Exosomes in nanomedicine: a promising cell-free therapeutic intervention in burn wounds

Abstract

Burn injuries are serious injuries that have a big impact on a person’s health and can even cause death. Incurring severe burns can incite an immune response and inflammation within the body, alongside metabolic changes. It is of utmost importance to grasp the fact that the effects of the burn injury extend beyond the body, affecting the mind and overall well-being. Burn injuries cause long-lasting changes that need to be taken care of in order to improve their quality of life. The intricate process of skin regeneration at the site of a burn wound involves a complex and dynamic interplay among diverse cells, growth factors, nerves, and blood vessels. Exciting opportunities have arisen in the field of stem cells and regenerative medicine, allowing us to explore the development of cell-free-based alternatives that can aid in the treatment of burn injuries. These cell-free-based therapies have emerged as a promising facet within regenerative medicine. Exosomes, also referred to as naturally occurring nanoparticles, are small endosome-derived vesicles that facilitate the delivery of molecular cargo between the cells, thus allowing intercellular communication. The knowledge gained in this field has continued to support their therapeutic potential, particularly in the domains of wound healing and tissue regeneration. Notably, exosomes derived from mesenchymal stem cells (MSCs) can be safely administered in the system, which is then adeptly uptaken and internalized by fibroblasts/epithelial cells, subsequently accelerating essential processes such as migration, proliferation, and collagen synthesis. Furthermore, exosomes released by immune cells, specifically macrophages, possess the capability to modulate inflammation and effectively diminish it in adjacent cells. Exosomes also act as carriers when integrated with a scaffold, leading to scarless healing of cutaneous wounds. This comprehensive review examines the role of exosomes in burn wound healing and their potential utility in regeneration and repair.

Graphical Abstract

Introduction

Burn wounds represent a significant global public health concern, affecting millions of people annually. These injuries can occur due to various causes such as thermal, electrical, chemical, or radiation exposure, and their severity can range from minor superficial burns to life-threatening injuries. They inflict immense pain and debilitation, impacting nearly all organ systems and leading to a spectrum of complications, such as infections, scarring, multi-organ failure, and enduring disability. Burn injuries encompass a spectrum of severity, ranging from superficial epidermal burns (first-degree) to profound injuries that breach deep tissues (fourth-degree). The classification of burn injuries into different degrees is essential in determining the appropriate treatment and wound management options (Fig. 1). Superficial burns, termed first-degree, exclusively affect only the outermost layer of the skin (epidermis), causing erythema and discomfort. This typically requires minimal treatment, focusing on pain relief and infection prevention. Second-degree burns breach the dermal layer, resulting in vesicle formation, heightened pain, and potential scarring. This necessitates more attentive wound care, such as cleaning, dressing changes, and potential topical treatments to promote healing and prevent infection. Third-degree burns extend into subcutaneous tissues, inducing pallor or eschar formation, often accompanied by nerve impairment. Fourth-degree burns, the most severe, transgress deeper structures like muscles and tendons, frequently mandating surgical intervention. As the complexity and severity of tissue damage increase significantly, surgical interventions, such as debridement (removing dead tissue) and possibly skin grafts, become necessary to facilitate healing and prevent complications. The level of burn injury also guides pain management strategies. Deeper burns are often more painful due to nerve involvement, requiring stronger pain medications and specialized approaches for relief. Furthermore, the potential for scarring and long-term functional impairments escalates with the depth of the burn injury. Therefore, wound management strategies vary based on the degree of burn to optimize healing, minimize complications, and promote the best possible outcomes for patients.

Fig. 1

Classification of burn wounds on the basis of severity of damage to the skin

Both major and minor severe burn injuries trigger a complex wound-healing process encompassing distinct but highly interconnected and overlapping phases. The initial phase, hemostasis, involves vasoconstriction and platelet aggregation, inducing clot formation to stem the bleeding. Subsequent to this, the inflammatory stage ensues, marked by the infiltration of leukocytes and the release of cytokines and chemokines, which initiate immune responses and facilitate the removal of debris. Concurrently, angiogenesis is prompted, and fibroblasts migrate to the wound site, heralding the onset of the proliferative phase (Fig. 2). Here, fibroblasts play a pivotal role in facilitating collagen synthesis, while keratinocytes migrate to re-epithelialize the wound [1]. Neovascularization, a hallmark of this stage, is crucial for the supply of oxygen and nutrients. Simultaneously, collagen fibrils undergo organization, enhancing tissue integrity in the remodeling phase. Extracellular matrix turnover, modulated by matrix metalloproteinases, refines tissue architecture. These stages are intricately governed by complex signaling networks, comprising regulatory interactions among cells and cytokines. Ultimately, this orchestrated progression culminates in wound closure and the subsequent formation of scars. Rigorous scientific investigation into these intricate processes continues to unveil novel therapeutic strategies aimed at optimizing outcomes in burn wound management.

Integral to the understanding of burn wound healing is an appreciation of the skin’s structural composition. Comprising a mosaic of cells, including keratinocytes, fibroblasts, and endothelial cells, as well as a dynamic extracellular matrix (ECM), the skin orchestrates a complex interplay of cellular and molecular events. The ECM, characterized by a network of proteolytic enzymes and secretory macromolecules, serves as a reservoir for critical growth factors such as transforming growth factor beta (TGF-β), epidermal growth factor (EGF), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and vascular endothelial growth factors (VEGFs). These growth factors have the capacity to be released in a localized manner, disengaging from their insoluble anchorage within ECM proteins such as collagen, fibronectin, heparins, and proteoglycans. This liberation occurs as a result of proteolytic enzyme-mediated degradation in response to wounds, which enhances the healing process [2].

The body experiences a detrimental and enduring reaction to severe burns, impacting both its overall metabolic activity and the healing process at the site of the burn. The pharmacokinetics and pharmacodynamics of drugs used to treat severe burn injuries are greatly influenced by these pathophysiological processes. Insufficient or excessive healing responses, such as those seen in hypertrophic scars, cause mucosal wound healing to fail, resulting in a poor clinical outcome [3]. Early burn wound excision and skin grafting are well-established clinical procedures that have enhanced the outcomes of patients with severe burns by lowering mortality and shortening hospitalization [4]. The impact of burn injuries extends beyond mortality rates and encompasses long-term physical, functional, cosmetic, and psychological traumas that can significantly impact the quality of life and overall health outcomes. Thus, ensuring effective management becomes critical in the context of patient well-being [5]. Tackling these intricate challenges necessitates the integration of innovative therapeutic modalities, meticulous scientific investigation, and a comprehensive patient-centered approach.

