The skin’s ability to heal wounds results from the body’s innate ability to repair and regenerate damaged tissues (Lau et al., 2017). It is a common occurrence in people due to accidents, surgeries, burns, and long-term illnesses. Various wound therapies have been suggested, such as pharmacotherapy (Jackson et al., 2012; Abdullahi and Jeschke, 2014), cellular therapy (Nauta et al., 2013; Morimoto et al., 2015), extracorporeal shock wave therapy (Mittermayr et al., 2012; Webster et al., 2012), negative pressure wound therapy (NPWT) (Webster et al., 2012), electrical stimulation therapy (Thakral et al., 2013; Baleg et al., 2015), and light therapy (Haubner et al., 2012; Lau et al., 2015).

The use of mesenchymal stromal/stem cells (MSCs) to treat skin wounds has gained popularity. MSCs offer a reasonable, safe, and successful therapeutic method due to their differential competency and other features, including ease of collection, low immunogenicity, and their essential role in native wound healing physiology (Zou et al., 2012; Markov et al., 2021; Hassan et al., 2014). In fact, MSCs promote the migration of skin cells, the development of blood vessels, the re-epithelialization of wounds, and the production of granulation tissue, thereby accelerating the healing process. Remarkably, they foster a regenerative wound-healing environment instead of a fibrotic one (Zahorec et al., 2015; Yao et al., 2016). While clinical studies have provided evidence suggesting the safety, practicality, and efficacy of MSC-based therapies (Huang et al., 2020; Gentile and Garcovich, 2021; Mahjoor et al., 2023), more evidence is needed due to small sample sizes and a lack of long-term follow-up. Multipotent stem cells (MSCs) exert therapeutic effects through various cell type-specific and wound-healing phase-specific functions, from hemostasis to remodeling (Hocking and Gibran, 2010).

Many adult organs, including bone marrow, adipose tissue, and umbilical cords (UCs), contain mesenchymal stem cells (MSCs) (Pereira et al., 2013). MSCs typically undergo two distinct environments along their journey from isolation to engraftment. Both the in vivo or physiological setting and the in vitro culture setting (from isolation to transplanting) are considered (before isolation and after transplantation). Cells are exposed to 20% O₂ during most MSC growth techniques nowadays, which is around 4–10 times higher than the concentration of O₂ in their natural habitats (Chow et al., 2001; Antoniou et al., 2004). MSCs cultivated in vitro may be stressed by the increased oxygen levels. Moreover, various studies have provided significant evidence in recent years, indicating the deleterious effect of ambient O₂ concentration on MSCs, including early senescence, prolonged population doubling time, DNA damage (Fehrer et al., 2007; Estrada et al., 2012), and poor engraftment upon transplantation (Sch¨achinger et al., 2006a). These findings highlight the critical influence of O₂ concentration on the biology of MSCs. Over the past 2 decades, in vitro research examining the pathways involved in stem cell preservation has proliferated. However, the effects of normoxic (often 20%–21% O₂ concentration) and hypoxic (typically 2%–9% O₂ concentration) environments on stem cell development have been largely overlooked (Simon and Keith, 2008). This article compares and contrasts MSC culture under normoxic and hypoxic in vitro settings. Lastly, this research discusses how MSCs cultivated in vitro under hypoxic settings might serve as a therapeutic option.

Wounds on the skin can be classified into two categories: those requiring tissue regeneration and those that do not. Open wound edges are brought together, and the wound is mostly closed through localized matrix remodeling after surgery or a minor incision. However, a complicated chain reaction can occur when tissue loss is significant in wounds, such as burns, abrasions, and traumatic wounds.

The first phase involves hemostasis and inflammation, which helps rebuild a sterile barrier and reduces exposure to the outside world. Subsequently, the wound undergoes tissue replacement (scar formation). If difficulties arise during the early phases of healing, such as an inadequate immune response or impaired angiogenesis, wounds can become chronic or non-healing (Lazarus et al., 1994; Yates et al., 2011; Frykberg and Banks, 2015; Mahmoudvand et al., 2023). Chronic wounds encompass vascular ulcers, pressure ulcers, and diabetic ulcers, and each patient requires a customized treatment plan based on the severity of their condition. Lack of wound healing progress poses risks of infection, tissue spread, and potential amputation.

