Wound pathophysiology and healing dynamics for stem cell-mediated therapeutics in different skin wounds

Abstract

Skin wound healing is a multifaceted and highly regulated biological event, which includes inflammation, cell proliferation, extracellular matrix deposition, neovascularization, and tissue remodeling. The underlying pathophysiological and healing dynamics of skin injuries vary significantly depending on the nature, depth, and etiology of the wound. Full-thickness excisional wounds, acid-induced chemical burns, and cold-induced burn injuries have distinct pathological patterns, each characterized by differential inflammatory cascades, oxidative stress levels, cellular infiltration, vascular compromise, and tissue regeneration dynamics. Understanding this heterogeneity necessitates the development of personalized therapeutic interventions that can address the specific challenges associated with each wound type. This review aims to explore experimental evidence of mesenchymal stem cell (MSC)-based therapies across various wound models and to highlight their therapeutic potential and optimization strategies. MSCs have emerged as promising candidates for regenerative wound therapy due to their multipotency, paracrine activity, and immunomodulatory functions. This review highlights the remarkable potential of MSC-based treatments to accelerate healing, reduce inflammation, and promote scar less tissue regeneration in different experimental models, including full-thickness acid burn wounds, heat burn wounds, cold-induced injuries, and chronic vs acute excisional wounds. The therapeutic efficacy of MSCs has been substantially enhanced through various preconditioning strategies, such as chemical preconditioning with α-terpineol, isorhamnetin, quercetin, and rutin, as well as their incorporation into biocompatible scaffolds and hydrogel matrices. These approaches have improved stem cell viability, survival under hypoxic and oxidative conditions, engraftment efficiency, and functional integration into the host tissue. This review also presents a comparative analysis of the wound healing mechanisms and pathological features associated with different skin injury models while emphasizing the mechanistic role of MSCs in modulating these responses. It further explores the impact of stem cell preconditioning and advanced biomaterial-assisted delivery systems in optimizing regenerative outcomes. Collectively, the findings underscore the necessity for context-specific therapeutic strategies and confirm the translational potential of MSC-based interventions as adaptable, targeted, and clinically viable solutions for complex skin injuries. Moreover, the integration of biomaterials and preconditioning strategies offers promising avenues to overcome current limitations in MSC therapy, ultimately resulting in the advancement of regenerative dermatology.

Key Words: Wound healing; Mesenchymal stem cells; Regenerative medicine; Preconditioning; Biomaterials; Clinical translation

Core Tip: Skin wound healing is a complex process affected by the type and severity of injury. This review provides a comparative overview of the pathophysiology of excisional, thermal, chemical, and cold-induced skin wounds while highlighting mesenchymal stem cell (MSC)-based regenerative strategies. It explains how MSCs modulate inflammation, promote angiogenesis, and support tissue remodeling through paracrine and immunomodulatory mechanisms. Additionally, it emphasizes optimizing MSC therapy via preconditioning (chemical, hypoxic, or genetic) and combining it with biomaterials like hydrogels and scaffolds to improve survival, engraftment, and functional recovery. The review also addresses translational barriers and the need for standardization in clinical applications, underscoring the potential of MSC-based treatments as future-oriented options for complex skin injuries.

INTRODUCTION

Skin wound healing is a dynamic and highly synchronized biological process that involves a series of overlapping phases, including hemostasis, inflammation, cellular proliferation, extracellular matrix (ECM) production, angiogenesis, and tissue remodeling. Although these stages form a shared network, the biological responses driving repair can vary significantly depending on the type, cause, and severity of the injury. Different categories of skin wounds, such as full-thickness excisional defects, acid burns, thermal burns, and cold injuries, exhibit unique pathological features that influence the course and efficiency of healing. These differences result from varying inflammatory responses, levels of oxidative stress, patterns of immune cell infiltration, and context-dependent vascular and stromal damage. Therefore, understanding the heterogeneity among wound types is crucial for developing effective therapeutic strategies that address the specific biological challenges associated with each injury type.

Among emerging therapies, mesenchymal stem cells (MSCs) have become a prominent focus of regenerative dermatology due to their multipotent differentiation ability and strong paracrine activity. Many experimental studies show that MSCs influence several key aspects of wound healing, including attenuating excessive inflammation, regulating macrophage polarization, enhancing angiogenesis, accelerating fibroblast proliferation, and activating endogenous repair pathways. These functional features allow MSCs to improve outcomes in various wound models, from acute excisional injuries to chronic non-healing ulcers, as well as chemically and thermally induced burns. Their wide therapeutic potential highlights the importance of understanding how MSCs interact with the distinct microenvironments created by different types of skin damage.

Despite their therapeutic potential, MSC efficacy is often limited by the harsh environment of injured tissue, which is typically characterized by hypoxia, high levels of reactive oxygen species (ROS), pro-inflammatory cytokines, and damaged vasculature. These conditions decrease MSC survival, hinder engraftment, and reduce functional activity after transplantation. To address these challenges, recent research has focused on enhancing the survival and reparative potential of MSCs through various preconditioning strategies. Chemical and pharmacological preconditioning with compounds (small molecules) has been shown to improve cell viability, increase resistance to oxidative stress, enhance paracrine signaling, and strengthen the capacity to regulate inflammatory responses. These approaches aim to improve the therapeutic performance of MSCs before their application to different wound environments.

More advancements in biomaterials have further enhanced the applicability of MSC-based therapies. Biocompatible scaffolds, hydrogel matrices, and engineered delivery platforms have become effective tools for improving cell retention, promoting sustained release of bioactive molecules, and facilitating the structural integration of transplanted cells within the wound bed. These systems provide a supportive three-dimensional microenvironment that mimics native tissue architecture, enhances cellular survival, and potentiates regenerative signaling cascades. The combination of preconditioned MSCs with advanced biomaterial-assisted delivery methods represents a promising strategy to overcome the limitations of conventional cell transplantation and achieve more consistent therapeutic outcomes.

Considering the diversity of skin injuries and their distinct pathological mechanisms, a comparative analysis of different wound models is necessary to elucidate how MSCs exert therapeutic effects in each condition. Such analysis provides a relevant understanding of damage-specific mechanisms of action, including regulation of immune responses, enhancement of angiogenic pathways, suppression of oxidative stress, and promotion of ECM reorganization. Understanding these mechanisms also enhances the development of personalized regenerative approaches that are aligned with the unique pathophysiological characteristics of each wound model.

Overall, MSC-based therapy is a versatile and clinically relevant option for treating complex skin injuries. The integration of stem cell preconditioning with innovative biomaterial platforms has opened new avenues for improving the efficacy, precision, and translational potential of MSC interventions. As research advances, MSC-based regenerative strategies play a significant role in developing personalized wound management and the broader field of regenerative dermatology (Figure 1).

Figure 1 MSC-based regenerative strategies. MSCs: Mesenchymal stem cells.

BIOLOGICAL BASIS OF CUTANEOUS WOUND HEALING

Phases of wound repair

Wound repair involves a coordinated, overlapping sequence of biological events typically described as hemostasis, inflammation, proliferation, and remodeling (Figure 2). Immediately after tissue injury, hemostasis is triggered by vasoconstriction, platelet adhesion and aggregation, and fibrin clot formation; the clot provides hemostasis and acts as a temporary scaffold that concentrates growth factors and chemokines at the wound site[1]. The inflammatory phase is initiated by a rapid influx of neutrophils, followed by monocyte-derived macrophages. Neutrophils help in early microbial clearance by releasing proteases and ROS; macrophages coordinate the process toward repair through the clearance of debris, the secretion of growth factors [e.g., transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF)], and a phenotype switch from a pro-inflammatory (M1) to a pro-resolving (M2) state. Persistent or dysregulated inflammation hinders the transition to healing and increases the risk of chronic wounds[2]. During the proliferative phase, keratinocytes migrate and proliferate to re-epithelialize the wound surface, while fibroblasts expand and synthesize ECM components, mainly type III collagen and fibronectin. Neoangiogenesis, driven by VEGF and angiopoietins, restores blood flow and promotes the formation of granulation tissue. Myofibroblast differentiation aids wound contraction. These processes are highly interconnected and are regulated over time by paracrine growth factor signaling and the local ECM environment[3]. The remodeling phase involves replacing early ECM (type III collagen) with stronger type I collagen (Col-I), as well as crosslinking and reorganizing collagen fibers, and removing excess cellular components through apoptosis or clearance. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) coordinate ECM turnover; an imbalance can lead to either excessive scarring (hypertrophic/keloid formation) or matrix degradation and chronic non-healing. Tensile strength gradually increases but often never reaches uninjured levels[4].