Fig. 2

Stages of burn wound healing: (1) Inflammatory Response: pro-inflammatory macrophages clear the wound from bacterial infection (2) Immune Recruitment: neutrophils and monocytes provide signal molecules important for wound healing (3) Healed wound: construction of granulation tissue contracts the wound

Current treatments available for burn wounds

The delayed process of wound healing, infection, pain, and hypertrophic scarring still pose a significant challenge to burn management and research, even though the current treatments are effective. Clinically accepted treatments include:

• 1) Skin Grafting: In cases of burns in which the dermis and all the other skin layers are destroyed, it becomes difficult to close the wound by suturing or through the process of primary healing [6]. It requires further surgical processes. One of the procedures used is skin grafting (Fig. 3). Skin grafting is the process of transplanting healthy skin from the patient’s undamaged donor site to the wound site. The major disadvantage of using skin grafting is that it can cause infection at the donor or recipient site, resulting in poor wound healing.

• 2) Skin Substitutes: When the donor skin is limited, the large burn wounds can be protected through the process of skin substitutes which not only enhances the wound healing but also reduces the inflammatory response, further increasing the dermal component of the wound that is healed and subsequently decreasing the chances of scarring [7, 8]. The process of applying skin substitutes can carry a high risk of complications at the donor site such as scarring, infections, abnormal pigmentation, and chronic pain.

• 3) Wound Dressings: Several wound dressings and antimicrobial agents are applied to the burns which are responsible for preventing infection, re-epithelialization, skin desiccation, and any other skin damage. Biological dressings include allograft, xenograft, and human amnion which are required temporarily for covering the wound to re-epithelialize. Despite having some advantages, wound dressings also hold some disadvantages such as bandages do not adhere to the wounds, cannot hold fluids, causing skin irritation, and leading to more pain for the patient.

• 4) Negative Pressure Wound Therapy (NPWT): NPWT, also called vacuum-assisted closure, topical negative pressure therapy, or microdeformational wound therapy is being utilized for both huge and small burns. Compared to the other pharmacological means that are available, NPWT has proven to be effective in halting the partial thickness of wound progression [9]. Various case-control studies have shown that there has been reduced wound infection in burn injuries due to the NPWT treatment which is used with the improved skin graft and dermal substitute as compared to the normal treatment [10].

Fig. 3

Conventional methods of burn wound management

In the multifaceted arena of burn wound management, a spectrum of approaches augments conventional treatments. These encompass hydrogel dressings fostering a favourable healing environment; biologic dressings such as cultured epithelial autografts and artificial skin substitutes; topical antimicrobial agents like silver sulfadiazine and honey-based dressings; phototherapy, notably low-level laser therapy, for mitigating pain and inflammation; biosynthetic dressings merging natural and synthetic components; hyperbaric oxygen therapy amplifying oxygen delivery; electrical stimulation methods like pulsed electromagnetic field therapy and microcurrent therapy.

PRP and HPE for burn wound healing

Platelet-rich plasma (PRP) is a form of plasma with an elevated levels of platelets that is produced by centrifugation of whole blood [11]. The platelet concentration in PRP is 3–5 times higher than in ordinary plasma. This increased platelet count allows PRP to have a rapid effect at the site of damage, replicating and outperforming the body’s natural reaction to stress. This causes the production of an elevated level of growth factors, which stimulates tissue regeneration, relieves pain, and lowers blood loss [12]. A rising number of studies have focused on the use of autogenous PRP to treat burns, including the randomized controlled trials (RCTs) [13]. Although most RCTs affirm PRP’s effectiveness and safety in treating burn wounds, there are currently few high-quality RCTs, and more standardization of PRP preparation and usage criteria is needed. A meta-analysis was conducted on the treatment of burn wounds with PRP. When compared to conventional therapy, the analysis of patients with burn wounds showed PRP therapy dramatically improved healing results. However, there was no significant difference between PRP and conventional treatment in terms of graft take ratio or infection incidence. Despite these positive findings, the analysis revealed a limitation: the majority of the included studies had small sample sizes, and certain comparisons were based on a small number of studies, necessitating caution when interpreting the results [14].

Human placental extract (HPE) has been used as a therapeutic agent. It contains a unique pharmacological characteristic that promotes wound healing. The HPE contains high quantities of growth factors, anabolic cytokines, nucleic acids, and necessary amino acids, all of which may promote tissue regeneration, as well as anti-inflammatory proteins that reduce inflammation [15]. Placental extract possesses immunotropic properties at the cellular level. It boosts cellular biosynthesis. It can be used as a growth factor to treat chronic wounds and burns. It serves as an angiogenic mediator, promoting angiogenesis [16]. Research has demonstrated that applying human placental extract directly to the margins of full-thickness wounds speeds up the healing process by raising TGF-β levels early on, encouraging the invasion of inflammatory cells, and raising VEGF levels later on, which promotes the formation of new blood vessels. Furthermore, the features of placenta, such as low immunogenicity, anti-inflammatory, and anti-scarring, make it a perfect alternative to treat full thickness skin wounds [17]. It should be mentioned that current research on the favorable effects of placental components in the treatment of a wide range of conditions must be tested over time and statistically to evaluate their potential/real therapeutic efficacy. Furthermore, undesirable effects and contradictions should be thoroughly researched and examined in both the short and long term [18].

Exosomes from MSCs

The selection of interventions pivots on burn severity, patient specifics, resource availability, and medical expertise. Concurrently, a variety of stem cell investigations are being conducted with promising outcomes in domains ranging from oncology and hematology to organ transplantation and wound healing. Stem cells, originating from diverse sources such as adipose tissue, bone marrow, umbilical cord, and embryos, have been harnessed for their regenerative potential across various wound types in the realm of wound healing [19, 20]. Particularly noteworthy is the application of MSCs as a promising alternative to conventional treatment methods. MSCs, due to their inherent regenerative properties and ability to modulate immune responses, offer a unique approach to burn wound healing. These cells not only facilitate tissue repair but also hold the potential to mitigate scar formation, providing a holistic solution to the challenges posed by severe burns. As we delve deeper into the realm of stem cell-based therapies, it becomes increasingly apparent that harnessing the capabilities of mesenchymal stem cells presents a paradigm shift in burn wound management, offering novel avenues for optimizing healing outcomes and improving patient well-being.

The discovery of stem cells of mesenchymal origin is an ongoing effort for several decades [21]. The biological significance and clinical applications of MSCs have proved to be a prominent issue in the field of research. Numerous studies have demonstrated the positive effects of MSCs in many clinical applications, and the pioneer transplantation of MSCs has been confirmed [22, 23]. The compelling attributes of MSCs – their capacity for self-renewal, multilineage differentiation, and immunomodulation – have heightened interest in their application within pathologies characterized by inflammation and degeneration [24,25,26]. However, the propensity of transplanted MSCs to differentiate unpredictably or uncontrollably within the host has led to still unspecified complications for the receiver, which has delayed the otherwise valuable prospects of the cell-based interventions [27, 28]. Consequently, researchers have embarked on investigating better options and shifting the focus toward in vivo cell-free therapies [29]. It’s noteworthy that only a minor portion of engrafted MSCs effectively reach target sites within damaged tissues; instead, a paracrine effect predominantly arises from the release of extracellular vesicles (EVs) [30]. MSC-EVs may be able to travel to distant sites and mediate immune responses or tissue regeneration through a variety of diverse mechanisms of action, including interactions with membrane-specific receptors and/or direct membrane fusion with the target cell [31, 32].

