The Role of Stem Cell on Wound Healing After Revascularization—Healing Following Revascularization—Unlocking Skin Potential

Abstract

Wound healing is a complex and dynamic process involving a series of cellular and molecular events. Revascularization, the restoration of blood flow to ischemic or damaged tissue, is a key step in wound healing. Adequate vascularization has been recognized as a necessary factor for successful tissue regeneration. In the later stage of revascularization and tissue remodeling in wound healing, stem cells regulate other repair cells and matrix formation by influencing the maturation of blood vessels. The reductive oxidation (REDOX) state may be a key mechanism through stem/progenitor cells to influence endothelial cells to mature blood vessels and improve the quality of healing. Mitochondria may play an important role in this process.

Wound healing is a complex and dynamic process involving a series of cellular and molecular events. Revascularization, the restoration of blood flow to ischemic or damaged tissue, is a key step in wound healing. Adequate vascularization has been recognized as a necessary factor for successful tissue regeneration since only newly formed microvessels can provide the right amount of oxygen, abundant nutrients, and large amounts of growth factors/cytokines or chemokines needed to ensure the perfect repair of damaged tissue. Without a new vascular system to provide adequate oxygen and nutrients, tissue expansion replacement is limited. Therefore, poor revascularization is often associated with delayed wound healing and scarring, which is a critical factor in poor clinical outcomes. Repair cells with normal function are the basis of tissue repair and regeneration. In all the processes of tissue repair and regeneration, the window to repair cells is firstly to effectively reduce ischemia-reperfusion injury in the early stage of trauma, and secondly to remove reactive oxygen species (ROS) in the wound bed immediately after debridement and maintain a well-wound microenvironment. ¹

Since new angiogenesis and its maturation is a particularly not only rapid but also very persistent process, it is very important to realize the effect of its internal mechanism on repair cells to deeply understand the potential of tissue repair. Wound healing is a complex biological process supported by countless cellular activities that must be closely coordinated to achieve their goals. The role of stem cells in wound healing has received increasing attention and could regulate other repair cells. However, previous studies on the involvement of stem cells in wound healing mostly focused on the early and middle stages of wound healing: inflammation and proliferation(including angiogenesis), which indeed play a role in the initiation and maintenance of tissue repair and regeneration. The cells regulate inflammation and immune response and promote the formation of new angiogenesis, which is a pivotal node of wound healing. As a unique cell type, stem cells have the ability to differentiate into various cell types and promote tissue repair and regeneration by the ability of secretory and paracrine.^2,3 Therefore, in the later stage of revascularization and tissue remodeling in wound healing, stem cells regulate other repair cells and matrix formation by influencing the maturation of blood vessels, the effect of stem cells on the quality and outcome of wound healing after revascularization is attracting more and more attention. Understanding the functional characteristics of stem cells and their potential mechanisms will be of great significance to the basic and translational research of wound healing. Once a breakthrough is made in the exploration of relevant mechanisms, it will play a great role in promoting the clinic application of skin and soft tissue injury repair and regeneration.

In most clinical settings, closing wounds is considered the end point of wound healing, but wounds can continue to undergo remodeling or tissue maturation for months or even years. This is the final stage of wound healing, and since tissue remodeling and cell survival and apoptosis are largely dependent on the supply of oxygen, nutrients, and other factors from the vasculature, delayed or insufficient revascularization within damaged or implanted tissues can impair the healing process and may result in poor integration of the transplanted tissue.

In fact, the reconstruction stage also includes the degeneration of new blood vessels, followed by granulation tissue remodeling to form scar tissue. The granulation tissue is mainly composed of type III collagen, which is partially replaced by stronger type I collagen as the wound is remodeled. This process is the result of simultaneous type I collagen synthesis and type III collagen degradation, followed by the reorganization of the extracellular matrix (ECM). Once reepithelialization occurs, myofibroblasts within the granulation tissue continue to synthesize matrix metalloproteinases (MMPs) and their respective inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Matrix metalloproteinases degrade specific components of ECM and are an important step in its remodeling. Imbalanced expression of TIMPs and MMPs can lead to abnormal modification of ECM and even scar or nonhealing wound.