Figure 2  Schematic illustration of the four sequential and overlapping phases of wound repair.

Molecular and cellular regulators

Wound healing is controlled by a network of soluble mediators, cell-cell interactions, and ECM signals. Key growth factors include TGF-β (the main regulator of fibroplasia and scarring), PDGF (a chemotactic signal for fibroblasts and macrophages), epidermal growth factor (which promotes keratinocyte proliferation), and VEGF (involved in angiogenesis). The signaling pathways that integrate these signals include members of the mitogen-activated protein kinase (MAPK) family (extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38), nuclear factor kappa B, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and TGF-β/Smad cascades; disruption of any of these pathways can affect cell proliferation, differentiation, and apoptosis within the wound bed[5]. Immune cells have dual roles: Neutrophils and proinflammatory macrophages remove pathogens and debris but produce proteases and ROS that can damage viable tissue; timely switching to M2 macrophage phenotypes that release reparative cytokines [interleukin (IL)-10, TGF-β] is crucial for healing. Resident stromal cells (fibroblast subpopulations), endothelial cells, pericytes, and epidermal stem cells each contribute discrete reparative functions; single-cell transcriptomic studies have revealed previously unrecognized fibroblast heterogeneity and state transitions crucial for effective remodeling[2]. Oxidative (redox homeostasis) and lipid mediators also influence repair: Controlled ROS levels serve as signaling second messengers (for example, to stimulate angiogenesis). In contrast, excessive oxidative stress impairs cell migration and elevates MMP activity. Pro-resolving lipid mediators (resolvins, maresins) actively terminate inflammation and promote tissue regeneration[2]. Collectively, wound repair is a complex, multi-phase process that relies on coordinated interactions between immune cells, stromal cells, growth factors, and ECM signals. Effective healing depends on balanced inflammation, controlled ROS activity, and timely activation of proliferative and remodeling phases. Disruptions in these regulatory networks can hinder tissue regeneration, resulting in chronic wounds or pathological scarring.

PATHOPHYSIOLOGY OF DIFFERENT SKIN WOUNDS

Mechanical wounds

Wounds are generally classified based on the extent of damage caused to the skin by any object. This category includes abrasions, crush injuries, contusions, puncture wounds, avulsions, bite injuries, lacerations, and high-pressure injection injuries. Each type of wound necessitates specific management and closure methods. All these types are detailed in Table 1[6,7].

Table 1 Types of mechanical wounds and wound management[6,7].

Mechanical wounds	Overview	Wound management and concerns
Abrasions	Superficial damage to epidermis, may involve upper dermis	Rarely require sutures
Avulsions	Tearing away of the skin and underlying soft tissue. Irregular wound edges and flaps, exposing underlying structures. Prone to ischemia	Associated with a higher infection risk. Often require layered closure or delayed primary repair
Contusions	Blunt-force injuries damage skin and underlying tissue without necessarily breaking the epidermis	May evolve into hematomas or mask deeper injuries such as fractures or compartment syndrome
Lacerations	Linear or jagged tears in the skin caused by sharp or blunt trauma. Depth and location determine risk for nerve, tendon, or vascular involvement	Often present with well-defined edges commonly managed with primary closure
Puncture wounds	Caused by narrow, pointed objects that penetrate deep into tissue. External opening may be small, but underlying structures can be injured	Risk of infection is high, especially with contaminated or deep punctures
Bite injuries	Bites pose a significant infection risk due to oral flora from humans, dogs, or other animals	Human bites especially hands are considered at high-risk and require prophylactic antibiotics and delayed closure
Crush injuries	Prolonged pressure or force damages skin and underlying tissue. May cause compartment syndrome or rhabdomyolysis	Concerns are tissue viability, decompression, and infection
High-pressure injection injuries	Injuries from paint guns, air, solvents, gasoline, hydraulic fluid, grease, and water. Wound may appear minor, but damage can be severe	X-ray to check if a foreign body is suspended. Antibiotics for contaminated wounds, delayed presentation
Incision wounds	Cuts caused by sharp objects like glass, razor blades, and knives or as a result of surgery. The surface of the skin is where the cutting edge tangentially moves. Often, an incised wound is longer than it is deep	Prone to infection, prophylactic intraoperative incisional wound irrigation is required, in which debris, metabolic waste, and exudate are washed away just before wound closure

Full-thickness excisional injuries

Full-thickness excisional wounds cut through the epidermis and dermis, often removing appendageal stem-cell niches such as hair follicles and sebaceous glands that ordinarily accelerate re-epithelialization. Losing these reservoirs slows down epithelial regeneration and increases dependence on edge keratinocyte migration or graft-based treatments. Extensive dermal loss triggers significant inflammation, heavy fibroplasia, and a higher risk of contracture or hypertrophic scarring. In experimental models, full-thickness wounds are used to test grafting methods, biomaterials, and cell therapies as they recapitulate reparative challenges present in clinical injuries[8]. Clinical management emphasizes timely debridement, infection control, moisture balance, and when indicated, autologous skin grafting (split- or full-thickness) or engineered skin substitutes; regenerative strategies aim to restore appendage structures, limit fibrosis, and accelerate functional recovery. Recent advances highlight scaffold design, growth factor delivery, and cell-based therapies (e.g., MSCs, hair follicle-derived epithelial progenitors) to improve outcomes[9].

Thermal (heat) burn injuries

Thermal burns cause immediate coagulative necrosis in the injury zone and secondary injury in surrounding zones of stasis and hyperemia; the outcome of tissue in the zone of stasis depicts lesion progression. Severe burns trigger systemic responses marked by a hypermetabolic state, immune system dysfunction, capillary leakage, and significant muscle catabolism, which together drive morbidity and hinder wound healing. Early wound excision and grafting lower infection risks and reduce metabolic burden; control of inflammatory and oxidative pathways in the zone of stasis has been shown experimentally to improve tissue salvage[10]. At the molecular level, burn injury triggers the release of circulating damage-associated molecular patterns (DAMPs), activates innate immune receptors, and induces cytokine storms that influence remote organ effects. Treatment strategies supported by clinical and preclinical evidence include targeted resuscitation, nutritional and metabolic support, and strategies to attenuate exaggerated inflammatory responses (e.g., immunomodulatory agents and antioxidants)[11].

Cold-induced burn injuries (frostbite/freezing injury)

Cold-induced injuries occur through physical ice-crystal formation, osmotic stress, and direct membrane disruption during freezing, followed by reperfusion injury upon rewarming. The pathophysiology involves intense vasoconstriction, endothelial damage, microthrombus formation, and sterile inflammation driven by ROS and DAMPs; reperfusion can amplify inflammatory cascades and extend tissue necrosis after initial freezing. Clinical severity ranges from superficial, reversible injury to deep necrosis requiring amputation. Rewarming protocols, thrombolysis for severe cases, and measures that limit reperfusion injury (antioxidants, anti-inflammatory agents) influence outcomes[12]. Many studies emphasize the importance of early recognition and interventions that decrease microvascular thrombosis and oxidative damage. Several centers report improved limb salvage with early tissue plasminogen activator in selected cases of severe frostbite, highlighting a vascular-centric pathogenesis[13].