The composition of EVs is determined by the parental MSCs, which modify their secretome in response to the inflammatory or non-inflammatory parameters of the local environment. In response to pro-inflammatory mediators, these MSCs adopt a non-inflammatory stance, releasing EVs that effectively suppress and restrain inflammation by inhibiting M1 macrophages, dendritic cell antigen presentation, natural killer cells, and various T cell subsets that contribute to inflammatory responses (i.e., Th1, Th17, and cytotoxic T cells) [24, 33, 34]. Conversely, in an anti-inflammatory milieu, MSCs generate pro-inflammatory EVs to stimulate and sustain the recruitment of cells capable of mounting and maintaining an immune response. MSC-EVs exhibit therapeutic effects that are equivalent to or even better than transplanted MSCs, according to preclinical investigations [35, 36].

The application of human umbilical cord MSC-EVs has demonstrated their potential to reduce burn-induced inflammation in a severely burned skin rat model. The infusion of EVs reduced the production of TNF-α and IL-1β production in macrophages while increasing IL-10 levels and suppressing the TLR4 signaling pathway and the inflammatory response, which was attributed to the transfer of miR-181c from EVs to macrophages [37]. Co-culturing adipose tissue-derived MSC-EVs with keratinocytes and fibroblasts in a scratch wound healing assay demonstrated enhanced cell migration and proliferation [38]. Furthermore, the effect of EVs on wound healing was studied in an excisional rat wound model, where topical treatment of MSC-EVs caused the process to be accelerated, thus contributing to the regeneration of skin fibroblasts [39]. These studies have unraveled the complexities of intercellular communication, a phylogenetically conserved mechanism integral to defense mechanisms like viral attacks, genetic material exchange, and biological material transfer [40, 41]. Through fractionation of extracellular components, it has been ascertained that exosomes are the predominant mediators of the MSCs’ paracrine effect [42]. Exosomes are a population of nano-sized vesicles with a diameter of up to 150 nm, positive for Alix, CD81+, and CD9+, capable of mediating MSC regenerative effects in a variety of diseases. Exosomes have been tested in vitro and in vivo for a variety of clinical disorders, including neurological injury, renal injury, diabetes, myocardial infarction, retinal ischemia, hepatic injury, and cartilage repair [43,44,45,46,47]. Exosomes produced from MSCs may provide a promising possibility for cell therapy applications in wound healing [48, 49].

Exosomes, microvesicles derived from the endosomal membrane, function as pivotal communication regulators among cells [50]. Regardless of their origin, exosomes have exhibited beneficial effects on burn wound healing and scar mitigation. These microvesicles are adeptly delivered to burn wounds, harnessing their physiological therapeutic potential. When comparing exosome therapy to cell-based approaches, exosomes offer numerous advantages: they exert enhanced biological effects through direct fusion with target cells, exhibit ease of storage and transport (at -70 °C for extended periods), allow straightforward management of concentration, administration route, and timing, while presenting minimal risks of immune rejection and transformation [51]. The diversity of exosome-delivered components may lead to varying signaling outcomes across different injury models. Several studies underscore that the efficacy of wound healing and tissue regeneration observed in stem cells is primarily attributed to their capacity for releasing secretome and paracrine factors rather than their potential to differentiate into skin cells at the site of injury [44, 52, 53]. This underscores the pressing need for in-depth investigation into the effects of exosomes derived from stem cells’ secretome on wound healing.

Exosomes: origin and composition

Exosomes, initially discovered as small vesicles released by sheep reticulocytes, share overlapping density, composition, and size characteristics with various subtypes of EVs such as ectosomes, prostasomes, microvesicles and endosomes. Among these, exosomes stand out, possessing a diameter of 40–150 nm and originating within multivesicular bodies (MVBs) before being secreted upon MVB fusion with the plasma membrane [52]. Exosomes have demonstrated remarkable significance in mediating cell-to-cell interactions and communications, wielding their influence not only in normal physiological processes but also in the progression and development of diseases [54]. The cargo content of extracellular vesicles is dictated by their cellular source, encompassing proteins, lipids, mRNA, and miRNA (Fig. 4). The functional transport of exosomal cargo across cells influences protein expression within recipient cells [55, 56].

Fig. 4

Structure and composition of exosomes. The exosome comprises a phospholipid bilayer membrane. The composition of exosomes is influenced by the host’s health, the cell type from which it is obtained, and external stimuli. The proteins annexins, tetraspanins, Alix, TSG101, MHC molecules, Rab proteins, cytoskeletal proteins, enzymes, growth factors, cytokines, and signal transduction proteins are all found in exosomes along with miRNA, mRNA, DNA and other molecules

Notably, exosomes harbor components that foster multivesicular body biogenesis and the fusion of these bodies with cell membranes, including membrane transport proteins like GTPases, Annexins, flotillin, and Rab proteins. Exosomes carry a spectrum of molecules beyond proteins, encompassing tetraspanins (CD9, CD63, CD81, and CD82), adhesion molecules (CD11b and CD54), and heat shock proteins (HSP) such as hsp70 and hsp90, which indicate cellular responses to environmental stress [57]. Lipid-related proteins, like cholesterol, sphingomyelin, ganglioside GM3, and internalized phosphatidylserine, also find a place within exosomes, contributing to their structure and function [58, 59]. The maintenance of exosomal structure and function involves the interplay of lipid-related proteins, phospholipases, and the selective trafficking of specific proteins into exosomes.

The selective packaging of RNA into exosomes constitutes a multifaceted process, encompassing diverse forms of RNA such as miRNA, small noncoding RNA (sncRNA), natural antisense RNA, fragments of transfer RNA (tRNA), mRNA, ribosomal RNA (rRNA), and long non-coding RNAs (lncRNAs) [60]. These intricate components collectively underline the remarkable versatility and impact of exosomes in intercellular communications and their potential applications in various scientific domains.

Exosome Biogenesis

Biogenesis is a complex and orchestrated process that involves the formation, packaging, and release of these small extracellular vesicles. It originates within the cell’s endosomal system and encompasses multiple stages of intracellular trafficking and sorting. It involves the sorting of proteins associated with the lipid membrane by ESCRT (endosomal sorting complexes required for transport)–dependent and ESCRT–independent mechanisms, involving trafficking of intracellular exosomes and endocytosis of exosomes by the target (recipient) cells [61]. To derive the exosomes, the endosomal membranes form an internal bud from the multivesicular endosome (MVE), forming intraluminal vesicles (ILV). An individual exosome is released when MVE fuses with the plasma membrane [62, 63].

