Skin, as the largest organ of the body, possesses various physiological functions including barrier, absorption, sensation, thermoregulation, metabolism, and immunity. Skin injuries caused by large-area burns, benign and malignant skin tumors, and pigmentary skin diseases severely impact patients' health. Among these, skin burns not only compromise the skin's barrier function but also alter its sensations of pain, temperature, and touch. Developing functional tissue-engineered full-thickness skin containing skin appendages is an effective approach to addressing large-area skin defects and holds significant research value. In recent years, with the rapid advancements in tissue engineering, mesenchymal stem cells (MSCs) have been identified as ideal seed cells for constructing full-thickness skin, demonstrating promising applications in the treatment of skin trauma.

1. Skin Healing Process and Mechanism

The repair of skin injury necessitates intricate orchestration, integration, differentiation, migration, proliferation, and apoptosis of skin cells to achieve the regeneration of the skin's multi-layered structure. The healing process of skin damage can be broadly categorized into four stages:

① Blood Coagulation Stage: Initially, platelets and coagulation factors are activated, initiating both intrinsic and extrinsic coagulation pathways to achieve hemostasis.

② Inflammatory Response Stage: Neutrophils, monocytes, and macrophages increase, regulating the inflammatory response through phagocytosis of foreign particles and modulation of inflammatory factor release.

③ Cell Proliferation Stage: This involves the formation of granulation tissue mediated by fibroblast proliferation, re-epithelialization mediated by keratinocytes, and angiogenesis mediated by endothelial cell proliferation.

④ Tissue Remodeling Stage: With the secretion of extracellular matrix (ECM) and the proliferation of granulation tissue, fibroblasts divide and synthesize a significant amount of collagen fibers. The number of newly formed blood vessels increases. In the later stages, under the action of matrix metalloproteinases (MMPs), ECM is degraded, and collagen undergoes metabolic turnover, ultimately leading to scar formation.

2.Mechanisms of MSCs in Promoting Wound Healing and Inhibiting Scar Formation

① Immunomodulatory Effects: Following tissue injury, the inflammatory environment at the wound site directly stimulates MSCs to exert their unique immunomodulatory effects. This includes enhancing cyclooxygenase-2 (COX-2) activity and upregulating prostaglandin E2 (PGE2) levels. Under the influence of PGE2, T cells and macrophages undergo reprogramming and begin to express high concentrations of IL-10, which research has shown to be essential for scarless healing. Overexpression of IL-10 significantly inhibits scar formation. MSCs also prevent neutrophils from infiltrating the wound site, thereby preventing the release of reactive oxygen species (ROS) by neutrophils, which can further cause oxidative tissue damage. IL-10 achieves anti-fibrotic effects by directly downregulating TGF-β1 expression in macrophages and T cells, reprogramming fibroblasts, upregulating matrix metalloproteinases (MMPs) expression, and downregulating collagen expression to promote extracellular matrix remodeling. Additionally, IL-10 inhibits excessive collagen deposition by reducing the expression of proinflammatory factors IL-6 and IL-8.

② Secretion of Nitric Oxide to Neutralize Reactive Oxygen Species (ROS): MSCs can promote the production of nitric oxide synthase, effectively inhibiting the formation of fibrotic tissue by altering the balance between ROS and reactive nitrogen species (RNS). During the initial inflammatory phase of wound healing, neutrophils secrete excessive amounts of ROS, such as superoxide, hydrogen peroxide, and alkyl peroxides, which are potent enhancers of collagen deposition. The primary role of ROS is to induce oxidative damage to membrane phospholipids and promote fibrosis through the induction of TGF-β1. MSCs can convert ROS into RNS, such as peroxynitrite, slowing down the damage to DNA and membrane phospholipids, thereby inhibiting fibrosis.

③ Secretion of Anti-Fibrotic Growth Factors: Research has shown that MSCs can secrete various cytokines and growth factors, some of which exhibit anti-fibrotic properties, such as hepatocyte growth factor (HGF), IL-10, and adrenomedullin. The mechanisms by which HGF alleviates fibrosis and inhibits skin scar formation include:

Regulation of Fibroblast Function: In the presence of HGF, the HGF receptor binds and neutralizes downstream signaling cascades of the TGF-β/Smad signaling pathway, inhibiting the pro-inflammatory transcriptional activity of SMAD3 protein. This leads to downregulation of TGF-β1, type I, and type III collagen expression in fibroblasts. Additionally, HGF stimulates the upregulation of MMP family members MMP-1, MMP-3, and MMP-13 in fibroblasts, promoting ECM remodeling.