Vascular maturation after revascularization is also very important. When the cells in the granulation tissue do not undergo apoptosis during the wound remodeling stage, they will become hypertrophic scars. In the process of remodeling, the new blood vessels are pruned to generate stable and well-flowing blood vessels, restore and maintain homeostasis, and form static endothelial cells. ⁴

Currently, there is less research on angiodegeneration compared to angiogenesis, This is most likely because the formation of new blood vessels has been studied easily in in vitro, but the negative feedback mechanisms of vascular degeneration have been difficult to model and observe. Revascularization is a rapid and long-lasting process that is reflected in the oxidative stress following injury and the subsequent tissue reductive stress. Therefore, the REDOX(Reduction-oxidation) state may be a key mechanism through stem/progenitor cells to influence endothelial cells to mature blood vessels and improve the quality of healing. Cellular REDOX homeostasis derives from the evidence that cells constantly generate free radicals both as waste products of aerobic metabolism and in response to a large variety of stimuli. Free radicals, once produced, provoked cellular responses (redox regulation) against oxidative stress transducing the signals to maintain the cellular redox balance. ⁵

Tissue-resident stem cells, including mesenchymal stem cells (MSCs), vascular progenitor cells, and other tissue-specific progenitor cells, are responsible for tissue regeneration. Studies have shown that blood vessels contain a variety of “life sources” involved in tissue development and renewal of resident stem cells and have become pivotal candidates for the development of regenerative medicine, including tissue engineering. ⁶

Circulating and Perivascular Stem/Progenitor Cells

Stem cell-based tissue repair and regeneration is considered promising because stem/progenitor cells have several advantages, including differentiation into desired cell lines, secreted cytokines/growth factors, involvement in immunoregulation, tissue remodeling post-injury, and initiation of endogenous repair/regeneration mechanisms.

Bone marrow mesenchymal stem cells (BM-MSCs) can be mobilized from BM or other reservoirs and transported through blood vessels to the site of injury. Subsequently, These MSCs (mesenchymal stem cells) will release kinds of factors that inhibit scarring (tissue fibrosis), promote vascular formation and maturation, and stimulate host progenitor cells to divide and differentiate into functional units for regenerative and growth. ⁷

On the other hand, due to the wide distribution of pericytes around human microvascular, they are able to participate more effectively in multiple important stages of regenerative repair. In addition to adult stem cells localized to the injury area, most mesenchymal stem cells can be produced by perivascular cells (pericytes), which are released from damaged or inflamed blood vessels at the injury site. ⁸

In recent years, with the deepening of people's understanding, some scholars have proposed to retain the name of MSCs (ie, retain the abbreviation of MSC) for mesenchymal stem cells, but change to the abbreviation of medicinal signaling cells (MSCs). ⁹ Some experimental studies have shown that circulating stem/progenitor cells may not only necessarily participate in the formation of new microvascular but may act as supporting cells or differentiate into fibroblasts, immune-like cells, and pericytes and produce a variety of growth factors and chemokines to participate in the tissue repair process by paracrine. ¹⁰ Experiments using cell line tracing have confirmed that these signals play a central role in repairing the origin of cells. Although no evidence has been obtained in skin and soft tissue repair and regeneration, it has shown great potential in skeletal muscle regeneration and promoting the recovery of heart and kidney function. ¹¹

Revascularization and Oxidative Stress

As an essential metabolic requirement for wound healing, oxygen needs to be delivered to all kinds of repair cells, and the blood vascular system is necessary to transport oxygen and other nutrients. One of the biggest challenges for perfect tissue repair is to establish a functional vascular system to overcome the abnormal healing caused by oxygen and nutrient deficiency in the tissue structure. ¹² Oxygen is also a molecule with biological functions. When revascularization is complete, readjusting the REDOX balance is critical to the outcome of tissue repair. ¹³ Because discomfort and decreased oxygen tension can lead to metabolic inadequacy and disturbances in the formation of ROS, tissue repair may be impaired. The role of ROS in mediating cell fate depends on stimulus intensity, cell surrounding environment, and metabolic state. Reactive oxygen species acts primarily through multiple targets, such as kinases and transcription factors, and plays different roles in different stages of initiation, differentiation, and maturation after injury. ¹⁴ Particular attention is paid to the role that ROS plays in differentiation and transdifferentiation (direct reprogramming) of cells after injury. ¹⁵