Chemical burns (acid-induced burn injury)

Chemical burns occur from corrosive substances, such as acids or alkalis. Acids generally cause coagulative necrosis and eschar formation, which can limit deeper penetration, while alkalis result in liquefactive necrosis that can penetrate more deeply and cause progressive tissue destruction. Severity depends on factors such as agent concentration, pH, contact time, and penetration characteristics (e.g., HCl can cause systemic toxicity). Immediate first aid (thorough irrigation) and prompt surgical treatments are essential for management[14]. Pathogenically, chemical agents provoke protein denaturation, disrupt lipid membranes, and cause rapid cell death, leading to a localized inflammatory response; prolonged exposure may result in secondary infection and persistent necrosis. Recent research emphasizes topical neutralizing agents, barrier dressings, and targeted methods to reduce secondary inflammation and enhance healing outcomes[15]. Overall, excisional, thermal, cold-induced, and chemical burns each damage tissue integrity through different mechanisms, yet converge on pathways of inflammation, vascular injury, and impaired regeneration. Effective wound management relies on early intervention, limiting secondary damage, and using strategies that restore vascular function, modulate inflammation, and promote tissue repair. Advances in biomaterials, stem-cell-based therapies, and targeted molecular approaches are continually improving outcomes for these diverse injury types.

CHRONIC VS ACUTE WOUNDS

Wounds are categorized as acute or chronic based on their healing physiology and/or pathophysiology. An acute wound heals in an orderly manner, replacing the damaged skin with scarred tissue with minimal fibrosis. Upon encounter with the subendothelial matrix, platelets become activated, contribute to clot formation and secrete various cytokines to recruit leukocytes at the injury site. Platelets secrete growth factors that stimulate keratinocytes and fibroblasts to proliferate and secrete anti-bacterial peptides. PDGF stimulates the production of endothelial cells to repair the blood vessel wall. Once sufficient clot is formed, the coagulation process is switched off. Injured tissue or necrotic cells stimulate inflammation through DAMP and pathogen-associated molecular patterns. These molecular signals activate tissue-resident immune cells, which in turn release pro-inflammatory cytokines, including IL-1 and tumor necrosis factor (TNF)-α, to attract circulating leukocytes, such as neutrophils. Neutrophils phagocytize pathogens and participate in ROS production. This hypoxic environment signals various cellular processes involved in cell proliferation and tissue repair. Along with neutrophils, monocytes infiltrate the damaged site where they differentiate into macrophages. Macrophages exhibit a spectrum of phenotypes, with major types being pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. The local tissue milieu determines macrophage phenotype. Once the inflammatory phase is over, M2 macrophages initiate re-epithelization, fibroplasia, and angiogenesis. The coordinated actions of macrophages facilitate the removal of debris, bacteria, and pro-inflammatory cells, while simultaneously promoting reparative processes that enable effective wound healing[16,17]. The normal function of inflammation in an acute wound is to prepare the wound bed for healing, as well as to recruit and activate fibroblasts. Inflammatory phase is the curtail phase, which, if prolonged, halts the physiological healing process. When a wound does not heal in an orderly and timely manner, it turns into a chronic wound. Pathophysiology of chronic wounds includes continuous inflammation, necrosis, prolonged hypoxia, and infection. All of these are interlinked and form the basis for chronic wound characterization. Chronic wounds contain higher numbers of M1 macrophages, while M2 macrophages are comparatively scarce. These macrophages have limited capacity to remove dead neutrophils, fueling persistent inflammation. Excess inflammatory signals, including TNF-α and IL-1β, sustain a pro-inflammatory environment. In the wound milieu, macrophages secrete MMPs, especially MMP-2 and MMP-9, which degrade the ECM and hinder the transition from the proliferative phase[17,18].

Ischemic ulcers

An ischemic ulcer or arterial ulcer develops due to the narrowing of the arterial lumen, which reduces the blood supply to the tissue, resulting in localized ischemia and hypoxia. Thromboembolism, atherosclerosis, diabetes, high cholesterol, hypertension, tobacco consumption, and aging are the causes of lumen narrowing and arterial insufficiency[16,17].

Diabetic wounds

Nearly 0.5 billion people have diabetes globally. Diabetes types I and II are the most prevalent, exhibiting hyperglycemia in patients. The resulting glycation from hyperglycemia leads to the accumulation of advanced glycation end products. Diabetes-linked advanced glycation end products buildup promotes oxidative stress, harms skin and immune cells, and stiffens the ECM. This chronic, low-grade inflammation can sustain itself, contributing to often deadly foot ulcers. Neuropathy, ischemia, and trauma form the classic pathogenic triad. In diabetes, a numb, structurally weakened foot is more likely to develop ulcers from repetitive mechanical stress and reduced blood flow. Nerve dysfunction common in neuropathy can impair sensation and movement, causing foot deformities such as hammerhead or claw toes[16,17].

Pressure sores

Pressure sores, also known as pressure ulcers, are caused by prolonged and localized pressure on tissues. Continuous localized pressure blocks blood and lymphatic vessels, leading to ischemia and hypoxia at the site, which can cause tissue necrosis. Compared to the epidermis, the subcutaneous tissue and muscles are more vulnerable to pressure-induced damage. These ulcers commonly develop over bony prominences, where cone-shaped damage extends from the bony area to the dermis. In addition to consistent pressure, factors such as moisture, friction, and shear stress significantly contribute to the development of pressure ulcers. The National Pressure Ulcer Advisory Panel classifies pressure ulcers into four categories based on the depth and severity of the damage. The first stage presents as non-blanchable erythema. In the second stage, the epidermis and dermis are affected to a lesser extent. The third stage shows atrophy of the subcutaneous tissue, sparing the fascia, while the fourth stage involves full-thickness skin loss, with damage extending to muscles, tendons, and bones. Altered physiology - such as fever, anemia, malnutrition, and endothelial dysfunction - also plays a role in pressure ulcer development. Additionally, comorbid conditions like diabetes, cardiovascular diseases, and lung diseases can accelerate the pathological condition[17,19].

SKIN WOUND HEALING AND REGENERATIVE APPROACHES

Regenerative medicine-based methods are becoming more popular for skin regeneration among the public and medical professionals. These methods are classified as either cell-free or cell-based treatments and are considered both safe and efficient. MSCs, tissue-induced pluripotent stem cells (iPSCs), blood-derived and fibroblast-based product treatments, such as those that use platelet-rich plasma products, are examples of cell-based therapies. MSC-derived extracellular vesicles (EVs), or exosomes, are the main components used in cell-free treatments. Before transplantation, MSCs are propagated ex vivo from various tissues, including bone marrow, fat, umbilical cord (UC), and fetal skin[20]. Regenerative medicine seeks to enhance the tissue regeneration process by addressing physiological deficiencies using a multifaceted problem-solving method associated with cutaneous wound healing[21]. These possibilities include the direct administration of biomaterials, growth factors, and stem cells to promote regeneration, alter the wound environment, and facilitate more efficient healing. MSCs promote all stages of wound healing and lessen scarring by migrating to the site of skin damage, reducing inflammation, and increasing the proliferative and differentiation capacity of endothelial cells, fibroblasts, and epidermal cells[22]. Regenerative medicine-based therapies for various pathologies, including skin pathologies, are generally regarded as safe[23]. However, some limitations remain, particularly those related to donor factors, tissue origin, isolation, and culture procedures, risk of adverse effects such as tumorigenicity, and some ethical regulatory restrictions.