To combat the severe consequences of chronic wounds, scientists and doctors are diligently developing and implementing innovative biological treatments. Abnormalities in the last two phases of wound healing, such as keloid or hypertrophic scarring, may result in extensive scarring in contrast to chronic wounds. Although the epithelium may regenerate, and the wound may appear healed, the underlying dermis in these scars is damaged and dysfunctional. Improperly healed wounds tend to be structurally weaker and more susceptible to re-ulceration.

Healing a wound involves a complex synchronization of several cell types, signaling pathways, and microenvironmental adjustments across three overlapping stages of repair: hemostasis/inflammation, tissue replacement, and resolution. The initial step is the hemostasis/inflammation phase, during which the focus is on stopping bleeding from broken blood vessels and preventing the spread of pathogenic infection. Platelets in the bloodstream join with enzymatically transformed fibrin to create a clot that covers the wound and acts as a temporary matrix (Barker and Engler, 2017). This fibrin clot is physiologically active and recruits pro-inflammatory macrophages (M1) and leukocytes, triggering a localized immune response in addition to preventing infection and clearing the wound of cellular and extracellular debris. The tissue replacement phase begins when specialized cells, such as fibroblasts, endothelial cells, epidermal cells, and progenitor cells, enter the wound bed. Fibroblasts produce collagen III-rich granulation tissue to temporarily replace the missing extracellular matrix (ECM), endothelial cells generate new blood vessels through angiogenesis, and epidermal cells migrate beneath the scab to permanently seal the wound surface and restore the epidermis. At this stage, pro-inflammatory macrophages (M1) decrease and are replaced by wound-healing macrophages, categorizing the lesion as sterile (M2). These M2 macrophages facilitate additional vascular healing by producing and modulating the granulated ECM.

The resolution phase involves the permanent replacement of the wound bed with mature ECM, removal of up to 90% of the surplus blood vessels, and cessation of any remaining proliferation and migration signals, replaced with stop signals, such as those operating via CXCR3 (Bodnar et al., 2013; Martins-Green et al., 2013; Mirshekar et al., 2023). Transdifferentiation of fibroblasts into myofibroblasts causes the wound bed to contract by reorganizing and restructuring juvenile collagen III into mature collagen I, thereby restoring the skin’s tensile strength (Sahota et al., 2003; Mahjoor et al., 2021). If everything proceeds as expected, the final result will be little more than a barely noticeable scar.

Various types of stem cells, including bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord MSCs, adipose-derived stem cells (ASCs), molar MSCs, and amniotic fluid ASCs, have been investigated as potential sources of MSCs, which hold significant promise in the field of stem cell therapies. These MSCs, regardless of their source, facilitate the healing process through two main processes: paracrine effects and direct development into skin cells (fibroblasts and keratinocytes) (Martins-Green et al., 2013; Kinnaird et al., 2004).

Miao et al. conducted a comparison between placental-derived MSCs and bone marrow-derived MSCs, assessing their morphology, growth, membrane markers, and differentiation potential (Yao et al., 2016; Lau et al., 2017). Placental MSCs exhibited similar morphology and growth characteristics, as well as the presence of markers CD105, CD29, and CD44. Notably, they lacked expression of hematopoietic markers CD34, CD45, and HLA-DR. Furthermore, the study demonstrated that placental MSCs could differentiate into endothelial and neuronal cells (Tsai et al., 2012). In another recent study focusing on venous leg ulcer treatment, allogeneic AM transplantation proved highly beneficial for serious wounds, showing antiadhesive properties, wound protection, and effective re-epithelialization (anti-scarring) (Martin-Rendon et al., 2007a).