Formation of Exosomes by ESCRT-Dependent Pathway

The ESCRT complex consists of the four soluble proteins, namely ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, and the associated ATPase Vps4 complex [64]. To sort the selected proteins into the ILV, the ESCRT complex is recruited. Ubiquitination of endocytosed receptors is done by ESCRT-0, which forms the cargo clustering. These cargoes contain proteins that will be incorporated into the released exosomes. The bud formation is induced when ESCRT-II is activated by forming a complex between a component of ESCRT-I known as tumor susceptibility gene 101 (TSG 101) and a ubiquitinated protein. The two additional proteins associated with the ESCRT complex are mammalian hepatocyte receptor tyrosine kinase substrate (Hrs) and vacuolar protein sorting-27 (Vps-27) that can detect monoubiquitinated cargoes and stimulate their transit into the MVB compartment. Protein clustering and aggregation are two additional mechanisms for protein sorting that occur independently of monoubiquitination. Protein aggregation results in the formation of new physical features and acts as a microdomain in this way. In turn, this microdomain can interact with ESCRT and travel inward to MVBs. Before sorting the cargo into the ILVs, the complex formed involves the isolation of the MVB proteins and the employment of a de-ubiquitination enzyme to remove the ubiquitin from the cargo protein. Vacuolar protein sorting-associated protein 4 (Vps4) adenosine triphosphate disassembles the ESCRT-III complex in the last stage [65].

Formation of Exosomes by ESCRT Independent Pathway

Despite the inhibition of the ESCRT complex, several studies have shown the formation of exosomes, recommending the presence of an ESCRT-independent pathway that involves lipids, heat shock proteins, and tetraspanins [63]. ILV is formed through the inward curvature of the limiting membrane of MVB with the help of lipids such as ceramide. Cholesterol and phosphatidic acid are also responsible for the formation of exosomes. In addition, proteins such as tetraspanins are also associated with cargo sorting for exosomes, and incorporation of melanosomal proteins into ILVs also occurs in an ESCRT-independent manner [66].

Methods for exosome isolation

Traditional techniques for isolating exosomes include ultracentrifugation, size-based approaches (size-exclusion chromatography and ultrafiltration), precipitation, and immunoaffinity capture. The final technique is mostly focused on the particular interaction between antibodies or aptamers and exosomal signature proteins, whereas the previous three methods primarily rely on the size and density of exosomes. Differential ultracentrifugation is regarded as the “gold standard” approach for exosome separation, since it successfully isolates minute particles like bacteria, organelles, and exosomes [67]. The process of separating extracellular components (such as exosomes, apoptotic vesicles, and protein aggregates) using density gradient centrifugation takes a long time since high-density medium (like sucrose or iodixanol, etc.) is introduced to the layer [68]. Exosomes isolated from MSCs may be extracted by a single-step sucrose cushion ultracentrifugation process [69]. Studies have demonstrated that ultrafiltration coupled with size-exclusion chromatography can minimize the quantity of impurity cytokine (interleukin-10) in isolated exosomes. The combination of diverse approaches may successfully minimize the presence of nanoscale pollutants, increase separation purity, and preserve the natural features of the exosomes [70, 71]. Membrane particle precipitation-based EV isolation is an appealing approach due to its simple process and high yield. Currently, there are various ways for isolating plasma EVs. Precipitation of membrane particles is the basis for a number of commercial EV isolation kits, including the ExoQuick, Invitrogen Total Exosome Isolation Reagent and miRCURYTM Exosome Isolation Kit. The purity of EVs remains a worry with this approach since it may precipitate viruses and a few proteins in the sample [72, 73].

Immunological separation techniques, which utilize antibodies that precisely target exosomal markers including CD63, CD81, CD9, and other proteins located on the exosomal membrane, offer sophisticated ways for separating exosomes [74]. The proteins promote excellent immunoaffinity binding for exosome capture, which is notably useful when extracting from antigen-presenting cells (APCs) with antibody-coated magnetic beads. The bead-based approach is flexible since it may be utilized with both western blotting and electron microscopy. It gives a new option to ultracentrifugation by utilizing magnetic beads for a more homogeneous population of vesicles [75]. An alternative technology, the lipid-nanoprobe (LNP) system, rapidly tags and separates EVs using magnetic enrichment in 15 min, equivalent to normal ultracentrifugation, and is effective for testing nucleic acids and proteins for finding mutations in patients with non-small cell lung cancer [73]. New strategies for isolating exosomes have been developed to address the limitations of traditional procedures such as ultracentrifugation, which is slow and frequently produces contaminants. Recent advances in microfluidic technology have made it possible to manage small quantities of fluids in microchannels, allowing isolation and identification to be performed on the one chip [76]. The utilization of microfluidic devices, as well as signal-detecting platforms, has enhanced exosome isolation by employing immunoaffinity capture, size-exclusion chromatography and ultracentrifugation techniques [77]. Several popular techniques employed in microfluidic devices are dielectrophoretic forces (DEPs), acoustic waves, deterministic lateral displacement (DLD), asymmetric flow field-flow fractionation (AF4), inertial lift force, viscoelastic flow, filtration, and immunoaffinity-based exosome separation methods [77,78,79].

Exosomes are characterized using a range of advanced techniques to determine their size, shape, and biomarker expression. Flow cytometry is used to evaluate surface markers, nanoparticle tracking analysis (NTA) to determine particle size and concentration, and dynamic light scattering (DLS) to determine particle size distribution. High-resolution imaging methods like transmission electron microscopy (TEM) and atomic force microscopy (AFM) provide comprehensive morphological insights, while resistive pulse sensing (RPS) allows for exact size measurements. Surface plasmon resonance (SPR) identifies biomolecular interactions, making these technologies indispensable for thorough exosome research [80, 81].

MSC-derived exosome therapy

In recent years, mesenchymal stem cell-derived exosome therapy has been frequently used to promote the process of wound healing in various skin conditions particularly in skin burns [82, 83]. MSC exosome not only inhibits pro-inflammatory processes but also suppresses fibrosis, further facilitating tissue regeneration [42]. Although the clinical trials for cell-free based therapy in burn wound healing are yet limited, there is a clinical trial (NCT04235296) based on MSC conditioned medium derived pleiotropic factors containing IGF-1, TGF-β, VEGF, etc. The trial has confirmed the role of MSC–conditioned media in modulating wound inflammation, repairing damaged cells, and promoting the regeneration of wounds. Though MSCs can modulate immune response, their capacity to grow and differentiate uncontrollably in the host has hampered their beneficial effects [28]. Since only a small part of injected MSCs reach the target site, numerous research investigations have demonstrated how they exert their positive effects through the paracrine function by releasing extracellular vesicles like exosomes.

Therapeutic potential of MSC-derived exosome therapy in burn wound healing

MSC-derived exosomes are known to have positive effects on wound healing and scarring, regardless of their identity or [84]. When we compare cell-based therapy and exosome therapy, exosomes exert a much greater biological effect because they directly get fused to the damaged target cells, exosomes are easy to store (at -80 °C for a long time), their concentration, route, and time of use are easy to manage, and there is the least risk of immune rejection and any type of transformation [51]. Exosomes have been applied to wounds to exploit their physiological therapeutic actions in wound healing such as cellular proliferation, migration, and differentiation [84] (Fig. 5).