Promotion of Keratinocyte Migration and Proliferation: HGF enhances keratinocyte migration and proliferation and upregulates the expression of vascular endothelial growth factor-A (VEGF-A), resulting in the formation of high-quality, well-vascularized granulation tissue at the wound site, which facilitates epithelialization.

Inhibition of Myofibroblast Differentiation: HGF inhibits the differentiation of fibroblasts into myofibroblasts at the wound site, thereby preventing the excessive accumulation of contractile myofibroblasts that contribute to scar formation.

④ Regulation of Skin Fibroblast Quantity and Function: MSCs modulate the production of extracellular matrix (ECM) by fibroblasts at the injury site, enabling skin repair that more closely resembles normal skin. During wound healing, the participation of fibroblasts is essential for restoring skin integrity, but these cells can produce excessive ECM, leading to scar formation. MSCs at the wound site secrete bioactive molecules, including HGF and PGE2, which can inhibit endothelial-to-mesenchymal transition (EMT) in vascular endothelial cells and suppress the differentiation of myofibroblasts. Additionally, during the repair process, MSCs can promote fibroblast function through paracrine secretion of bioactive factors.

⑤ Enhance vascular regeneration and stabilization in tissues. The inflammatory phase, epithelialization period, and maturation stage during wound repair rely on the proliferation and repair of newly formed capillaries and connective tissues, namely granulation tissue. During the proliferative phase, vascular regeneration provides nutrients for the formation of granulation tissue. In the process of vascular system remodeling, in addition to macrophages and fibroblasts, MSCs also secrete angiogenic cytokines, such as fibroblast growth factor and VEGF-A, which promote the proliferation, migration, and differentiation of microvascular endothelial cells. Furthermore, MSCs can promote vascular stability and protect the vascular system through paracrine secretion (e.g., secretion of adrenomedullin).

⑥ MSCs have the potential to differentiate into various skin cells. MSCs exhibit typical differentiation capabilities into osteoblasts, adipocytes, and chondrocytes, which is similar to the phenotypic transformation of fibroblasts from various sources into myofibroblasts at the wound site. Research indicates that MSCs can differentiate into various skin cells, including epidermal cells, keratinocytes, and small blood vessel endothelial cells, directly participating in the regeneration process of the epidermis and dermis, thereby promoting skin wound healing.

3.Effective Approaches to Enhance the Efficiency of MSCs in Repairing Skin Injuries

Currently, the primary methods of utilizing MSCs to promote skin injury repair are local injection and intravenous injection. Local injection typically involves administering MSCs around the wound site, but due to difficulties in controlling the injection area and depth, it fails to ensure direct and even distribution of MSCs across the wound surface. Intravenous injection, on the other hand, cannot guarantee that all MSCs will participate in skin repair, and the exosomes secreted by MSCs are rapidly cleared from the body, making it challenging to maintain high concentrations and necessitating multiple administrations. Additionally, both injection methods can inflict secondary damage to the skin, posing challenges for treatment. These issues limit the full therapeutic potential of mesenchymal stem cells, hindering their optimal repair effects. Consequently, significant research efforts have been directed towards exploring ways to enhance the repair efficiency of MSCs, yielding fruitful advancements.

① Biological scaffolds. Biological scaffolds possess excellent biocompatibility and biodegradability, enabling the delivery of stem cells to wound sites. Commonly used biological scaffold materials for drug delivery to promote skin injury repair can be categorized into natural scaffolds and synthetic scaffolds. Natural scaffold materials include hyaluronic acid, chitosan, collagen, alginate, among others. Synthetic scaffold materials, on the other hand, comprise polyethylene glycol, poly(lactic-co-glycolic acid), and others. Currently, hydrogel scaffolds are the most widely used scaffold form for skin injury applications. They stabilize the concentration of exosomes after local application to the skin, delaying their release and enhancing wound healing capabilities. Hydrogels are stable hydrophilic networks formed through physical, chemical, or enzymatic crosslinking. Featuring excellent biocompatibility, biodegradability, and water retention properties, hydrogels have proven to be the most user-friendly, economical, and readily available materials, demonstrating immense application potential in clinical medicine. Hydrogel-loaded exosomes derived from mesenchymal stem cells (MSC-EXOs) can address the issue of rapid exosome clearance in vivo caused by intravenous injection methods. By placing the exosome-containing hydrogel directly on or near the target site, it ensures a more concentrated dose of exosomes while simultaneously leveraging the wound healing effects of both exosomes and the hydrogel.