Tissues and organs maintain a stable balance between oxidants and antioxidants. Oxidants are compounds that produce ROS such as free radicals, while antioxidants scavenge radicals and prevent other compounds from being oxidized. The reactions induced by oxidants and antioxidants are collectively referred to as REDOX reactions, which include both oxidative stress and reduction reactions, respectively. ¹⁶

At the early stage of injury, due to the destruction of the vascular system, tissues undergo the processes of ischemia and hypoxia, hypoperfusion and metabolic disorders, and oxidative stress leads to tissue repair. Subsequently, the damaged tissue enters REDOX and revascularization gradually completes the reconstruction of the new vascular system. ¹⁷ Revascularization after injury is a common pathophysiological process necessary for the repair of all damaged tissues and is crucial for maintaining metabolic homeostasis and function. ¹⁸ After tissue injury, the tissue is hypoxic before revascularization. Repair cells have many endogenous antioxidant systems that are mobilized to ensure a balance between ROS and antioxidants, and if this balance is disrupted by factors such as high levels of ROS resulting from prolonged hypoxia, tissue damage, and dysfunction can occur. It manifests as incomplete regeneration, that is, excessive or insufficient deposition of collagen leads to excessive scar tissue or nonhealing.^19,20 In other words, perfect healing depends on a balance of REDOX. After revascularization, oxygen tension could change the biological properties of endogenous stem cells and play a vital role in wound tissue remodeling. ²¹

Stem Cell Niches and Revascularization

Stem Cell Niches and REDOX

In 1978, the concept of stem cell niches was first proposed by Schofield, which is a physiologically restricted local microenvironment that maintenance the activity of stem cells. ²² The stem cell niches can be defined as specific anatomical locations that regulate stem cells involvement in tissue maintenance, repair, and regeneration. The stem cell niches is a complex, heterogeneous, dynamic system that includes supporting ECM, adjacent niche cells, secreted soluble signaling factors (such as chemokines, cytokines, and growth factors), physical parameters (such as strain, shear stress, substrate rigidity, and topography), and environmental factors (hypoxia, inflammation, metabolites, etc). Stem cells, blood vessels, nerves, ECM, and the 3-dimensional spaces that make up this unit provide a highly specialized microenvironment. Crosstalk between these elements is essential for stem cell self-renewal and cell fate regulation, thereby enabling tissue homeostasis and regeneration. ²³

The stem cell niche acts as a dynamic and stable system that continuously assists body development, tissue maintenance, and regeneration. The stem cell niches hypothesis has provided a preliminary framework for defining cell types and factors that regulate stem cells behavior and function. Under steady-state conditions, there is a symbiotic relationship in the niche. However, the role of stem cell niches after tissue injury, especially revascularization and remodeling response remains unclear. ²⁴

Under physiological conditions, vascular homeostasis is a fine balance between stem cells and the microenvironment. ²⁵ The pathological changes after injury make each other interact with each other, and these interactions contribute to the repair, forming, and remodeling of blood vessels after injury. Since REDOX level may be control the regenerative potential after skin and soft tissue injury, the influence of REDOX on stem cells has been a hot topic in this field in recent years.^26,27 Previous studies have shown that stem cells/progenitor cells have multidirectional differentiation potential and regenerative ability, which is the key to ensure wound-perfect healing. ²⁸ Many functions of stem cells are regulated by the niches in which they live, and the dynamic microenvironment surrounding vascular reconstruction is an important component of the niches of stem cells, maintaining the characteristics of stem cells and regulating the activities of these cells. ²⁹ Reactive oxygen species are a group of small reactive molecules in the niches regulation mechanism. It is well known that ROS are increasingly involved in the physiological regulation of crucial developmental processes. There is also growing evidence that ROS are involved in many different levels of biological processes, from gene expression and protein translation to protein-protein interactions. ³⁰ As a cell signaling, transmitting information from one tissue to another, and converting environmental cues into cellular responses to balance cellular inputs, such as nutrients and cytokines, and conduct appropriate cellular responses. Reactive oxygen species act as a rheostat to coordinate various cellular processes and regulate cell activity to adapt to available bioenergy sources. ³¹ With advances in genomics and proteomics, there is also growing information about the various ways ROS balance and control cellular processes. In stem cells in particular, changes in stress states, also known as REDOX regulation, may be responsible for communication between mitochondria and the nucleus. ³⁰ REDOX-mediated mitochondrial nuclear cross-talking may explain the coordination between cell metabolism and chromatin remodeling, gene expression, cell cycle, DNA repair, and cell differentiation. ³² Because tiny changes in ROS levels can have profound effects on the fate of stem cells. The physiological elevation of ROS can lead to cell proliferation, while the larger increase in ROS may induce cell differentiation. Resting stem cells in the hypoxic niche exhibit low of levels ROS due to the well-organized antioxidant defense system that protects stem cells from external oxidative stress, while high levels of ROS could promote stem cell/progenitor cell differentiation or migration. ³³ However, the signaling mechanism of microenvironment regulation on stem cells remains to be further elucidated.