Fig. 5

Role of exosomes on the skin and wound healing

Effect of exosomes on proteomics of cells participating in burn wound healing

The exosome contains proteins involved in the activation of granulocytes and leukocytes and the regulation of cell migration involved in the immune response. The proteomic analysis of exosomes revealed that there are various factors responsible for the process of wound healing, such as overexpression of miR-30b, enhances the angiogenic capacity of exosomes [85]. Recently, a study conducted on iPSCs-KCs-Exos discovered numerous miRNAs, with miR-762 being the most abundant. Additionally, their findings revealed that miR-762 promoted the migration of endothelial cells and keratinocytes, comparable to the treatment involving iPSCs-KCs-Exos. This implies that the presence of miR-762 possibly contributes to the advantageous effects of KCs-Exos on the recovery of wounds [86].

Effect of exosomes on the phases of wound healing

Exosomes are known to have a significant effect on tissue repair and skin regeneration in the three main stages of burn wound healing i.e., inflammation, proliferation, and remodeling.

Inflammation phase

A well-regulated inflammatory reaction is a normal part of the body’s self-defense against harmful stimuli, but an extended and irregular inflammatory response can slow wound healing and worsen fibrosis, excessive scarring, or re-epithelialization inhibition [87, 88]. Furthermore, cytokine overproduction may result in tissue damage [89]. The two phenotypes, pro-inflammatory M1 and anti-inflammatory M2 macrophages, play a crucial role in cutaneous regeneration and function in nearly every step of wound healing. Dysregulation of macrophages can lead to excessive inflammation or fibrosis [90]. Exosomes produced from M2 macrophages (M2-exosomes) are administered subcutaneously into mice wounds, which significantly reduces the local population of M1 macrophages and increases the M2 macrophages, resulting in the effective conversion of M1 to M2 macrophages [91].

Exosomes from various types of MSCs can control inflammation by downregulating pro-inflammatory enzymes like cyclooxygenase (COX-2) and iNOS, as well as cytokines and chemokines like tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and monocyte chemoattractant protein (MCP-1). Numerous studies have demonstrated that MSC-derived exosomes can increase the levels of IL-10, an anti-inflammatory cytokine thought to be crucial in the regulation of cutaneous wound inflammation and scar formation [84].

Studies have shown that exosomes have an immunomodulatory effect because of their specific miRNA content [92]. The LPS conditioned exosomes have enhanced macrophage polarization regulating capacities and can resolve chronic inflammation by shuttling miR let-7b; as a result, these exosomes offer significant immunotherapeutic potential for wound healing. Human umbilical cord MSCs (hUCMSCs) exhibit the largest concentration of three miRNAs (miRNA-21, miRNA-146a, and miRNA-181c) directly connected to the control of immune response and inflammation, according to analysis of miRNA expression patterns [93]. The hUC-dMSCs exosomes contain miR-181c, inhibiting the TLR4 expression and suppressing the inflammatory reaction associated with the burn injury [37]. Overall, more studies are required to pinpoint the precise molecular mechanisms by which MSC-exosomes reduce inflammation in the context of skin regeneration and wound healing.

Proliferative phase

The primary processes during the proliferation phase include contraction of wounds, neo-angiogenesis, deposition of collagen, formation of granulation tissue, and re-epithelialization. The macrophage-derived exosomes have strong angiogenesis-promoting and proliferative effects on diabetic wound healing by inhibiting the release of pro-inflammatory cytokines and enzymes. Pro-angiogenic microRNAs are more abundant in exosomes produced by CD34 + cells, and they both incite angiogenesis in vitro and in vivo [94]. Exosomes derived from human umbilical cord blood endothelial progenitor cells (hUC-bEPC) increase the proliferation, migration, and tube formation of vascular endothelial cells and are known to accelerate wound healing in diabetic rats in vitro. Furthermore, these exosomes induce the production of angiogenesis-related molecules in endothelial cells, such as FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, iNOS, and IL-8. In the endothelial cells stimulated with endothelial progenitor cell-exosomes, the mRNA levels of matrix metalloproteinase (MMP-9) were remarkably decreased [95]. Therefore, the pro-angiogenic effect exerted by EPC-exosomes may be due to the suppression of MMP-9 protein because a higher level of MMP-9 is associated with poor wound healing.

Skin fibroblasts play a role in cutaneous regeneration and tissue repair by participating in wound closure, ECM deposition, and tissue remodeling [96]. Cell proliferation and skin re-epithelialization are crucial for cutaneous regeneration and can be well modulated by giving exosomal therapy [84]. The MSCs-derived exosomes while transporting their contents to the cytoplasm, regulate the proliferation and migration of skin fibroblasts by regulating the expression of growth factors and their related genes such as TNF α, IL 10, collagen, Il-6, Ccl2, Cd206, Ccl7, and Ccl17 [97]. These exosomes participate in the formation of granulation tissue and the synthesis of collagen providing structural support for wound repair [98]. Exosomes produced from human fibrocytes include proteins and miRNAs with a variety of biological functions and are known to have enhanced wound healing by stimulating skin cell migration and proliferation in a diabetic rat model [99]. Studies have found that hUC-dMSCs-derived exosomes promote skin cell proliferation in a dose-dependent manner by promoting re-epithelialization in a rat deep second-degree burn injury model by activating the Wnt/β-Catenin signaling pathway [100]. The human aortic endothelial cells (hAECs)-derived exosomes taken up by the fibroblasts promote their migration and proliferation and down-regulate the expression of ECM such as collagen-I and III. The high concentration of hAECs-derived exosomes, however, minimize the fibrotic growth [101].

Remodeling phase

Remodeling, which entails maximizing the tensile strength by reorganizing, disintegrating, and re-synthesizing the extracellular matrix (ECM), is the last stage of wound healing. During this stage, the body strives to restore normal tissue structure by progressively remodeling granulation tissue, the formation of scar tissue, and collagen fibers [102]. Collagen, non-collagen (fibronectin and laminin), elastin, proteoglycans, and amino glycans are the major components of the ECM, and dysregulated ECM formation can cause the wound surface not to heal and prevent scar formation (REF). Recently, it’s been reported that exosomes from different sources can benefit wound healing including the remodeling phase. The production of type I collagen and elastin can be encouraged by exosomes produced from hUC-dMSCs [103]. Exosomes produced by human induced pluripotent stem cells (hiPSCs) that have developed into dermal mesenchymal stem cells (dMSCs) have also been shown to increase the expression of type I collagen, type III collagen, and elastin mRNAs as well as the production of type I collagen, type III collagen, and elastin proteins [104]. Exosomes produced from ADSCs (Adipose-derived stem cells) control collagen synthesis at every step of wound healing by increasing collagen I and III production in the early stages of wound healing and decreasing it in the later stages, reducing scar formation [105].