MSCs secrete a myriad of active compounds, such as insulin-like growth factor, hepatocyte growth factor, transforming growth factor beta-1, vascular endothelial growth factor (VEGF), keratinocyte growth factor, fibroblast growth factor 2, platelet-derived growth factor, and collagen I9, which play critical roles in wound healing by stimulating angiogenesis, skin cell proliferation, and ECM formation (Fui et al., 2019). The precise mechanism of direct differentiation of MSCs into specific skin cells remains incompletely understood. However, it is well-established that MSCs have the capacity to differentiate into various types of resident cells when introduced into a wound site. The primary mechanism through which stem cells differentiate into specialized cell types, such as endothelial cells, fibroblasts, and keratinocytes, is through the local release of multiple chemicals. Thus, strategies aimed at augmenting the paracrine MSC secretion within the wound microenvironment hold the potential to enhance the efficiency of wound healing. One promising approach could involve modifying the composition of the suspension by utilizing bubbles encapsulating a thoughtfully selected secretome, which can then be applied to the wound to potentially enhance wound healing outcomes. However, randomized trials confirming the effectiveness and safety of MSC use in wound treatment are still needed. Clinical trials involving MSCs are currently underway, and their findings are awaited to further validate the effectiveness and safety of MSC-based treatments for wound healing (Kyung-Chul et al., 2019).

In the process of wound healing, MSCs originating from subcutaneous fat tissue and the blood supply play a role in regulating early and mid-phase inflammation, as well as contributing to the restoration of dermal tissue. Upon infiltrating the wound, MSCs initiate the secretion of proinflammatory cytokines, which recruit neutrophils and M1 converted macrophages, aiding in the degradation of damaged tissue during the hemostasis/inflammation phase. Additionally, MSCs help in controlling the overall inflammatory response by reducing the number of activated T cells, neutrophils, and macrophages. During the tissue replacement phase, MSCs guide the polarization of monocytes into proreparative M2 macrophages, facilitating the clearance of cellular debris and modifying the temporary matrix. In the resolution phase, MSCs continue to regulate the matrix by releasing matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), while also maintaining a balance between transforming growth factor-b1 and transforming growth factor-b3 to prevent hypertrophic scarring (Yates et al., 2011).

A unique delivery system employing fibrin glue was investigated in both acute and chronic wounds by Falanga et al. (Sch¨achinger et al., 2006b). They topically applied bone marrow-derived MSCs combined with a fibrin spray up to three times. The study observed that surgical defects from excision of nonmelanoma skin cancers healed within 8 weeks, suggesting that MSCs contributed to accelerated resurfacing. Additionally, chronic lower-extremity wounds present for longer than 1 year significantly decreased in size or healed completely by 20 weeks. The study also found a correlation between the surface density of MSCs and the reduction in ulcer size. Another study by Yoshikawa et al. included 20 patients with various nonhealing wounds who were treated with autologous bone marrow-derived MSCs cultured on a collagen sponge (Chavakis et al., 2010). It was observed that 90% of the wounds healed completely when treated with the cell composite graft, and the addition of MSCs facilitated tissue regeneration.

In 1958, Cooper et al. and Zwartouw & West-wood noticed that certain cells grew more quickly at low O₂ tension levels compared to standard air levels (Cooper et al., 1958; Zwartouw and Westwood, 1958). This prompted them to conduct the first experiments to evaluate the effects of varied O₂ tension levels on MSC growth. Hypoxia has been identified as a key regulator of MSC recruitment, migration, and differentiation (Raheja et al., 2010a; Raheja et al., 2011). A study by Rochefort et al. demonstrated that hypoxia mobilized MSCs from the bone marrow into the peripheral blood but not hematopoietic progenitor cells (Rochefort et al., 2006). Additionally, stem-cell quiescence and plasticity have been linked to low O₂ tension levels (D’Ippolito et al., 2006; Liu et al., 2011). Researchers have been interested in finding the appropriate O₂ tension levels for in vitro cultivation (Das et al., 2010). Raheja et al. (2010) conducted an experiment in which they seeded and stimulated MSCs for differentiation in different oxygen concentrations. Results indicated that 21% oxygen was optimal for MSC development into osteoblasts, whereas oxygen levels below 5% significantly slowed the differentiation process. At 5% and 21% O₂, however, no significant change in osteogenic markers was detected (Raheja et al., 2010b). Early passaged (P2) MSCs at 5% O₂ concentration showed enhanced osteoblastic and adipogenic differentiation capacity (Basciano et al., 2011a). Recent studies have shown that MSCs may survive in an oxygen-depleted (1%–5% O₂) environment (Grayson et al., 2007a; Nekanti et al., 2010; Holzwarth et al., 2010a; L´opez et al., 2011). It has also been observed that MSCs isolated from adipose tissue that has been precultured in a hypoxic environment have enhanced adipogenic and osteogenic differentiation potentials (Valorani et al., 2012) (Table 1). Nevertheless, when MSCs were maintained and driven for differentiation in 1% O₂ concentration, only a small number of studies demonstrated a loss in differentiation potential (Yang et al., 2011; Hung S.-P. et al., 2012). Several clinical trial data indicate that transplantation of MSCs cultivated under an ambient environment has a limited ability for engraftment (Mohamadnejad et al., 2010; Sch¨achinger et al., 2006b).