In addition to their role in REDOX regulation, ROS may also alter the epigenetic landscape, which plays a particularly relevant role in regulating the fate of stem cells. ³⁴ Many metabolic intermediates are essential substrates for posttranslational modifications of histones, which together establish the epigenetic of stem cells. Since glycolysis and oxidative phosphorylation activities can directly affect ROS, leading to changes in the concentration of various metabolic intermediates, this may be an underlying mechanism of ROS-mediated epigenetic regulation. On the one hand, ROS can change protein function, and on the other hand, a growing number of protein networks have been shown to regulate the level of ROS. Many of these REDOX sensor proteins are directly regulated by ROS in response to oxidative stress and stem cell fate. ³⁰

Cellular metabolism is the sum of catabolic and anabolic processes, and the balance between 2 changes depending on the cellular process being performed. One of the main ways metabolism affects signaling pathways is by altering ROS levels. In sequence, ROS can react directly with various proteins such as kinases, phosphatases, or transcription factors to alter processes that regulate cell cycle progression, apoptosis, quiescence, or differentiation. In addition, ROS can directly modify metabolic enzymes or proteins involved in nutrient sensing pathways, thereby directing metabolic flux. ³⁵ In these cases, ROS can be thought of as signaling molecules involved in the cross-talking between metabolism and stem cell fate determination. It should be noted, of course, that metabolism can also influence cell fate through a variety of mechanisms unrelated to ROS or through pathways where the metabolic effects of ROS are less obvious. ³⁶ However, these methods of cross-talking between metabolism and cell fate have not been well demonstrated in stem cells compared to the role of ROS.

One of the ultimate applications of ROS as stem cell destiny and reprogramming stem cells biology is to produce healthy differentiated cells to repair damaged or deteriorating tissues and organs. ³⁷ Given that ROS may affect many biological processes, and our knowledge of the microenvironment is limited, exploring how ROS metabolism can be applied to generate stem cells and influence its fate involved seems to be a huge challenge. Although the exact effect of ROS on the signaling pathway during reprogramming has not been evaluated, the ROS level seems to change during reprogramming, and the change can at least affect the reprogramming efficiency. ³⁸

The Influence of REDOX Signals on Mitochondria may be an Important Path to Reveal the Mechanism of Wound Healing

