Introduction

During the past decade, mesenchymal stem cells (MSCs) have emerged as a promising therapy for the treatment of many pathologies. Clinical trials of MSCs to treat chronic wounds that do not heal are underway. There is also interest in their therapeutic potential to accelerate the closure of burn wounds and treat autoimmune disease damage. Many studies show the positive effects of MSC therapy. These positive results are not due to MSCs’ differentiation to replace damaged skin. Their therapeutic effects derive from the secretion of soluble factors that regulate cellular responses to skin injury.

Despite the potential of MSC therapy, the field remains in its infancy, with many challenges to be addressed before it can be used effectively in clinical practice. Studies to determine the interactions between MSCs and different cell types in wounds are urgently needed. Such studies must also identify the MSC-derived factors that are responsible for regulating local cellular responses to injury and how the wound environment affects MSCs. This paper will review the current understanding of MSCs for wound healing.

Skin and wound healing

Skin plays an exquisite, crucial role that involves different biological functions. It is the largest organ of the human body; in adults, it has an area of approximately 1.5 – 2 m and accounts for about 15% of body weight1, 2. The skin consists of different receptors to sense temperature, moisture, pain, and texture3. The skin regulates body temperature, stores water, and prevents dehydration to maintain the body’s internal balance and protect the body from negative conditions, such as extreme temperatures. The skin is also significant in metabolic processes, notably vitamin D synthesis and lipid storage2, 3. One of the most important functions of the skin system is to act as a protective barrier between the external and internal environment of the body2. Mechanically, the skin is constantly replenished to maintain a balance between cell death and regeneration. Sweat glands, sebaceous glands, and skin flora embedded within the skin layer also contribute greatly to the overall function of this organ4. The skin faces challenges due to aging (chronic) and wounds (acute).

Skin aging refers to the functional and aesthetic deterioration of the skin, diminishing its capacity to protect and regulate the body. Skin aging is characterized by the accumulation of damaged macromolecules in cells, diminution of regenerative capacity, and loss of physiological function5. Some discernible features of skin undergoing early again are thickening, deep wrinkles, spotting, and roughness6, 7.

Two types of skin aging occur simultaneously: aging over time and aging due to the effects of intrinsic factors (, genetics, cell metabolism, hormones, c.) and extrinsic factors (., habitat, dust, radiation, toxins, chemicals, .). Over time, the accumulation of factors leads to molecular damage, including DNA mutation, telomere shortening, epigenetic alterations,., and cellular disorders, such as oxidative stress, cellular senescence, autophagy, proteostasis, inflammation, deficiency of the immune system, .8, 5. Thus, molecular changes due to genetic and epigenetic aging ultimately result in the aggregation of worn-out cells and the deterioration of tissue homeostasis and healing capacity9.

The extracellular components in the skin encompass collagen, elastin, fibrillin, and proteases. During aging, the ratio of type III collagen to type I collagen increases due to the loss of collagen I, which involves the down-regulation of the TGF-β/Smad signaling pathway and connective tissue growth factors; consequently, the architecture of the skin is poorly reconstructed10, 11. Moreover, the family of various matrix metalloproteinases (MMPs) exhibits a considerable impact on skin balance and anti-aging. MMPs originate from a common family of endopeptidases that decompose ECM proteins and, thus, promote the degradation of the skin12. This family has also been shown to increase with age, while endogenous MMP inhibitors decline correspondingly13, 14. Furthermore, reactive oxygen species (ROS) augment MMPs with age15. These elements could be synthesized internally as metabolized oxidizing precursors, or derive from external ultraviolet exposure. ROS activate the mitogen-activated protein kinase family (MAPK), which then induces MMP transcription factors16, 17. UV is associated with the NF-κB pathway, which is responsible for regulating MMP in skin fibroblasts16, 18.