NEED FOR STEM CELL-BASED TREATMENTS

Cell-based treatment, as a form of regenerative medicine, is one of the most promising areas in modern science and medicine. This powerful technology offers unlimited possibilities for revolutionary and potentially curative treatments for some of humanity’s most lethal diseases. Regenerative medicine is rapidly emerging as a major technology in healthcare, focusing on repairing or replacing damaged cells, tissues, or organs to restore normal function. The hope that regenerative medicine could serve as an alternative to traditional drug therapies is becoming more realistic every day, fueled by dedicated research exploring its potential in various diseases, including neurodegenerative disorders and diabetes[24].

Stem cell-based treatments are defined as any treatment for a disease or medical condition that uses viable human stem cells, such as embryonic stem cells (ESCs), iPSCs, and adult stem cells, for both autologous and allogeneic applications[25]. Stem cells are ideal for tissue and organ transplantation due to their ability to differentiate into the specific cell types needed for repairing damaged tissue. For example, a case of epidermolysis bullosa showed skin regeneration after treatment with keratinocyte cultures derived from epidermal stem cells[26]. Additionally, transplanting patient-derived iPSC-derived pigment epithelial cells into the retina resulted in significant vision improvement in patients with macular degeneration[27]. However, due to the complexity of stem cell-based treatments, researchers are seeking stable, safe, and widely accessible stem cell sources capable of differentiating into multiple lineages. Therefore, it is crucial to carefully identify the type of stem cells suitable for therapeutic use[28,29].

TYPES OF STEM CELLS IN CUTANEOUS REGENERATION

ESCs

ESCs can differentiate into many different lineages. The inner cell mass of pre-implantation embryos is often the source of ESCs. They are used in a wide range of biomedical applications due to their exceptional developmental potential and unlimited self-renewal capacity. To obtain and maintain ESCs in a naive pluripotent state, various culture media have been developed[30]. These stem cells are only accessible during the early stages of development and can differentiate into any cell type. ESCs can be used to grow skin, nerve, and liver cells for transplantation[31].

iPSCs

iPSCs are generated from adult somatic cells that have been genetically reprogrammed to resemble ESCs by inducing the expression of specific genes and other components necessary to maintain the characteristics that differentiate ESCs[32]. Human and mouse fibroblasts and other somatic cells have been reprogrammed into iPSCs using different combinations of transcription factors (KIf4, Sox2, Oct4, and Myc)[33].

MSCs

MSCs, also called adult stem cells, can be obtained from both human and animal sources. Human MSCs (hMSCs) are multipotent, non-hematopoietic stem cells (HSCs) capable of differentiating into ectodermal (neurocytes), endodermal (hepatocytes), and mesodermal (osteocytes, adipocytes, and chondrocytes) lineages. MSCs do not express CD14, CD34, CD45, or human leukocyte antigen (HLA-DR), but they do express cell surface markers such as CD105, CD90, CD73, CD44, and CD29. Initially, these cells are isolated from bone marrow. Afterward, hMSCs have been derived from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, UC, and Wharton’s jelly which contains potential MSCs. Long-term culture of hMSCs in specific media has been achieved without causing significant abnormalities. Additionally, MSCs exhibit immunomodulatory properties, secrete cytokines, and possess immune receptors that influence the microenvironment in host tissues. Their multi-lineage potential, immunomodulation capabilities, and production of anti-inflammatory chemicals make them a valuable tool for treating chronic diseases[34].

Bone marrow-derived MSCs

Bone marrow-derived MSCs (BM-MSCs) are postnatal stem and progenitor cells that may self-renew[35] and have the ability to develop into many mesenchymal lineages, including cartilage, bone, tendon, muscle, fat, and marrow stroma, which promote hematopoiesis. Their differentiation capacity makes them ideal for cell-based treatment techniques for mesenchymal tissue injuries, both locally and systemically[36]. BM-MSCs have been extensively used in tissue engineering approaches[35].

Adipose-derived MSCs

Adipose tissue develops from the mesoderm during embryonic development and is found in all mammalian species. It acts as an endocrine organ that regulates energy metabolism by storing lipids. There are two types of adipose tissues - brown and white - but white adipose tissue produces the most studied adipose-derived stem cells (ADSCs). These stem cells are highly promising for regenerative medicine and tissue engineering. ADSCs can be used in various applications due to their widespread availability and ability to differentiate into mesodermal tissues, including bone, cartilage, muscle, and fat. They have been used in research studies related to heart diseases, diabetes mellitus, osteoarthritis, and soft tissue repair, including facial repair and mastectomy[37].

UC-derived MSCs

Human UC has great potential as a source of MSCs. Apart from their promising benefits, such as a painless collection process and rapid self-renewal, human UC-derived MSCs (hUC-MSCs) have been shown to differentiate into three germ layers, aggregate in injured or inflamed areas, stimulate tissue regeneration, and modulate immune response. Since UC is regarded as medical waste and hUC-MSCs are collected noninvasively, access to these cells is not hindered by ethical concerns. Like MSCs derived from other sources, hUC-MSCs possess a specific potential for self-renewal while maintaining multipotency, i.e., the ability to develop into adipocytes, osteocytes, chondrocytes, neurons, and hepatocytes[38]. These cells could also be used to treat various diseases, including diabetic wound healing[39], cardiovascular conditions[40], and liver diseases[41].

Placenta- and amniotic-derived MSCs

A human placenta is the first structure to develop during pregnancy, and an embryo cannot survive without it. Therefore, the human placenta plays a vital role in fetal growth, nourishment, and the mother’s ability to maintain immune tolerance[42]. Recently, the placenta has garnered more attention because it contains a diverse pool of stem cells, including MSCs[43]. Placentas are typically discarded after birth, making them easy to obtain. The human placenta contains tissues of both fetal and maternal origin[44]. It is divided into two types: Fetal (amniotic fluid, amniotic membrane, and UC/Wharton’s jelly) and maternal (decidua parietalis). MSCs derived from various placental sections (PMSCs) are an abundant, allogeneic, and long-lasting source of MSCs compared to BM-MSCs. PMSCs can be preserved after delivery for future use in allogeneic and autologous disease therapies. They can be observed as a bridge between BM-MSCs and ESCs. PMSCs do not carry the ethical concerns associated with ESCs. Many diseases, including cancer, neurological, cardiovascular, and bone disorders, are treated using PMSCs[45].

Other stem cell sources (epidermal stem cells, hair follicle stem cells)

In general, epithelial stem cells come under the larger category of adult tissue-resident stem cells. They are classified into two main types: Hair follicle stem cells and interfollicular epidermal stem cells, and each has unique properties. The basal layer of the epidermis contains interfollicular epidermal stem cells, which are thought to be capable of self-renewal. It has been hypothesized that some epidermal basal cells exhibit stem cell-like characteristics, as epidermal renewal occurs continuously throughout life[46]. These epidermal stem cells replicate a few times after injury before differentiating. As a result, these cells are unipotent and differentiate to form the mature epidermis of the adult skin[47]. In contrast, it has been shown that hair follicle stem cells from the bulge region can both aid in epidermal regeneration in response to trauma and regenerate hair follicles[48].

MSC-MEDIATED THERAPEUTIC MECHANISTIC PATHWAYS

MSCs exert their therapeutic effects through a complex interaction of cells, mediators and cellular processes, i.e., immunomodulation, paracrine secretion, differentiation, ECM remodeling, and signaling pathway activation (Figure 3). These processes vary across different wound types (acute, chronic, burn, diabetic, etc.), but many converge on common molecular and cellular mechanisms. The main therapeutic pathways MSCs use to promote tissue regeneration and wound healing are outlined here.

Figure 3 Mechanistic overview of the role of mesenchymal stem cells in wound healing. ECM: Extracellular matrix; MSCs: Mesenchymal stem cells; EVs: Extracellular vesicles.