Reductive oxidation signaling is an important part of precise regulation of cell metabolism, controlling cell proliferation, differentiation, apoptosis, and death. Recent advances in stem cell biology have revealed the physiological importance of REDOX signaling in stem cell maintenance and tissue regeneration. Stem cells are mostly at quiescent state to preserve themselves, but proliferate and differentiate for tissue repair and regeneration, the key to this shift is metabolic switches. ³⁹ Mitochondria are organelles with this regulatory capacity that serve many important cellular, including energy production, REDOX signaling, and metabolism. These functions are closely related to mitochondrial morphology, which is highly dynamic and capable of rapidly and briefly changing cell function in response to environmental cues and cells need. Mitochondrial morphology and activity are critical to a variety of physiological processes, including wound healing. Mitochondria are considered to be one of the main sources of ROS, so they are actively involved in the regulation of cellular REDOX and ROS signaling. ⁴⁰ The cellular function of mitochondria is highly cell/tissue specific and can be heterogeneous even within the same cell due to the presence of mitochondrial subpopulations with different functional and structural properties. ⁴¹ However, the interactions between the different functions of mitochondria are not fully understood. Mitochondrial function may change with changes in cell metabolism. On the other hand, some factors and feedback signals of mitochondria may affect the physiology of the whole cell. There are many interactions between mitochondria and other parts of the cell, various cytoskeletal proteins, endoplasmic reticulum, and other cellular elements that can actively participate in the regulation of mitochondrial and cell metabolism, as well as stem/progenitor cells. ⁴² Resting stem cells rely on glycolysis to produce energy and are not affected by mitochondrial oxidative phosphorylation and free radical production. ⁴³ However, proliferative stem cells are involved in mitochondrial oxidative phosphorylation to meet increasing energy demands, which is accompanied by free radical production. Stem cells have evolved complex mitochondrial protection programs to ensure that only stem cells with intact mitochondria proliferate with enough energy to pass the cell cycle, while stem cells with damaged mitochondria remain stationary until the damage is repaired, or cause cell death when the damage can't be repaired. Therefore, the influence of REDOX signals on mitochondria is an important molecular mechanism that determines the fate of stem cells. ³²

Stem cells, whether pluripotent stem cells or adult tissue stem cells (ASCs), require unique metabolic programs and REDOX states to maintain proliferation and, at same time, maintain pluripotency and/or specific differentiation.^44,45 After activation by kinds stress, proliferating ASCs increase oxygen consumption by affecting growth factor kinase signaling, altering metabolite levels and REDOX status of cells, reducing expression of antioxidant enzymes, and activating ROS signaling. Revascularization can regulate the release of tissue repair potential. Therefore, the metabolic status and REDOX profile of stem cells can be used as indicators of their self-renewal, pluripotency, and differentiation. ⁴⁶ Reductive oxidation biology and metabolic programming-related genes are the most abundant transcription factors and proteins in stem cells, consistent with an important role in energy regulation in stem cells survival and function. ⁴⁷

Brief Summary

Work over the past few years has revealed the importance of REDOX signaling in stem cell biology, revealing that ROS signals the metabolic state of stem cells and thus influences the fate of stem cells. Whether ROS affects the stem cell epigenome has not been proven. But given the ability of metabolic intermediates to alter epigenetic mechanisms, this seems to foreshadow the possibility that ROS may alter stem cell epigenomes. The exact underlying mechanism of epigenetic metabolic regulation of stem cells is yet to be explored. An emerging theme is that metabolic pathways can also transmit cues of change in the external environment to regulate the inner fate of cells. Stem cell metabolism is a prominent representative of the combination and balance of internal metabolic needs and external metabolic limitations. Studies have shown that mitochondria are important mediators of platelets promoting healing of mesenchymal stem cells.^48,49

With an in-depth knowledge of modeling-fibrotic processes and the cellular basis, revascularization of stem cell REDOX stress may be used to modify critical targets for fibrosis and scarring, or refractory ulcers. Although the application of stem cells in clinical practice is still in its infancy, further research is needed to fully elucidate the beneficial potential of stem cells before and after wound revascularization and to optimize new approaches and strategies for their clinical application.

On the other hand, one of the biggest challenges facing tissue engineering technologies that are expected to make breakthroughs in perfect tissue repair is also related to the design and formation of vascular systems in the construction of their systems. ⁵⁰ A perfect vascular system is a necessary structure for transporting oxygen and other nutrients and is also the basis for the function of seed cells and biological scaffolds. Its lack limits the size of tissue-engineered structures and is a major obstacle to their successful regenerative function.

The metabolic processes of stem cells represent a good balance between the intrinsic demands of the cell state and the influences exerted by external nutrient and oxygen levels. A comprehensive understanding of these needs and constraints will improve the possibility of manipulating them. For example, ROS management in stem cells; The use of mitochondrial activity and its regulation (through mitochondrial clearance, generation, separation, and transfer) to achieve the homeostasis control of stem cells will enable us to better grasp the fate of stem cells and give full play to their potential, both in vivo regenerative medicine and in vitro tissue engineering will make breakthroughs, and finally achieve the perfect repair of damaged tissues.