Aging cells typically present a low expression of β-galactosidase, particularly those that are about to shift into senescence. Additionally, senescence-related gene expression, including CHEK1 and cyclin-dependent kinase inhibitor p16, is relatively down-regulated during aging9. Meticulous studies have revealed the dysregulation of microRNAs (miRNAs) as aging progresses. MiRNAs (short noncoding RNAs that bind to the 3’ untranslated region of target mRNA to prevent its gene expression) primarily serve as regulators for cell survival, proliferation, differentiation, and senescence19, 20. Age-dependent defectiveness restricts repair genes9.

The processes of skin regeneration and post-traumatic wound healing are indispensable, especially in the epidermal and dermal layers, as they help reduce the risk of infection that is associated with a high mortality rate21. This healing reaction occurs in three stages: the inflammatory phase, the proliferation phase, and the remodeling phase.

Skin injury triggers the inflammation response, which initiates coagulation, providing a fibrin network alongside vascular constriction to close the wound and preserve its integrity. This fibrin meshwork not only balances endogenous conditions and fights invading microorganisms, but also provides the site for cell migration and the collection of growth factors, including transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF)22. White blood cells are recruited into the site of injury within the first 24 hours and remain there for 2 to 5 days23, 24. White blood cells release protease and ROS to directly destroy bacteria, digest mortified tissue, and engulf the remnants of cell debris. Meanwhile, neutrophils appear to release cytokines, including necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, to amplify the inflammatory response25.

The proliferative phase occurs 48 after the wound occurs and can last for up to 14 days26 after the remission of inflammation, resulting in a miniature lesion when the contraction and formation of filaments induces horn cells26. This phase is characterized by granulation tissue formation to replace the provisional wound matrix and vascular network recovery of the sources of cytokines and growth factors, notably transforming growth factor-beta (TGF-beta, including TGF-β1, TGF-β2, and TGF-β3), interleukin (IL), and vascular formation factors27, 24. In response to tactile inhibition and the physical strain of endoplasmic lesions, horn cells and epithelial stem cells from hair follicles are triggered to migrate and proliferate to cover the wound margin. Furthermore, macrophages send nitric oxide28 signals or other cell types release epidermal growth factors (EGF), keratinocyte growth factors (KGF), insulin-like growth factor 1 (IGF-1), and neural growth factor (NGF), which appear to trigger the reepithelization process29, 30. Additionally, angiogenesis occurs to enable the transportation of nutrients and oxygen that favors wound healing. During the formation of new blood vessels, endothelial cells are activated by vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and thrombin serine protease31. The final step of this process is marked by the formation of granulation tissue leading to the infiltration and proliferation of fibroblasts into the wound edge. Granulation tissue consists of loosely organized fibroblasts, macrophages, capillaries, granular leukocytes, and bundles of type III collagen3, 32.

The remodeling phase completes the healing process, beginning within 2 to 3 weeks of the injury and potentially lasting for a year33. The process of controlling the balance between the degeneration and synthesis of new tissue is immensely rigorous and any disruption or error results in the formation of chronic wounds24, 32. TGF-β1 promotes fibroblast differentiation into myofibroblasts34. The extracellular matrix (ECM) is remodeled and synthesized to become more robust, characterized by the degeneration of type III collagen over time and its gradual replacement with type I collagen32. However, some skin components, such as hair follicles or sweat glands, cannot recover from severe skin damage35. Thus, wound healing terminates with the formation of scars, which are closely related to inflammation34.

Mesenchymal stem cells and their bioeffects on skin regeneration

Mesenchymal stem cells and their extracellular vesicles

MSCs have been found and isolated from various somatic locations36. Their biological functions have been assessed differently, as well as being isolated in different places with different methods; no distinct indication helped define the characterization of MSCs. To circumvent this problem, the International Society for Cellular Therapy suggested minimum standards to identify MSCs in humans37: First, MSCs must have a fibroblast shape, adhesive capacity, and maintain their shape under standard culture conditions. Second, MSCs must express CD73, CD90, and CD105 but not CD14, CD11b, or CD79α, CD19, CD34, CD45, and HLA-DR. Third, MSCs must differentiate into osteocytes, chondrocytes, and adipocytes37. Although scientists recently discovered that MSC populations do not thoroughly comply with these minimal criteria—for instance, some fail to express CD10538 —these rules are still ubiquitously used to compare and exchange data among MSC studies (Figure 1).