The uMSC-derived exosomes comprise an array of particular microRNAs (miR-21, miR-23a, miR-125b, and miR-145) that have been shown to play important roles in preventing the development of myofibroblasts by inhibiting excessive α-smooth muscle actin and collagen deposition linked to the activity of the TGF-β/SMAD2 signaling pathway, thereby preventing scarring [106]. Studies have shown that ASC-derived exosomes decreased the scar size and increased the ratio of collagen III to collagen I in murine incisional wounds when given intravenously. It enhanced the ratio of TGF-β3 to TGF-β1 and prevented the development of fibroblasts into myofibroblasts. Additionally, by activating the ERK/MAPK pathway, these exosomes enhanced the production of matrix metalloproteinases-3 (MMP-3) in skin dermal fibroblasts. This resulted in a high ratio of MMP-3 to tissue inhibitor of matrix metalloproteinases-1 (TIMP1), which is favourable for the remodeling of the ECM [107].

Mechanism of exosomes in healing of wounds

Exosomes promote cell migration and proliferation, enhance the development of new blood vessels, and reduce inflammation to aid in the healing of wounds. They carry RNAs, lipids, and proteins that initiate growth pathways, encourage the formation of new blood vessels, and control the expression of certain genes. Exosomes also play a role in matrix remodeling and protecting cells from apoptosis, which ultimately promotes effective tissue healing.

Exosomal MicroRNAs (miRNAs)

Exosomes are rich in miRNAs, which are small non-coding RNAs that regulate gene expression after transcription. These miRNAs can influence a variety of biological functions, including inflammation, angiogenesis, and tissue repair, all of which are critical for burn wound healing [108]. Exosomes from MSCs are known to carry miRNAs that regulate key inflammatory pathways. It has been previously reported that miR-19b promotes wound healing by reducing inflammatory damage and the breakdown of extracellular matrix. The study found that the exosomal miR-19b from ADSCs directs Ccl1 mRNA in keratinocytes, which enhances skin defect healing through modulation of the TGF-β pathway [109]. Zhang et al. investigated the effect of exosomes from human umbilical cord mesenchymal stem cells (UCMSCs) on wound healing in normoxic and hypoxic environments. The research suggests that exosomes generated by hypoxic UCMSCs have a stronger effect on promoting angiogenesis and wound healing. Specifically, the miR-125b present in these exosomes plays a critical role in directing and regulating the synthesis of VEGF in endothelial cells, an essential function that supports angiogenesis during wound healing [110]. Ma et al. observed that in skin fibroblasts and vascular endothelial cells, miR-126-3p directly targets the mRNA that encodes the PI3K regulatory subunit 2. This targeting promotes the formation of new blood vessels and the accumulation of collagen, both of which are essential for repairing skin problems. Their findings demonstrate the potential of miR-126-3p to enhance wound healing through activation of the PI3K/AKT/mTOR pathway [111]. Yuan et al. discovered that miR-29a expression was much lower in human burn scars and hypertrophic scar (HTS) fibroblasts than in normal tissue and fibroblasts. This suggested that miR-29a might aid in reducing burn scarring and accelerating wound healing. The study found that exosomal miR-29a inhibited the activation of the TGF-β/Smad3 pathway, which in turn caused α-SMA, collagen-I, and collagen-III levels in scar tissues to drop. Furthermore, the use of a TGF-β agonist reversed the inhibitory effect. These findings suggest that by interfering with the TGF-β/Smad3 pathway in fibroblasts, miR-29a plays a critical role in inhibiting the formation of hypertrophic scars [112]. Further, it has been found that miR-542-3p targets the mRNA-coding angiopoietin-2, a protein that promotes angiogenesis in endothelial cells. In comparison to mice with normal skin, Xiong et al. observed a significant drop in miR-542-3p expression in the skin of injured mice. They exploited the ability of miR-542-3p to promote angiogenesis and aid in tissue repair to enhance wound healing [113, 114].

It has been shown that MiR-146a promotes vascular growth and inhibits the negative impacts of macrophages to aid in the healing of diabetic ulcers. It uses two main mechanisms to function. Nuclear factor kappa B (NF-κB) and TNFR-associated factor 6 (TRAF6) are indirectly suppressed by miR-146a when it binds to the mRNA for IL-1R-associated kinase 1 (IRAK1) in macrophages. By inhibiting the synthesis of inflammatory cytokines, this approach reduces inflammation. The mRNA encoding Smad4 in keratinocytes is bound by miR-146a, resulting in an upregulation of VEGF expression. This process promotes angiogenesis and vascular regeneration during wound healing [115, 116]. Exosomes derived from umbilical cord blood and enriched with miRNA-21-3p were administered topically to mice skin wounds, promoting faster reepithelialization, decreasing scar depth, and increasing angiogenesis. MiR-21-3p primarily functions by suppressing PTEN and sprouty RTK signaling antagonist 1 (75). MiR-146a-3p from ADSC-exosomes may promote fibroblast migration and proliferation by upregulating the expression of p-ERK2 and serpin family H member 1, which would accelerate wound healing and improve angiogenesis in rats [108].

Exosomal proteins

Exosomes can carry a large amount of cargo and protect its contents from harmful substances and enzymes, which makes them ideal for delivering nano-treatments. They are also perfect as carriers for transferring genetic material, drugs, and other therapeutic materials to certain cells, boosting their efficacy in therapeutic applications due to their exceptional durability and compatibility [117]. Exosomes have been modified through the use of bioengineering techniques, yielding higher concentrations and better stability of exosome particles with specific therapeutic effects. These modifications increase the therapeutic impact of native exosomes and increase their targeting efficiency. Li et al. demonstrated that Nrf2-overexpressing ADSC-derived exosomes (ADSC-exos) might lessen stress-induced early aging of endothelial progenitor cells (EPCs) in a high-glucose (HG) environment. This was achieved by lowering the levels of inflammatory cytokines and ROS and raising the levels of VEGF, SMP30, and VEGFR2 phosphorylation [118]. Furthermore, the overexpression of Nrf2 by ADSC-exos was shown to accelerate the healing of diabetic wounds in a rat model of diabetic wounds by increasing the production of granulation tissue and angiogenesis and reducing inflammation and oxidative stress-related proteins. Growth factors like VEGF and bFGF that are secreted by platelets can be encapsulated in exosomes. These growth factors activate the PI3K/Akt signaling pathway when they are delivered to the target cells, which promotes the development of new blood vessels in healthy endothelial cells. This process is critical for the angiogenesis and wound repair [119]. Zhao et al. showed that augmenting hucMSC-exos with high amounts of eNOS using genetic engineering and optogenetic approaches (hucMSC-exos/eNOS) can accelerate wound vascularization by activating the PI3K/Akt/mTOR or Fak/Erk1/2 signaling pathways. Additionally, hucMSC-exos/eNOS affected the immune microenvironment for diabetic wound healing by promoting autophagy, M2 polarization, Treg cell aggregation, and TRM cell residency [120].

