluable potential as a potential method for treating keloids.

Acknowledgement

The authors want to thank Dr.Matetei Pachuau for language help.