MSCs have been attested to work through the paracrine system via extracellular vesicles (EVs)39, 40. MSC-EVs comprise a variety of vesicles of varying sizes and are described as miniature prototypes of the cells that secrete them41. EVs can be divided into three main types by origin: apoptotic bodies (1000–5000 µm), microvesicles (1000 µm), and exosomes (30 – 150 µm)42. EVs seem to be more stable in the living body than foreign elements43. Immunoregulatory factors such as IL-10, TGF-β, INF- γ, IDO, and prostaglandin E2 persist within EVs44, 45. Their chief components include proteins, lipids, and nucleic acids. These components mediate many effects in recipient cells and are enclosed within a phospholipid bilayer. Furthermore, at least 730 different proteins have been shown to be enriched in exosomes derived from mesenchymal stem cells46. Additionally, 171 miRNAs have been pinpointed in EVs derived from MSCs47 and play pivotal roles in gene expression. For example, miR-130a-3p triggers cell proliferation, angiogenesis, the inhibition of programmed death, or the regulation of immune cell survival (https://www.ncbi.nlm.nih.gov/gene/406919). Exosomes have gradually revealed their potency and paved the way for the fourth generation of stem cell therapy.

Perspectives

Because MSCs and MSC exosomes manifest hypoimmunogenic properties, they are hopeful choices for treating chronic wounds, injuries, and plastic surgery incisions. This has huge potential in the field of tissue remodeling and engineering. Since the Physiology Nobel prize in 2013 was awarded to the three Laureates who presented insights into exosomes, studies of MSC exosomes have increased at a dizzying rate. According to Mordor Intelligence, the exosome market was reportedly worth 174.04 million USD in 2020 and is predicted to increase by 27.89% in this year (https://www.mordorintelligence.com/). The growing demand for anti-aging therapies drives much of the market. Furthermore, the availability of various methods for exosomal isolation and purification helps fuel further studies of exosome treatments.

Most research now focuses on proving the utility of MSC exosomes as a single treatment. Clinically, MSC exosomes should be used in combination with other therapies, such as laser, topical medication, surgery, etc. Consequently, more studies are needed to demonstrate the synergistic or inhibitory effects of MSC exosomes in combination with conventional treatments. Researchers are optimistic about the development of MSC therapy. Similarly, exosomes as a drug delivery system are a bright research path. For almost a decade, there has been tremendous progress in our understanding of all aspects of exosomes. Further improvements in drug-loading strategies and the optimization of these therapies are promising for future clinicians.

The limitations of MSC-EV use

The extracellular secretions of MSCs, especially the exosomes, are being intensively studied for their notable features. MSCs’ impact via exosomes is significant in tissue repair. MSC therapy is gaining popularity and is known as fourth-generation stem cell therapy—the new cell-free therapy106. MSCs not only play an essential role in the treatment of wounds, skin regeneration, and anti-aging but also contribute to the treatment of immune-related diseases, tumors, and neurological disorders and are the standard by which to diagnose and prevent disease. In addition, exosomes from MSCs can be used for drug delivery, encasing pharmaceuticals in a phospholipid bilayer that confers outstanding physiological advantages107.

In contrast, the considerations for using MSC exosomes include:

It is challenging to determine the composition of exosomes, such as their proteins, lipids, and, especially, miRNAs. All of these components derive from the cell culture process and are variable and heterogeneous. Qualitative studies generally seek to confirm whether an agent of interest persists within exosomes. The half-life of exosomes is also unknown. This is an important evaluation criterion to use exosomes for drug transport107. Drug efficacy is highly dependent on delivery time, so this is a major limitation of knowledge regarding the potential use of exosomes for transport.