Immunomodulation and macrophage polarization

MSCs release factors that change macrophage phenotype from a pro-inflammatory M1 state to an anti-inflammatory, healing M2 phenotype. For example, MSC-derived TNF-stimulated gene/protein 6, IL-6, and prostaglandin E₂ have been reported to promote M2 polarization, thereby reducing local inflammation and accelerating wound closure[49,50]. They can downregulate pro-inflammatory cytokines (TNF-α, IL-1β) and upregulate anti-inflammatory cytokines (IL-10 and TGF-β). In chronic wounds (e.g., diabetic wounds), this helps resolve prolonged inflammation that delays healing[51,52]. MSCs also suppress the proliferation and activation of effector T cells [e.g., T helper 1 (Th1), Th17], while promoting regulatory T cells and Th2 responses through secretion of immunosuppressive factors such as prostaglandin E₂, indoleamine 2,3-dioxygenase, TGF-β, IL-10, and via cell-cell contacts (e.g., programmed cell death protein 1/programmed death ligand 1 interactions). They modulate innate immune cells: For example, MSC-secreted factors can reduce neutrophil apoptosis, influence natural killer cell cytotoxicity, and inhibit dendritic cell differentiation or maturation, thereby dampening excessive immune responses and preventing additional tissue damage[53]. Through these immunomodulatory effects, MSCs create a microenvironment conducive to the transition from the inflammatory phase to the proliferative/regenerative phases of wound healing, a feature particularly beneficial in chronic or non-healing wounds.

Paracrine secretions: Promoting proliferation, angiogenesis, and cell recruitment

MSCs release various growth factors, including VEGF, basic fibroblast growth factor (FGF), FGF-2, keratinocyte growth factor, hepatocyte growth factor, PDGF, stromal cell-derived factor 1/C-X-C motif ligand 12, insulin-like growth factor, and angiopoietin-1[54]. These factors stimulate endothelial cell proliferation and migration, promote neovascularization, and support re-epithelialization and granulation tissue formation[55,56]. Besides soluble factors, MSCs secrete EVs (including exosomes and microvesicles) that contain proteins, mRNAs, and microRNAs (miRNAs), which act on neighboring cells to modulate proliferation, differentiation, and survival. These exosomes have been shown to enhance keratinocyte and fibroblast migration and proliferation and influence ECM remodeling[57,58]. MSCs (and their exosomes) influence several intracellular signaling cascades in target cells, including PI3K/Akt, MAPK, Wnt/β-catenin, and Notch pathways, which regulate key processes such as cell proliferation, migration, differentiation, and angiogenesis[59]. Through paracrine factors and exosomes, MSCs stimulate dermal fibroblasts (enhancing collagen, elastin, and fibronectin production), keratinocytes (for re-epithelialization), and endothelial cells (for new vessel formation), thereby accelerating wound closure and tissue regeneration[60]. This paracrine and EV-mediated mechanism is widely considered the primary way MSCs exert therapeutic effects in wound healing, often more than direct differentiation under many circumstances[61].

ECM remodeling and balanced matrix deposition

MSCs (via secreted factors or conditioned medium) promote the production of ECM proteins (e.g., collagen, elastin, fibronectin) by resident fibroblasts and simultaneously suppress matrix-degrading enzymes (e.g., MMP-1), thereby maintaining ECM integrity and preventing excessive degradation[60]. During the later remodeling stage, MSCs influence the balance between MMPs and TIMPs, supporting controlled ECM turnover (organized matrix deposition and maturation rather than disordered fibrosis). It is reported that upregulation of MMP-2 and MMP-9 alongside downregulation of TIMP-1 and TIMP-2 after MSC treatment, may aid in remodeling granulation tissue and achieving proper wound closure[52]. In some cases, (e.g., hypertrophic scars), MSCs or their derivatives can modulate profibrotic signaling. For example, hUC-MSCs inhibit hypertrophic scar fibroblast proliferation, migration, and fibrogenic activation through suppression of the TGF-β1/Smad3 signaling pathway[51]. Similarly, in other models, MSCs have been shown to inhibit the p38 MAPK signaling pathway (e.g., via release of proenkephalin), reducing excessive fibroblast proliferation and scar formation[50,52]. Through these ECM-modulating effects, MSCs contribute to the restoration of tissue architecture and mechanical integrity while minimizing fibrosis and pathological scarring.

Signaling pathway activation, exosome/miRNA-mediated regulation, and cellular crosstalk

MSC-derived signals activate pathways such as PI3K/Akt, MAPK, Wnt/β-catenin, and Notch, promoting the proliferation, migration, angiogenesis, and differentiation of target cells, including fibroblasts, keratinocytes, and endothelial cells[59]. MSC exosomes carry miRNAs that modulate gene expression in recipient cells; for example, some miRNAs suppress inflammatory signaling, such as downregulating Toll-like receptor 4, thereby decreasing inflammation and aiding wound regeneration[62]. MSCs also secrete TNF-stimulated gene/protein 6 and other factors that inhibit neutrophil extravasation, by interfering with IL-8/C-X-C motif ligand 8 signaling, reducing early tissue damage caused by excessive inflammation[63]. In some injury models, MSCs migrate to wound sites influenced by microenvironment cues, and may differentiate into relevant cell types such as endothelial cells, smooth muscle cells, and perhaps fibroblast or skin lineages, directly supporting tissue repair and vascularization[50]. In burn wounds or full-thickness skin injuries: MSCs (especially when preconditioned or combined with scaffolds/hydrogels) may promote neovascularization, granulation tissue formation, re-epithelialization, and ECM deposition through paracrine growth factors, exosome-mediated signaling, and possibly direct differentiation into endothelial or skin lineage cells[64,65]. In scar-prone wounds or fibrotic conditions, MSCs can reduce excessive fibroblast activation or pathological ECM deposition by modulating TGF-β1/Smad and MAPK/p38 pathways, thereby reducing fibroblast proliferation and collagen overproduction, thus lowering the risk of hypertrophic scar or fibrosis formation[66,67]. Collectively, the intricate interplay, paracrine signaling, exosome-mediated miRNA/protein transfer, activation of cellular pathways, immune regulation, and environment-dependent differentiation, allows MSCs to coordinate and orchestrate the wound healing process in a multifaceted manner.

ROLE OF OTHER STEM CELLS IN SKIN REPAIR AND REGENERATION

HSCs

HSCs are a vital source for producing all blood cell lineages, and their role in the immune and vascular systems is crucial for skin regeneration and repair. HSC-derived immune cells, such as neutrophils and macrophages, initiate the inflammatory phase during injury by fighting pathogens and debris, and release growth factors and cytokines that promote healing. Macrophages are particularly important as they support angiogenesis, activate fibroblasts, and promote keratinocyte proliferation when switching from pro-inflammatory to pro-repair phenotypes, by fostering the formation of new blood vessels and ensuring an adequate supply of oxygen and nutrients to healing tissues, HSC-derived endothelial progenitor cells further improve wound healing. Furthermore, these cells secrete cytokines that regulate collagen deposition and ECM remodeling, helping to restore the skin’s structural integrity[68].

iPSCs

The ability of iPSCs, which are reprogrammed from adult somatic cells to a pluripotent state, to produce nearly any cell type required for skin regeneration, including fibroblasts, keratinocytes, endothelial cells, and vascular cells, makes them highly promising for wound healing. Transplanting iPSC-derived keratinocytes has been shown to accelerate re-epithelialization and reduce scar formation in full-thickness skin wounds. It is suggested that iPSC-derived skin cells can directly assist in restoring epidermal structure. Besides directly replacing cells, iPSC-derived cells (or their secreted factors) promote angiogenesis, collagen deposition, and ECM remodeling all of which are essential for skin regeneration and wound closure. Cell-free approaches, such as using exosomes or EVs derived from iPSCs or iPSC-derived MSCs, have demonstrated efficacy in promoting wound repair by increasing keratinocyte and endothelial cell migration, reducing excessive inflammation, and increasing vascularization. These strategies may also lower the risk of tumorigenicity associated with whole-cell therapies[69,70].