Clinical studies utilizing MSCs have shown some promising therapeutic results, as documented in various review papers and meta-analyses (Abdel-Latif et al., 2007; Lipinski et al., 2007; Chavakis et al., 2010; Song et al., 2011). The engraftment and differentiation capacity to cardiomyocytes in vivo of first-passage mouse BM-MSCs was shown to be higher than that of fifth-passage mouse BM-MSCs (Jin et al., 2011). Moreover, murine MSCs pre-conditioned in a hypoxic environment demonstrated increased blood flow and vascular development at day 7 compared to MSCs maintained in a normoxic condition (Leroux et al., 2010). Additionally, hypoxia or a reagent that mimics the reaction to hypoxia increased the expression of the chemokine receptors CXCR4, CXCR7, and CX3CR1 (Hung S.-C. et al., 2007; Liu et al., 2012a; Saller et al., 2012; Tsai et al., 2012). Survival of MSCs in hypoxic environments depends on their capacity, shared by all cells, to switch efficiently from aerobic to anaerobic metabolic pathways (Das et al., 2010). An experimental investigation done with rats on the alterations in MSCs under serum-deprivation and hypoxia settings indicated that serum deprivation was the critical factor leading to ischemia-induced death of MSCs. Nevertheless, it also indicated that prolonged exposure to hypoxia led to mitochondrial malfunction and Caspase-3 activation, a crucial factor in apoptosis (Zhu et al., 2006). The buildup of lactate from glycolysis may become an inhibiting factor over the long term (Lord-Dufour et al., 2009; Das et al., 2010), despite the fact that MSCs have been proven to resist hypoxia (e.g., O₂ 1%) for at least 48 h (Martin-Rendon et al., 2007a). Hypoxia-inducible factor-1 alpha (HIF-1α), when stabilized, migrates into the cell nucleus and joins with hypoxia-inducible factor-1 beta (HIF-1β) to promote intracellular signaling pathways linked with cell survival. To regulate gluconeogenesis, hypoxia-responsive genes such as glucose-6-phosphate transporter (G6PT) bind to their promoter regions as dimeric complexes. There is evidence that the higher glucose level from gluconeogenesis aids MSC survival in hypoxic or serum-depleted environments (Ren et al., 2006a; Martin-Rendon et al., 2007a; Hung S. C. et al., 2007). In addition to HIF-1 upregulation, a rise in erythropoietin receptors and anti-apoptotic proteins Bcl-2 and Bcl-XL, followed by lower Caspase-3 levels, all contribute to a higher survival rate under hypoxia compared to normoxia. In addition, hypoxia stimulates the production of the proangiogenic molecules interleukin IL-6 and VEGF (Wang Y. et al., 2008) (Table 1). Several signaling pathways, such as the Akt and ERK pathways, control these positive effects.