Another important consideration is the exosome productivity of mesenchymal stem cells. The exosomes for this new cell-free therapy are usually obtained from MSC cultures. This means that cell therapy and exosome therapy are linked. A study has determined that producing sufficient exosomes to have the same effect as cell therapy requires 10 – 25 times the normal quantity of MSCs108. Significant development is needed before exosome therapy can be routinely applied. More research on a suitable culture medium to produce more extracellular vesicles is needed to address this limitation. The current cell culture abandonment medium does not meet the conditions required to develop a routine therapy.

There is also substantial concern about cell properties. Exosomes reflect the properties of their parent cells. Research shows that exosomes from young cells can reverse the aging of aging cells105. Therefore, more in-depth studies are needed on the relationship between MSC age and the exosomes they release. This greatly affects the potential of mesenchymal stem cells from different sites. If the assertions in105 are true, mesenchymal stem cells obtained from umbilical cords and placentas will be preferred.

Another problem in the production of exosomes is purity. MSCs do not express HLA-DR, so they should not induce an adverse immune response. However, an immune response has been observed when MSCs enter the body. This is associated with impurities in the injections. Ultracentrifugation is a standard method for exosome isolation and purification. It uses a density gradient, which may permit other agents or types of vesicles with the same density as the target components to remain. Developing an environment to eliminate, limit, or replace undesirable elements is one solution. However, the optimization of the culture and acquisition process is the ideal way to eliminate this problem.

Despite these limitations, the enormous potential of MSC exosomes is apparent. Exosomes have many advantages over mesenchymal stem cell therapy: They can be stored as proteins for short-term use without the concern of cell survival and using them after thawing is much easier. They are much smaller than MSCs and do not become trapped in small capillaries like cells do. One report shows that this is a serious limitation of cell therapy, as when intravenous administration results in pulmonary embolism109. However, exosome therapy cannot completely replace mesenchymal stem cell therapy because of their interrelationships.

Abbreviations

AMP: Adenosine monophosphate, bFGF: Basic fibroblast growth factor, CCL-2/ MCP1: Chemokine (C-C motif) ligand 2/ onocyte chemoattractant protein 1, CCL-5/ RANTES: Chemokine (C-C motif) ligand 5/ regulated on activation, normal T cell expressed and secreted, CXCR4 C-X-C: chemokine receptor type 4, CXCR5 C-X-C: chemokine receptor type 5, DCs: Dendritic cells, ECM: Extracellular matrix, EGF: Epidermal growth factor, EVs: Extracellular vesicles, HGF: Hepatocyte growth factor, HLA-5: Human Leukocyte Antigen - 5, HLA-DR: Human Leukocyte Antigen – DR isotype, IDO: Enzyme Indoleamine 2,3-dioxygenase, IGF-1: Insulin-like growth factor 1, IL: Interleukin, KGF: Keratinocyte Growth Factor, MAPK: Mitogen-activated protein kinase, MMP: Matrix metalloproteinase, MSC: Mesenchymal stem cell, NK cells: Natural killer cells, NGF: Nerve growth factor, PDGF: Platelet-derived growth factor, PGE-2: Prostaglandin E 2, ROS: Reactive oxygen species, TGF-β: Transforming growth factor beta, TGF-β1: Transforming growth factor beta 1, TGF-β2: Transforming growth factor beta 2, TGF-β3: Transforming growth factor beta 3, TNF: Tumor necrosis factor, Th1 cells: T helper cells, Th2 cells: T helper cells, Tregs: Regulatory T cells, VEGF: Vascular endothelial growth factor, IFN-γ: Interferons γ

Acknowledgments

This research is funded by Vietnam National University Ho Chi Minh City (VNU-HCM) under grant number 562-2020-18-03.

Author’s contributions

Phat Duc Huynh was responsible for the layout and content of the manuscript. Quynh Xuan Tran, Vy Quang Nguyen, and Sao Thi Nguyen equally contributed to this work. Ngoc Bich Vu conceptualized, coordinated and edited the article.

Funding

Vietnam National University Ho Chi Minh City (VNUHCM) under grant number 562-2020-18-03.

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Competing interests

The authors declare that they have no competing interests.