ADSCs

ADSCs are found in many skin layers, including the hypodermis and associated hair follicle regions. They play a crucial role in skin regeneration, aging repair, and wound healing by dividing into skin cells and releasing regenerative signaling chemicals. During injury, ADSCs quickly migrate to wound sites, where they can differentiate into fibroblasts, endothelial cells, and keratinocytes. They also function with dermal fibroblasts to produce ECM components required for tissue repair. Their secretome regulates inflammation by modulating macrophage activity, stimulating angiogenesis, and supporting granulation tissue development, all of which are vital during the proliferative and remodeling phases of healing. Due to these abilities, ADSCs are considered highly promising for therapeutic and regenerative applications, including emerging cell-free approaches based on ADSC-derived secretome or EVs, although further research is needed to optimize their clinical effectiveness[71].

OPTIMIZATION STRATEGIES FOR MSC THERAPY

Multiple in vitro studies have demonstrated the potential of MSCs across various therapeutic areas, including immunomodulation, cell proliferation, tissue regeneration, angiogenesis, anti-fibrotic, anti-inflammatory, and anti-apoptotic activities. However, MSC transplantation in animal models has shown limited therapeutic efficacy. Time and route of delivery, prior in vitro conditions, and the hostile host environment at the damage site may contribute to the compromised therapeutic effect of MSCs. Various adjuvant strategies have been explored to boost the therapeutic potential of MSCs, including genetic modification and priming or preconditioning. These priming methods involve pre-incubating MSCs with pharmacological agents, cytokines, trophic factors, or exposing them to hypoxic conditions to enhance their survival and regenerative capacity in challenging microenvironments[67].

Preconditioning approaches

Current research studies are now optimizing the methods to prepare MSCs to cope with the harsh microenvironment, as well as optimizing the time and dose of the therapy. Preconditioning approaches involve pretreatment of cells with cytokines, small molecules, or compounds, or providing specific culture conditions like hypoxia before transplantation. Table 2 compares the therapeutic aspects of each preconditioning strategy[72-78].

Table 2 Comparison of various preconditioning approaches.

No.	Preconditioning strategy	Mechanism of action	Consideration
1	Hypoxic preconditioning	Low O2 stimulates hypoxia-inducible factor production, which in turn stimulates the expression of genes involved in cell survival, proliferation, migration, angiogenesis, metabolism, and cell apoptosis[77]. Preconditioned cells secrete extracellular vesicles that contain proteins, lipids, nucleic acids, and metabolites, and induce therapeutic effects through paracrine signaling[78]	Time duration and %O2 provided for hypoxia need to be optimized. Mainly essential for cell survival after transplantation
2	Compound/small molecule preconditioning	Compounds or small molecules may possess immunomodulatory, anti-inflammatory, and antioxidant properties. They may induce cell proliferation and differentiation[72,73,78]	Cytotoxicity profile and effective dose optimization need to be done. Utilized when considering a specialized aspect or stem cell differentiation towards a specific lineage
3	Genetic engineering of MSCs	Genetic or epigenetic modification of MSCs is done to modify the expression of various genes involved in cell proliferation, differentiation or other immunomodulatory properties[74-76]	For specialized therapeutic aspects or stem cell differentiation towards a specific lineage. It involves genetic manipulation

Hypoxic preconditioning

Hypoxia preconditioning is one of the approaches to make cells adapt to a hypoxic environment. It is due to the phenomena that injured tissue suffers ischemia and hypoxia due to vascular damage, inflammation, and ROS production in the micro milieu. During in vitro culture, cells are exposed to 21% of O₂, but after transplantation, cells encounter a lower level of O₂, i.e., 1%-6% O₂. This abrupt change disrupts vital cellular processes, including proliferation, differentiation, metabolism, and senescence; as a result, it makes cell survival difficult and compromises therapeutic efficacy. In hypoxia preconditioning, the O₂ exposure level is reduced 15% to 1%, which helps cells adapt to the hypoxic environment[79]. Various studies have reported the effectiveness of hypoxia preconditioning. Recovery of structural and functional damage has been reported in multiple disease models, such as ischemic acute kidney injury, cardiovascular diseases, spinal cord injury models, and wound models[79-82].

Chemical preconditioning (α-terpineol, isorhamnetin, quercetin, rutin)

Stem cells are being preconditioned with small molecules, compounds, or cytokines having cytoprotective properties. Compounds and extracts from plants traditionally used to treat various diseases are used to prime MSCs[72,73]. Synthetic compounds and multiple cytokines and growth factors are also analyzed for preconditioning MSCs. One of the most important and promising groups of natural products for the treatment of skin lesions is the flavonoids. Flavonoids are phenolic compounds that can be found in fruits, vegetables, herbs, cocoa, chocolate, tea, soy, red wine, and other plant food and beverage products. These compounds are reported for their anti-bacterial, anti-fibrotic, antioxidant, and anti-inflammatory properties[83]. Many flavonoids have been characterized as potent ROS inhibitors, making them vital antioxidants. The use of antioxidants, such as flavonoids, is thought to accelerate wound healing by reducing oxidative stress[84]. Quercetin, one of the flavonoids, possesses potent antioxidant and anti-inflammatory effects, which support its potential use in wound healing. It forms o-quinones, which, upon treatment with a solvent containing water, restore the potent antioxidant activity of the quinone. Additionally, quercetin can reduce both acute and chronic inflammatory stages. Quercetin has also been reported to minimize fibrosis and scar formation during wound healing, promote fibroblast proliferation, reduce immune cell infiltration, and alter fibrosis-related signaling pathways[85,86].

Another flavonoid, rutin (quercetin-3-O-rutoside), has antioxidant, anti-inflammatory, neuroprotective, nephroprotective, and hepatoprotective effects. Chen et al[87] showed that rutin effectively upregulated nuclear factor erythroid 2 like 2 expression, increasing the production of antioxidant enzymes, and downregulated nuclear factor kappa B expression, reducing the production of MMPs, growth factors, and inflammatory cytokines in wounds. The mechanistic representation of rutin for promoting wound healing may be derived from the regulation of blood sugar and excellent antioxidant and anti-inflammatory effects.

α-terpineol (αT) belongs to a larger class of monoterpenes. Its pharmacological properties, including anti-inflammatory, antioxidant, and antimicrobial activity, makes it particularly valuable for wound healing[88,89]. Terpineols are unsaturated monocyclic monoterpenoid alcohols and can be found in flowers such as narcissus and freesia, in herbs such as marjoram, oregano, and rosemary, and in lemon peel oil. By reducing inflammation, oxidative stress, nitric oxide, and TNF-α, αT helps create an optimal environment for tissue repair and wound healing[90]. MSCs preconditioned with αT showed improved healing in full thickness rat burn wound[91].

GENETIC ENGINEERING OF MSCS

Genetic engineering involves genetic and epigenetic modifications, which have been employed to enhance the therapeutic potency of MSCs. Genetic modification consists of the change in the expression of various genes by inserting, deleting, and modifying them. Genetic expression of signaling molecules or proteins, which play a vital role in cellular processes such as cell proliferation, differentiation, or immunomodulatory properties, is being manipulated by DNA modification. The genetic modification can be done either by viral vectors, including lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses, and non-viral vectors such as DNA, RNA, protein, and protein-RNA complexes[74].