The Akt signaling pathway is phosphorylated in response to hypoxia and HIF-1 stabilization but is destroyed in the presence of normal oxygen levels. The expression of the pro-apoptotic protein Bax is downregulated, while the expression of the anti-apoptotic factor Bcl-2 is upregulated when Akt is active. Apoptosis or additional stabilization of HIF-1, leading to its translocation into the cell nucleus and activation of hypoxia-responsive genes like G6PT and angiogenesis-related proteins like VEGF and IL-6, may result from such overexpression interacting with the Bax accumulating in the mitochondria (Hill et al., 2009). The ideal culture time of MSCs in hypoxic circumstances, like the optimal O₂ tension level, is yet unknown. After just 10 min in culture, Wang et al. found that MSCs subjected to short-term hypoxia pre-conditioning showed improved cell survival and angiogenic characteristics (Wang J. A. et al., 2008). Cell growth was shown to be 30 times greater in low O₂ conditions (2% for 7 passes) compared to normoxic cultures by Grayson et al. (Grayson et al., 2007b). After 7 days in culture, Hung et al. found that MSCs could proliferate more effectively when exposed to hypoxia (1% O₂) (Hung et al., 2012b). An in vitro migration experiment was also carried out in this work, and the results revealed that MSCs’ migratory potential was improved by hypoxia (Hung et al., 2012c). Matin-Rendon et al. found that MSC proliferation increased even after just 24 h of exposure to 1.5% O₂ (Martin-Rendon et al., 2007b). The number of cells in the G2/S/M phase was shown to rise during hypoxia (Ren et al., 2006b), and D'Ippolito et al. demonstrated that a low O₂ tension level shortened the time required for the cell population to double when cultivated at 3% O₂ (D’Ippolito et al., 2006).

The opposite was found in research by Holzwarth et al., which compared the proliferation rates of MSCs after 7 days of culture at 21%, 5%, 3%, and 1% O₂. Strong proliferation rates were seen in their research when MSCs were cultured at 21% O₂, which is hyperoxic compared to the physiological environment in which MSCs dwell. After 7 days in culture, a significantly lower percentage of MSCs (1.37%) reached the G2/M phase in hypoxic cell cultures (1% O₂) than in cultures with 21% O₂. With fewer cells in the G2/M phase, the authors conclude that cell proliferation is inhibited under low- O₂ conditions (Holzwarth et al., 2010b). Via nuclear factor kappa-dependent processes (Crisostomo P. R. et al., 2008), hypoxia seems to control the concentrations of soluble factors (including VEGF, fibroblast growth factor-2 (FGF2), hepatocyte growth factor, and insulin-like growth factor 1 (IGF-1)). Results indicate that hypoxia may have an effect on the immunoregulatory capacities of MSCs. After 24 h of exposure to hypoxia, Wu et al. found that most genes were regulated using human MSCs. Nevertheless, matrix metalloproteinase-2 (MMP2) levels dropped after less than 4 h of hypoxia, but VEGF and membrane type 1-matrix metalloproteinase (MT1-MMP) levels increased (Wu et al., 2007). Increased VEGF expression was also reported by Muir et al. in hypoxic circumstances (Muir et al., 2006). After 48 h of cultivation with FBS under hypoxia, Potier et al. found that the expression of TGF-3, but not FGF2 or VEGF, was reduced. The levels of IL-6, IL-8, and MPC1 were not altered in that investigation (Potier et al., 2007). In contrast, Hung et al. reported that under hypoxia, the expression of IL-6, macrophage chemotactic protein (MCP1), and VEGF was elevated in MSCs grown in an FBS-free media (Hung S. C. et al., 2007).

Nakagawa et al. suggested that MSCs, together with bFGF in a skin defect model, accelerate wound healing and showed that the human MSCs transdifferentiated into the epithelium in rats (Valorani et al., 2012). Shumakov et al. showed that the transplantation of MSCs on the surface of deep burn wounds in rats decreased inflammatory cell infiltration and accelerated the formation of new vessels and granulation tissue (Hung S.-P. et al., 2012). The cells were also shown to produce bioactive substances that seemed to accelerate the regeneration process. Collectively, these data demonstrate that MSC treatment impacts all phases of wound repair, including inflammation, epithelialization, granulation tissue formation, and tissue remodeling.