Lnc RNA and cirRNA

The lncRNAs MALAT1 and H19, which are both abundant in exosomes produced from adipose-derived stem cells (ADSCs), are critical for wound healing. Through its association with miR-124 and activation of the Wnt/β-catenin pathway, MALAT1 increases the migration of human dermal fibroblasts and enhances the healing of ischemic wounds by promoting cell proliferation and migration. Similarly, lncRNA H19 found in ADSC-exosomes increases SOX9 levels by inhibiting miR-19b, which causes an increase in human skin fibroblast migration, invasion, and proliferation. Research on living subjects has demonstrated that ADSC-exosomes use these mechanisms to help skin wounds heal in mice [121, 122]. Exosomes produced by ADSCs contain significant concentrations of GAS5 lncRNA, which has been shown to reduce LPS-induced inflammation in human skin fibroblasts, suggesting a potential role in promoting the healing of chronic wounds. Furthermore, it has been demonstrated that HOTAIR, a kind of long noncoding RNA found in extracellular vesicles isolated from bone marrow stem cells (BMSCs), improves angiogenesis and healing in diabetic db/db mice [123, 124].

Circular RNAs play a major role in controlling disease environments. Exosomes enriched with mmu_circ_0000250 derived from ADSCs increase the survival of epithelial cells (EPCs) in high-glucose environments by promoting autophagy and reducing apoptosis. Mmu_circ_0000250-enriched exosomes stimulate skin angiogenesis and decrease apoptosis in diabetic mouse wounds by activating autophagy via suppression of miR-128-3p and increase in SIRT1. Similarly, circ-Gcap14 derived from hypoxic preconditioned ADSCs increases angiogenic factors and accelerates wound healing in diabetic mouse wounds by suppressing miR-18a-5p and augmenting HIF-1α levels [125, 126].

Exosomes from cells other than MSCs

Macrophage exosomes

The skin’s healing process can be delayed when it is damaged by accidental injuries, burns, or surgeries, which may result in infections or scarring. There is a pressing need to discover a solution that can expedite skin recovery. There are multiple factors that hold significance. M2 macrophages will be recruited to the wound site and their involvement will be essential in the time-restricted task of angiogenesis. It is crucial in medical practice to shorten the healing time and repair the skin’s structure following damage [127].

The role of macrophages in wound healing has been observed in numerous studies. Immune cells called M2 macrophages, also known as alternatively activated macrophages, assist in the process of healing. Furthermore, as the skin heals, special cells called wound macrophages increase the production of a protein called metallo-matrix protease (MMP). This protein helps reshape the area around the wound to avoid the formation of scars. Researchers made attempts to regulate the level of M2 at the wound site in order to enhance wound healing, resulting in highly promising outcomes. However, there have been few studies that have used M2-Macrophage exosomes for healing skin. Exosomes are like unique markers of cells and closely related to their parent cells. These special vesicles can cause specific biological changes at a particular location [128]. The introduction of M2 macrophage exosomes into the area just below the skin surrounding a wound can promote faster wound healing. These M2 macrophage exosomes change the activity of immune cells called macrophages in a way that improves angiogenesis, re-epithelialization, and the formation of collagen, which all help speed up the healing process [91].

M2 macrophages assist in the angiogenesis through various mechanisms. According to a recent study, M2-macrophage exosomes can encourage the in vitro ability of HUVECs to produce angiogenic factors. They demonstrated that miR-21-5p functioned as an important mediator within M2-macrophage exosomes, suppressing PTEN and activating AKT/mTOR signaling before affecting endothelial cell function. miR-21-5p was transported to HUVEC via exosomes from M2 macrophages. Additionally, the infusion of M2-macrophage exosomes into wounds on real murine skin sped up angiogenesis and skin healing [129].

Endothelial progenitor cells-exosomes (EPCs- exo)

Endothelial progenitor cells (EPCs) are the early-stage cells that eventually develop into vascular endothelial cells. EPCs, also known as endothelial progenitor cells, can be mobilized from the bone marrow to the bloodstream in response to illnesses or physical ailments, enabling them to repair injured blood vessels. Research has found that EPCs are important in diseases related to the heart, brain, blood vessels, tumors, and healing through their proliferation and differentiation [130]. EPC-Exos significantly increased the angiogenic activity of endothelial cells in vitro and that Erk1/2 signaling was the major mediator throughout this process. EPC-Exos promoted angiogenic responses in endothelial cells by stimulating Erk1/2 signalling, which ultimately speeds up the healing and regeneration of cutaneous wounds [131]. Exosomes from murine BM EPCs have been found to hasten the healing of skin wounds in both diabetic and control mice by transmitting the miRNA-221-3p [132]. According to Hassanpour et al., the diabetes condition can interfere with the CD63-Alix-Rab27a signaling pathway, which lowers EV production, transportation, and fusion in EPCs [133]. These findings highlight the function of EPC-exosomes and offer new possible avenues for treating diabetic skin lesions.

Exosomes from epidermal cells

Keratinocytes (KC) are highly specialized epithelial cells that play an important role in epidermal regeneration following injury by proliferating and re-epithelializing [134]. Keratinocytes interact with several other types of cells such as melanocytes, Langerhans cells, lymphocytes, and fibroblasts within the layers of our skin. Keratinocytes have the ability to produce exosomes, which allow some intracellular proteins to be externalized into the keratinocyte milieu, such as stratifin, which has matrix metalloproteinase (MMP)-1 activating action for fibroblasts [135]. Keratinocyte-derived EVs (KC-EVs) have important physiological effects on target cells because they contain a variety of bioactive chemicals, including nucleic acids, metabolic enzymes, cytoskeletal proteins, signaling proteins, trafficking proteins, and adhesion proteins [136]. They regulate various crucial cellular processes, such as cellular adhesion, migration, differentiation, apoptosis, and protein synthesis by interacting with integrin proteins found in our skin cells [137]. It is believed that throughout the healing process, integrins may be delivered to dermal fibroblasts by EVs released by keratinocytes and integrated into the ECM. Li et al. discovered that human keratinocyte-derived MVs supplied miRNA-21 and accelerated cutaneous wound healing in diabetic rats by promoting fibroblast activity and angiogenesis [138]. EVs can potentially have an impact on fibroblasts’ gene expression. For instance, in fibroblasts, EVs produced from epidermal cells significantly influence the genes that code for MMP-1 and MMP-3, IL-6 and IL-8, and genes involved in TGF-β signaling. Human keratinocytes and fibroblasts are able to migrate and proliferate more rapidly when HaCaT cell-derived EVs are present, which may hasten wound healing by triggering the mitogen-activated protein kinase pathway [139].