② Pretreatment. Certain pretreatment methods can improve the biochemical and biophysical properties of MSCs, thereby enhancing their repair functions. Studies have found that adipose-derived MSCs pretreated with endothelial cell culture medium exhibit enhanced angiogenic, proliferative, and endothelial differentiation capabilities both in vitro and in diabetic mouse wound models. MSCs can also undergo pretreatment with certain chemicals to improve their ability to repair skin injuries. For instance, treating adipose-derived MSCs with lecithin-emulsified emu oil and butylated hydroxytoluene significantly enhances their regenerative potential. Additionally, researchers have attempted to activate urine-derived MSCs using bioglass materials, enhancing skin healing in mice by stimulating paracrine effects between MSCs and recipient cells, as well as between fibroblasts and endothelial cells.

③ Hypoxic Environment. Studies have shown that hypoxia can enhance the skin wound healing capabilities of MSCs. When human amniotic MSCs were implanted in mouse wound healing models, it was found that compared to MSCs cultured under normal conditions, those cultured under hypoxic conditions exhibited higher survival and proliferation rates, with increased expression of vascular endothelial growth factor (VEGF). These MSCs also improved the viability and migration of human dermal fibroblasts, increased the expression of extracellular matrix, and accelerated wound healing. Furthermore, researchers discovered that under hypoxic conditions, mouse bone marrow-derived MSCs secreted more basic fibroblast growth factor (bFGF), VEGF-A, and interleukin-6 (IL-6) in vitro. In vivo experiments also revealed that transplanted bone marrow-derived MSCs under hypoxic conditions significantly accelerated skin wound contraction, with enhanced cell proliferation and neovascularization.

④ Gene Recombination. Genetic recombination of MSCs, using them as both seed cells and vectors to deliver therapeutic genes to wound sites, represents a promising approach for skin injury treatment. By genetically modifying MSCs to enhance their activity, researchers have aimed to improve their skin repair capabilities. For instance, bone marrow-derived MSCs overexpressing transforming growth factor-β3 (TGF-β3) have not only improved the wound healing process in rabbit ears but also reduced scar tissue formation. Researchers have also utilized recombinant lentiviral vectors to modify human amniotic MSCs to overexpress interleukin-10 (IL-10). These modified MSCs were found to significantly outperform unmodified MSCs in accelerating wound healing, promoting angiogenesis, modulating inflammation, regulating extracellular matrix (ECM) remodeling, enhancing wound closure, and improving healing quality in mice.

⑤ Cell Sheet Technology. Cell sheets represent a scaffold-free, densely packed cellular tissue. Since cell density is a crucial factor in enhancing the efficacy of cell transplantation therapy, compared to single-cell sheets, transplanted layered cell sheets can better promote tissue function restoration and regeneration. Studies have reported that using hematoporphyrin-encapsulated polyketone membranes as a material, bone marrow-derived MSCs were seeded onto the membrane and applied to mouse wounds. This method of transplanting and stacking three-layered cell sheets promoted angiogenesis and skin regeneration at the wound site, enhancing the repair of damaged skin tissue in mice.

Although the application of MSCs cannot replicate the embryonic-like scarless wound repair seen in fetuses, it still holds immense therapeutic potential for adult wound healing. In the process of skin wound repair and healing, inhibiting the formation of skin scars to improve wound healing quality has become a new goal in current skin regeneration and repair research. In this context, the vast therapeutic potential of MSCs emerges as a novel strategy to suppress skin scar formation, offering a fresh perspective and treatment approach for wound repair.

References

[1] Zhang Li, Wang Qiang. Research on the Role of Mesenchymal Stem Cells in Skin Injury Repair [J]. Chinese Journal of Clinical Medicine, 2015, 22(04): 571-574.

[2] Wu Yan, Zhang Zhe, Yang Lan, et al. Mechanism of Mesenchymal Stem Cells Inhibiting Scar Formation During Wound Healing [J]. China Medical Herald, 2016, 13(09): 47-50.

[3] Cheng Yansiwei, Song Guanbin. Mesenchymal Stem Cells and Skin Injury Repair [J]. Journal of Biomedical Engineering, 2021, 38(02): 387-392.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.