Epigenetic modification involves changes in chromatin state without altering the DNA sequence of a gene. These heritable changes affect gene function and occur through DNA modification, histone modification and the regulation of non-coding RNA. The mechanisms of epigenetics include, histone modification, and regulation of non-coding RNA[75]. A recent direct approach to DNA modification is CRISPR/Cas9. The CRISPR/Cas9 efficiently cleaves the target DNA sequence by creating double-strand breaks. This DNA is then edited and repaired by the intracellular DNA repair system[92]. Elevating the levels of factors, like VEGF, FGF, epidermal growth factor, stromal cell-derived factor 1, etc., could enhance MSC migration, vasculature-targeting activity, anti-inflammatory responses, and cell survival, thereby supporting greater recovery of renal function[76]. However, translating this approach to a clinical setting requires in-depth research and optimization, as overexpression of the genes also poses the risk of stem cell-derived tumors[93].

BIOMATERIAL-BASED DELIVERY SYSTEMS

Biomaterial-based cells and drug-delivery scaffolds are being used and studied to improve and sustain drug and cell release. For drugs or biomolecules, these supporting scaffold provide sustained and controlled release of therapeutic agents at the site of damage without any loss in the systemic circulation. In the case of cells, these scaffolds provide a three-dimensional (3D) architecture that supports the attachment and survival of transplanted cells at the injury site, enhancing therapeutic outcomes.

Hydrogels, scaffolds, nanomaterials

Hydrogels are 3D polymer-based structures having high water content and porosity. These structures possess characteristic chemical, physical, and mechanical properties, such as viscoelasticity, matrix modulus, dimensionality, porosity, and cell adhesion[94]. Water retention and porosity of hydrogels give them ECM-like features, facilitating the exchange of signaling molecules and nutrients and providing architecture for cell adherence. These hydrogels are derived from either naturally occurring polymers, such as chitosan, agarose, gelatin, alginate, collagen, fibrin, and hyaluronic acid, or synthetic polymers, such as polyethylene glycol, polyvinyl alcohol, and poly(2-hydroxyethyl) methacrylate[95]. A combination of natural and synthetic polymers is also being used to develop a hybrid hydrogel[95]. Different tissues are being used to develop an ECM-derived hydrogel that can mimic ECM more effectively. Various ECM-like structural components, including collagen, hyaluronic acid, and soluble and juxtacrine factors present in bioscaffolds, aid cell migration and differentiation. The composition of these ECM components varies depending on the type of tissue of origin[96]. The bio-scaffolds showed improved tissue repair in the dermis[97], muscle[98], heart[99] peripheral nerve[100], and bladder[101,102].

Combination therapies (MSCs and growth factors, MSCs and biomaterials)

Tissue-engineered and cell-based approaches are combined to enhance the therapeutic effects of all agents. Preclinical studies used cell-seeded scaffolds in combination with various drugs and biomolecules, such as growth factors, cytokines, and small molecules. Research has demonstrated a potential synergistic effect in different in vitro and in vivo disease models. Pullulan-collagen-based hydrogel seeded with MSCs showed significantly improved wound healing in an excisional wound model compared to transplanting MSCs alone[103,104]. A hybrid hydrogel derived from polyvinyl alcohol and tapioca pearl starch, loaded with αT, exhibited a comparatively higher healing rate by promoting angiogenesis, collagen and keratin deposition, re-epithelization, and enhanced wound closure in a chemical-induced burn wound model[105]. Another study using a gelatin-methacryloyl-based hydrogel loaded with VEGF showed potential for biocompatibility and improved healing quality both in vitro and in vivo in a porcine wound model[106].

Overall, biomaterial-based delivery systems provide controlled release of therapeutic agents and create supportive 3D microenvironments that improve cell retention, survival, and function at wound sites. Hydrogels, scaffolds, and nanomaterials mimic key ECM features and enhance regeneration across multiple tissues. Combination strategies integrating MSCs with growth factors or biomaterial platforms further boost reparative outcomes and represent a promising direction for advanced wound healing therapies.

CLINICAL APPLICATIONS AND TRANSLATIONAL CHALLENGES

Cell dose

Translating pre-clinical studies into clinical applications remains a challenge for stem cells (MSCs). Several factors hinder this translation[107]. First factor is the number of cells needed per therapeutic dose. The number of cells obtained from a specific tissue is limited. It has been reported that 1 mL of bone marrow contains only 1-100 MSCs, yet the desired dose requires 1-100 million cells. This cell count is achieved by in vitro culture, which takes time. Culture conditions can affect MSC characteristics, including the loss or decrease of pluripotency markers[108], unwanted differentiation[108], and replicative senescence[109,110]. Cell culture media often use fetal bovine serum or other xeno-products for MSC expansion, which poses a risk of host-vs-graft disease.

Route of transplantation and skin grafts

The other important aspect of the clinical use of MSCs is the route of transplantation and the efficient delivery and survival of MSCs at the injury site. In many studies, MSCs are delivered either systemically or locally. Through the intravenous route, the cells are observed to be accumulated in the lungs and phagocytosed by monocytes within 24 hours of transplantation[111,112]. Upon arrival at the local injury site, the cells encounter a harsh microenvironment; the presence of inflammation, ischemic tissue, and high ROS production makes cell survival difficult. It is reported that less than 5% of the transplanted cells were present at the site of injury a few hours after administration[113]. Thus, a local carrier or scaffold is needed for the survival, sustained release, migration, and engraftment of cells within the damaged tissue or wound bed[114]. Researchers are exploring skin grafts and natural or synthetic scaffolds for cell delivery, but these studies have various aspects yet to be explored regarding safety and efficacy. For the safe and standardized administration and release criteria of all forms of MSC therapeutics, immune compatibility and hemocompatibility necessitate the inclusion of HLA mismatch assessment and expression of pro-coagulant factors[115].

Another important factor is the mechanism of action of MSCs. To date, MSCs have been shown to exert therapeutic effects through engraftment, differentiation, or paracrine signaling. However, the timings and extent of these factors for an effective therapeutic effect is still unclear. Furthermore, the intrinsic properties of MSCs also vary depending on the source of isolation. Age, tissue type, and donor health condition all affect the intrinsic properties of MSCs[116]. There is a need for further exploration of the mechanism of action to understand which therapeutic aspect of MSCs would be best for specific pathological conditions, and what is the efficient source of cells for an optimal effect.

Establishment of good manufacturing practices

The application of MSCs falls under the category of advanced therapy medicinal product, which mandates strict regulations in accordance with good manufacturing practice (GMP) developed by the regulatory bodies such as the Food and Drug Administration, European Medicines Agency, or national authorities like the Drug Regulatory Authority of Pakistan. Mass production and scalability of MSCs are critical aspects that hinder their clinical translation. There is a lack of standardized protocols for MSC isolation and manufacturing. Manufacturing centers, stem cell banks, and laboratories are still working to optimize protocols in order to achieve the gold standard for scalable cell production. Differences in ex vivo expansion and manufacturing procedures affect the therapeutic potential of the cells[117]. The monolayer culture technique is inefficient for large-scale MSC production, prompting research into multilayer flasks, bioreactors, and microcarriers to enable higher yields[118,119]. However, it is still a debate as to what type of microcarriers, culture media, and methods are best for harvesting cells in their most unaffected form[120]. Quality control of the final product is an essential aspect; the entire process of cell production, expansion, and packaging must adhere to aseptic conditions[119]. Assays examining cell viability, microbial contamination, and the proliferation and differentiation potential of MSCs should be the integral components of standardized production.