Burn infection

Burns are injuries to the skin and internal organs brought on by chemical, electrical, and thermodynamic changes. The goal of treating patients with severe burns is to hasten wound closure in order to limit exposure to the environment [140]. Patients with severe burns are also more susceptible to infection and sepsis. The significance of exosomes from stem cells has received attention in the healing of burn wounds because stem cells significantly enhance wound healing [141]. Previous research has shown that exosomes can enhance burn wound healing. hucMSC-exos improve wound healing by stimulating Wnt4/β-catenin signaling. Excessive inflammation caused by burns is one of the primary reasons why burns are difficult to heal. Furthermore, the permeability of burns has a systemic impact on the body as opposed to the local harm brought on by mechanical injury. Exosomes can increase skin cell viability, encourage cell proliferation, and lessen cell damage. Li et al., 2016 revealed that hucMSC-exos might minimize excessive inflammation at burn sites by regulating microRNA-181c [37]. RNA-sequencing studies have discovered that the microRNA profiles in plasma exosomes change after a burn injury, which can lead to abnormal gene expression in the skin during the early stages of burn recovery [142]. Protein profiling research has revealed that burns can lead to noteworthy alterations in the functionality of serum exosomal proteins. This affects the activity of enzymes, the ability of proteins to bind to heparin, coagulation, and lipid transport in the body. Exosomes have the capability to enhance mRNA and protein levels, thus supporting the healing and restoration of impaired tissues to their regular state [143].

Visceral injury is a frequent burn complication that has an impact on wound healing and rescue. It is directly associated with the modification of exosomes. The secret to wound healing is also to keep internal organs functioning at their regular physiological level. According to one study on vascular permeability, serum exosomes containing S100A9 may play a role in increasing pulmonary microvascular hyperpermeability [144]. Injecting hucMSC-exos reduced acute lung injury brought on by burns through modulating microRNA-451 [145]. These investigations show that stem cell exosomes may repair internal organ injury and burn-induced exosome alterations are the key to inflicting systemic harm. Unfortunately, there aren’t many studies on exosomes for other visceral injuries, even though this might be a key area for burn research in the future.

Challenges in exosome therapy

There may be multiple challenges while using MSC exosome-based treatment in a clinical environment. The complex techniques needed in isolating, purifying, and scaling up exosome production pose a considerable barrier to advancing exosome treatment from experimental to clinical settings. Variations in the safety and quality attributes of exosome products arise from the lack of standardized procedures for exosome separation. The production of MSC exosomes as a clinical product may be acceptable in terms of pharmaceutical preparations, provided consistency and safety are ensured. Bioengineering technology may be used to change exosome phenotypes, allowing the introduction of particular biological substances transported by exosomes to boost therapeutic efficacy or decrease adverse effects [146, 147]. Furthermore, the use of biomaterials into MSC exosome-based therapies offers promise in overcoming these practical challenges, potentially imposing systemic effects on wound repair, increasing their efficiency and providing additional therapeutic advantages [148]. Although clinical studies have demonstrated the safety, feasibility, and efficacy of MSC-based therapies, it is critical to recognize the limitations of these trials, particularly the small sample sizes and the lack of longer follow-up data [147, 149]. The significance of investigating the therapeutic potential of MSC-secreted components, specifically exosomes, in the context of cutaneous wound healing is highlighted by these inherent difficulties and related conflicts. Emerging evidence shows that exosomes derived from MSCs may be a feasible therapeutic method in the absence of cells, with significant benefits over MSCs alone. The use of MSC-derived exosomes has been linked to a decreased risk of tumor development and immunogenicity. However, the long-term risks of MSC-exosome treatment are unclear, prompting an investigation of their impact on the immune system [150, 151]. Further, there has been an upsurge in clinical studies for MSC therapies for skin problems, with 96 trials listed on ClinicalTrials.gov. The studies focus on conditions such diabetic foot ulcers, burn injuries, psoriasis, and scleroderma, indicating an increasing interest in MSC-based therapies for a variety of skin ailments (Table 1). Despite the fact that exosomes could serve as a promising therapeutic intervention, the practical implementation of MSC-exosome-based therapies for skin regeneration faces several obstacles. These problems include inadequate targeting, rapid clearance dynamics, and a relatively limited half-life while working in the complex milieu of a wound [152]. In addition, their regulatory position is also being debated. The US FDA has yet to officially categorize exosomes for the pharmaceutical sector, leaving regulatory problems unresolved [153]. Further research in the quality control of exosome products is required, implying a lengthy journey before their application in clinical settings.

Table 1 Human trials on MSC exosomes as registered on ClinicalTrials.gov. (source: ClinicalTrials.gov [147])

Exosomes and future perspectives

Exosomes have garnered substantial attention in the realm of wound repair and cutaneous regeneration in recent decades. Notably, MSC-exosome-based therapy is emerging as a highly promising approach to enhance wound healing and diminish scarring. MSC exosomes offer a number of benefits as a cell-free alternative therapy, including ease of preparation, storage, and transportation, as well as ease of dosing and administration at the time of choice. They also seem to have high therapeutic efficacy and no risk of immunological rejection or carcinogenesis. As a result, MSC-exosomes have a high potential for cutaneous regeneration and may be able to successfully substitute complete MSC-based therapy. Additional research into the molecular processes by which MSC exosomes induce skin regeneration will provide further insight into their unique contents and functions. To conclusively establish the skin regeneration therapeutic potential of MSCs in a large number of patients, more clinical trials using exosomes of human origin are required as most of the mechanisms have been outlined and examined in rodents only. Yet, there are still several critical scientific difficulties to be overcome first.

It’s crucial to acknowledge that exosome therapy yields distinct biological effects at different phases of wound healing. The composition of MSC-exosomes secreted within diverse microenvironments varies considerably, thereby yielding differing components and effects. This important scientific aspect has often been overlooked in the investigation of MSC-exosomes. Being a novel kind of medicine, further investigation is also required to comprehend the regulatory implications of using exosomes.

Conclusion

Recent study suggests that exosomes can accelerate angiogenesis and have a significant regenerative effect on burned skin by encouraging keratinocyte and fibroblast migration and proliferation. A substantial stride has been taken in comprehending the cargo mechanism that conveys signals, orchestrating cell activity alterations via encapsulated miRNAs and proteins. The role of MSC exosomes in the management of pathology, immunomodulation, and regeneration is a significant function that could be crucial in burn wound healing. The replacement of traditional treatments for burn wounds with exosome-based treatments as a cell-free therapy may be of current importance in improving the efficiency of burn wound healing or regenerative therapy, given the purported advantages of exosomes compared to other cell types and molecular methods to regeneration. Stem cell treatment for burn wound healing is an emerging topic in the current field of study; it demanded an updated review that included recent breakthroughs in stem cell therapy for burn wound healing as well as pertinent experimental trials. We also anticipate that more researchers need to pay more attention to this scientific topic, which is to develop better and more appropriate ways for recreating the damaged site microenvironment of burns. Additionally, a more pronounced emphasis on recreating the compromised microenvironment of burn injuries is imperative. This review serves as an inspiration for researchers to explore novel avenues in exosome therapy for burn wound healing.