The chemokine receptors CX3CR1 and CXCR4 and the hepatocyte growth factor receptor cMet are all regulated by hypoxia, which also affects the production of soluble factors. These receptors enhance MSCs’ ability to migrate to and home in on injured cells (Das et al., 2010). Hepatocyte growth factor and its receptor cMet, the expression of which is elevated during hypoxia, may have a role, as discovered by Rosova et al. (Rosova et al., 2008a). Hung et al. demonstrated that MSCs’ migratory ability was enhanced by hypoxia, and Wang et al. demonstrated the same thing in the context of a brain injury (Hung et al., 2012d). The CXCR4 receptor was implicated in the capacity of these cells to move to injured tissue in the latter investigation (Wang Y. et al., 2008). In animal models, MSCs cultured under hypoxia appear to have improved immune regulatory performance, suggesting a possible role of hypoxia in cellular therapy strategies (Mao et al., 2004; Hu et al., 2008). Hypoxia has also been shown to influence the secretion of trophic factors and membrane markers associated with MSC migration and homing. Several cytokines, chemokines, and integrins may all play a role in migration. One chemokine that is widely expressed is stromal cell-derived factor-1 (SDF-1/CXCR4 receptor) (Liu et al., 2012b). Thus, the expression of CXCR4 on the surface of MSCs cultivated under hypoxia has a significant role in the migration of transplanted cells (Martin-Rendon et al., 2007b).

Others defined a hypoxia pretreatment of tissue-engineered substitutes, which are 3D biomimetic collagen-chitosan sponge scaffolds (CCSS), as biocompatible and biodegradable supports for the effective delivery, favorable adhesion, and survival of BM-MSCs to accelerate chronic wound healing by inhibiting inflammation and improving angiogenesis (Lazarus et al., 1994; Yates et al., 2012). 3D biomimetic tissue-engineered substrates have a great effect on the development of scaffolds for tissue engineering in chronic wound healing, particularly in combination with cells such as MSCs, fibroblasts, keratinocytes, and endothelial cells. Co-transplantation of CD341 cells with CD341-derived endothelial cells in a 3D fibrin scaffold improved wound healing by decreasing the inflammatory reaction and increasing neovascularization in diabetic rats. The relative secreted factors include VEGF, PDGF, bFGF, IL-10, and IL-17 (Lau et al., 2015). In addition, a similar tissue-engineered substitute using autologous fibroblasts and keratinocytes seeded in Hyaff-11, an ester of hyaluronic acid, has been reported to enhance wound healing in diabetics and is associated with enhanced revascularization (Barker and Engler, 2017).

Others found that the major inflammatory cell infiltrated the CCSS in the wound site, and the expression of TNF-a and IL-6 was significantly higher at early implantation. However, BMMSCs efficiently improved this effect and significantly suppressed TNF-a and IL-6 levels in the wound site, thereby contributing to improved wound healing, particularly, hypoxia pretreatment of the tissue-engineered substitute. They found that the expression of IL-10 was enhanced at the wound site and that IL-10 levels in the tissue-engineered substitute with hypoxia pretreatment were higher compared with other conditions. In addition, normoxic pretreatment of the tissue-engineered substitute also caused a significant increase in IL-10 levels when compared with CCSS only in the wound site.

In conclusion, the potential of mesenchymal stromal cells (MSCs) in advancing wound healing therapies is truly remarkable. Their paracrine interactions play a pivotal role in expediting the healing process, promoting angiogenesis, and facilitating skin regeneration, making them a promising solution for cutaneous wounds. As researchers strive to enhance the therapeutic efficacy of MSC-based treatments, inducing hypoxia has emerged as a powerful tool to further unleash their capabilities.

By subjecting MSCs to hypoxic pre-conditioning, we can better equip them to withstand the challenges of the wound microenvironment, minimizing cell death and maximizing their beneficial effects on tissue repair. The future of wound healing appears brighter than ever as we delve deeper into the transformative potential of MSCs under hypoxic conditions. Embracing this approach not only holds promise for improving patient outcomes but also paves the way for more effective and efficient wound healing strategies.

By harnessing the power of MSCs in the context of hypoxia, we are forging a path towards advanced regenerative therapies that will enhance the lives of patients and promote overall skin health. The journey to harnessing the full regenerative potential of MSCs in wound healing has just begun, and its impact on medical practice is bound to be profound.