Preclinical and clinical studies in various wound models

The well-established wound model for in vitro 2D culture is the scratch assay, which analyzes the migratory potential of cells. This model is developed by making a scratch in a monolayer of adherent cells, such as dermal cells (fibroblasts or keratinocytes) or other cells (MSCs), using a pipette tip. Cells migrate to fill the gap caused by the scratch, restoring cell-cell contacts, and the closure is measured by taking images at different time points[121,122]. Significant improvement in cell migration or wound-healing rates was observed when various cell types were primed with MSC-conditioned medium[123,124], MSC-derived EVs[125], or co-cultures of MSCs. In vivo studies, with or without priming, across various wound models, including excisional, ischemic, burn, and diabetic wounds, demonstrate comparable healing. In 2008, Debin et al[126] used autologous BM-MSCs in patients with type 2 diabetes, demonstrating relief of critical lower limb ischemia and accelerated ulcer healing. Another randomized clinical trial targeting non-healing ulcers with autologous BM-MSCs reported significant improvement in pain-free walking and reduction in ulcer size compared to the control group[127]. A summary of ongoing and completed clinical trials evaluating MSC-based therapies for various wound healing and regenerative conditions taken from the National Library of Medicine database is presented in Table 3.

Table 3 Ongoing and completed clinical trials using mesenchymal stem cells for wound healing and related disorders.

Wound/injury	Site of transplantation/implantation	Additional agents	Autologous/allogeneic	Cell source	Phase	NCT number
Mandible fractures	Local application on the fracture site during the surgical procedure	-	Autologous	Adipose tissue	3	02755922
Tibial closed diaphyseal fractures	Local application in the fractured site	-	Allogeneic	Adipose tissue	2	02140528
Tendon injury	Ultrasound-guided injection at the injury site	-	Allogeneic	Adipose tissue	1	01856140
Non union fracture	Local application	Biphasic calcium phosphate biomaterial granules and iliac crest graft	Autologous	Bone marrow	3	03325504
Second- or third-degree burns	Local application	Plasma fibrin hydrogel	Allogeneic	Adipose tissue	2	03113747
Heel injury	Local application	Skin graft	Allogeneic	Umbilical cord	1	04219657
Poor healing after uterus injury	Intrauterine injection	-	Allogeneic	Umbilical cord	1	03386708
Second degree burn wounds of less than 20% of the total body surface area	Local application	-	Allogeneic	N/A	1	02104713
Chronic wounds in diabetic foot syndrome	Direct application onto the prepared wound bed	Fibrin gel	Allogeneic	Adipose tissue	1-2	03865394
Non-union of long bone fractures	Local application in fractured zone	-	Autologous	Bone marrow	1	01206179
Non-united tibial and femoral fractures	Injection in non-union site	-	N/A	Bone marrow	2	01788059
Tendon injury	Local injection under ultrasound guidance	Fibrin glue + range of motion exercise	Allogeneic	Adipose tissue	2	02298023
Distal tibial fractures	Local implantation at the fracture site	MSC carrier	Autologous	N/A	1-2	00250302
Ocular corneal burn	Subconjunctival injection	-	N/A	Bone marrow	2	02325843
Fracture non-union healing	Local application of in vitro-expanded MSC	Carrier	Autologous	Bone marrow	N/A	02177565
Chronic ulcer wounds	Topical application of Wharton jelly MSC culture medium	Gel carrier	Allogeneic	Umbilical cord	1	04134676
Knee articular cartilage injury	Local application	-	Allogeneic	Umbilical cord	3	01041001
Deep second-degree burn wound	Local application	Hydrogel sheet	Allogeneic	Adipose tissue	1	02394873
Long bones non-union	Percutaneous application around fracture ends	-	Autologous	Adipose tissue	1-2	04340284
Knee articular cartilage injury or defect	Local application	-	Allogeneic	Umbilical cord	3	01626677
Support of autologous chondrocyte transplantation with instant MSCs	Filling of cartilage defect	Glue carrier, autologous chondrons	Allogeneic	N/A	1-2	02037204
Tibial shaft fracture	Local injection	-	Autologous	Bone marrow	N/A	00512434
Mandibular distraction osteogenesis	Local injection	-	N/A	Bone marrow	N/A	03861650
Severe epidermolysis bullosa	Serial infusions	Allogeneic hematopoietic stem cell transplant	Allogeneic (related)	N/A	2	02582775
Severe epidermolysis bullosa	Infusions	Allogeneic hematopoietic stem cell transplant	Allogeneic	N/A	1-2	01033552
Recessive dystrophic epidermolysis bullosa	Infusions	-	Allogeneic	Umbilical cord blood	1-2	04520022
Epidermolysis bullosa	Local application	Hydrogel sheet	Allogeneic	Adipose tissue	1-2	02579369
Epidermolysis bullosa	Local application	Hydrogel sheet	Allogeneic	Adipose tissue	1-2	03183934
Treatment-refractory chronic venous ulcers	Topical application	-	Allogeneic	Skin derived	1-2	03257098
Recessive dystrophic epidermolysis bullosa	Infusions	-	Allogeneic	Skin derived	1-2A	03529877

NCT: National clinical trial; N/A: Not applicable; MSCs: Mesenchymal stem cells.

Safety, ethical, and regulatory issues

For the production of clinical-grade MSCs, quality control checks are the priority. In this regard, the evaluation and control of contamination risks posed by microbial agents that could affect the quality of cells and the safety of patients have to be considered. All protocols have to be standardized for GMP of clinical-grade MSCs covering various aspects, starting from the initial source of MSC isolation, informed consent, and exclusion criteria, then ex vivo expansion of cells, testing for sterility, Gram stain, mycoplasma, or endotoxin contamination, and other microbial monitoring. Sterile packaging, shelf life control, and safer delivery are the other important aspects to be considered. Research is still going on to cover all these aspects and to establish a gold standard protocol for GMP[128].

Standardization and reproducibility in stem cell therapy

In stem cell research, reproducibility issues require the development and application of strong best practices and standards. A standard, according to International Organization for Standardization (ISO), is a consensus-based document that has been approved by an established organization and provides guidelines, specifications, and requirements to guarantee that materials, products, processes, and services are appropriate for their intended use and continuously satisfy quality, safety, and efficiency standards. The ISO has created official cell culture standards, some of which are particularly relevant to human stem cells. The International Stem Cell Banking Initiative’s earlier work served as the foundation for ISO 24603: 2022, which outlines the specifications for the bio-banking of human and mouse pluripotent stem cells, including biological material collection, establishment, characterization, and quality control (International Stem Cell Banking Initiative, 2009). Still there is a need of pre-normative research that would record and capture the best practices that researchers would conclude and communicate to build uniform criteria and standards.

CONCLUSION

MSCs have emerged as potent therapeutic agents for complex skin injuries due to their remarkable regenerative, paracrine, and immunomodulatory potential. Through the secretion of growth factors and cytokines, MSCs modulate inflammation, enhance angiogenesis, and promote ECM remodeling, thereby accelerating tissue repair. Their capacity to control immune cell activity, especially by inducing macrophage polarization toward an anti-inflammatory M2 phenotype, aids the transition from inflammation to regeneration. Recent advances in preconditioning methods, including hypoxic exposure and treatment with phytochemicals such as αT, isorhamnetin, quercetin, and rutin, have significantly enhanced MSC viability, oxidative resistance, and paracrine signaling, resulting in better wound healing. Simultaneously, genetic and epigenetic modifications have been used to boost pro-angiogenic and anti-apoptotic pathways, further enhancing their therapeutic potential. The integration of MSCs with biomaterial-based delivery systems, such as hydrogels, scaffolds, and nanocomposite matrices, has provided a biomimetic environment that supports cell survival, retention, and functional activity, offering a sustained release of bioactive substances and better regenerative results. Despite these advances, translational challenges persist, including donor-related variability, limited scalability, and the need for standardized GMP protocols to ensure safety, purity, and reproducibility of clinical-grade MSC products. Moreover, adherence to internationally recognized standards, such as those set by ISO and the International Stem Cell Banking Initiative, is crucial for regulatory approval and clinical application. Overall, the integration of optimized preconditioning techniques, bioengineered scaffolds, and molecular enhancements represents a transformative direction in regenerative medicine, with strong potential to achieve scar less, fully functional skin regeneration in the clinical settings.