Chronic refractory wounds are not yet clearly defined, but are usually understood as wounds that do not heal through the normal process of wound healing due to various intrinsic or external factors and enter a state of pathological inflammatory response, resulting in a long-lasting wound. Chronic hard-to-heal wounds are a long-standing therapeutic challenge in surgery, resulting in a high rate of disability. Some existing therapeutic measures are based on common trauma models rather than refractory wounds, and some related basic research is mainly focused abroad, with more domestic research at the clinical level. In recent years, the level of treatment for chronic refractory wounds has been gradually improved with the continuous improvement of basic research, including the study of the role and interaction of various cytokines and their receptors, and the application and maturation of various new therapeutic measures. In this paper, we review the mechanism of difficult-to-heal wounds and the progress of various therapeutic tools such as skin substitutes, growth factors, and negative pressure treatment techniques for wounds.
1. Normal wounds and chronic hard-to-heal wounds
1.1 The healing process of normal wounds
The healing process of normal wounds can be roughly divided into three stages: the local inflammatory response stage, the cell proliferation and differentiation stage, and the tissue plasticity and reconstruction stage. Each phase is regulated by paracrine or autocrine cytokines and growth factors. Growth factors are peptides present in the organism that have a significant regulatory effect on cell growth and differentiation and play a key role in normal wound healing. More than 18 different types of growth factors have been identified in wounds, including EGF TGF-α, FGF1-10, KGF, PDGF, IGF-1, VEGF, TGFβ1-3, IL-1α, IL-β, TNF-α, TNF-β, and HIF1-α.
Platelets in the early post-injury fibrous clot first release growth substances and cytokines including PDGF, which chemotactic more neutrophils to migrate to the injury, and a few days later neutrophils are replaced by activated macrophages, which release PDGF, TNF-α, IL-1, IL-6 and other cytokines to further amplify the inflammatory response to clear microorganisms and necrotic tissue.
When the inflammatory response decreases, the following growth factors gradually replace cytokines in the wound fluid: IL-1, TNF-α upregulate KGF expression by fibroblasts, KGF-1, 2 and IL-6 secreted by fibroblasts promote migration of keratin-forming cells and proliferation and differentiation into epidermal cells. IL-1, TNF-α, KGF, TGFβ-1 induce keratin-forming cells at the wound edge to synthesize VEGF. VEGF promotes the proliferation of dermal endothelial cells and plays an important role in neointima formation. pDGF promotes the proliferation of fibroblasts and induces the synthesis of extracellular temporary matrices such as dextran amines and fibronectin. tNF-α upregulates the expression of integrins by fibroblasts, which anchor cells to temporary matrices. tGF-β strongly stimulates the synthesis of collagen matrix by fibroblasts. .
During the tissue remodeling phase, collagen fiber strength increases, collagenase or other proteases degrade excess collagen fibers, excess capillary network subsides, and wound mucin and water decrease. imbalance of TGFβ expression causes abnormalities in this phase. Studies have shown that fibroblasts taken from keloid patients both overexpress TGFβ1 and TGFβ2. Fibroblasts in keloids are more sensitive to TGFβ2 compared to normal fibroblasts.
1.2 Mechanisms of refractory wounds
1.2.1 Molecular mechanisms of refractory trauma occurrence
In chronic wounds, tissue repair cells such as fibroblasts, epidermal cells, and vascular endothelial cells show typical manifestations such as nuclear fixation with nuclear chromatin margination. Fibroblasts are the main cells of myofibrogenic repair, and their impaired proliferation will lead to impaired synthesis of extracellular matrix, especially collagen, with the result that wound healing is delayed. The main focus of their research is on matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TiMPs). In refractory wounds, the persistent overproduction of inflammatory mediators and the accumulation of large numbers of neutrophils on the wound surface result in increased levels of MMPs and significantly lower levels of TiMPs in exudate from refractory wounds compared with acute wounds. The mechanisms by which various inflammatory mediators interact with MMPs and TiMPs are being investigated.
One possible mechanism is that tumor necrosis factor A (TNF2A) induces the expression of the membrane type 12 matrix metalloproteinase gene (mt12mmpgene) through the NF2JB pathway, which in turn activates pro-2 matrix metalloproteinase 2 (pro2MMP22) to increase matrix metalloproteinase 2 expression, and this effect must occur in the presence of collagen. TNF2A or collagen alone had little effect on fibroblast-mediated pro-matrix metalloproteinase 2 activation. Furthermore Stojadinovic et al. performed experiments with isolated human skin to study the role of B2catenin and oncogene c2myc in refractory wounds and found that B2catenin and c2myc levels showed overexpression in refractory wounds, thus suggesting that B2catenin may inhibit through several different mechanisms It is therefore proposed that B2catenin may inhibit the migration, growth and differentiation of keratin-forming cells through several different mechanisms, including activation of its downstream oncogene c2myc; blocking the action of epidermal growth factor (EGF); and acting as a coactivator of the glucocorticoid receptor (GR) along with ornithine methyltransferase (CARM21) allowing glucocorticoids to genetically inhibit keratin 6/keratin 16 (Keratin6/ Keratin16) expression at the gene level, which in turn affects cytoskeletal protein structure.
Overall, altered scaffolding of tissue repair cells, excessive apoptosis of repair cells, and signaling between growth factors and target cell receptors. Loss of coupling. and regulation of multiple inter-factor networks, loss of control, are all mechanisms that contribute to the development of refractory trauma on the body surface.
1.2.2 Pathophysiological mechanisms underlying the emergence of trauma healing is a dynamic, sequential and complex process that can usually be divided into four interrelated and overlapping processes: hemorrhage, inflammation, granulation tissue formation and tissue plastination. However, this orderly process is disrupted by various systemic or local factors, leading to the development of chronic hard-to-heal wounds. The factors responsible for such disruption are summarized in the following five points: malnutrition, poor tissue perfusion and ischemia-reperfusion injury, bacterial load, infection and necrotic tissue retention, diabetes mellitus, and cellular senescence. Under the influence of these factors, the ability of trauma repair is weakened and the injury factors dominate, ultimately leading to the formation of hard-to-heal trauma.
1.2.2.1 Malnutrition The increased nutritional and energy requirements of the body after trauma, when accompanied by poor tissue perfusion caused by vascular disease, hypovolemia or tissue edema, result in absolute and/or relative deficiencies of protein, energy and various micronutrients (usually various vitamins, trace minerals various essential amino acids such as arginine). These can lead to delayed or prolonged wound healing by mechanisms such as reduced synthesis of synthetic hormones, slowed rate of protein synthesis and accelerated catabolism, low immune function due to protein deficiency, and increased chance of infection. Malnutrition not only decreases the patient’s body mass, but acute trauma tends to become chronic, and Harris et al. showed that the incidence of decubitus ulcers increased by 74% under the combined effects of braking and loss of defatted body weight.
1.2.2.2 Tissue malperfusion and ischemia-reperfusion injury The role of tissue malperfusion in the formation of hard-to-heal wounds is widely recognized, including its triggering of ischemia and hypoxia, metabolite accumulation, and hypoxia-induced neutrophil hypofunction, all of which can contribute to delayed wound healing. However, the impact of ischemia2 reperfusion injury on the development of difficult-to-heal wounds has only recently gained attention, and Mustoe suggests that repeated ischemia-reperfusion injury based on tissue ischemia is also an important factor in the formation of difficult-to-heal wounds. After ischemia-reperfusion injury occurs inflammatory cells enter the tissue and release pro-inflammatory cytokines and oxygen free radicals under the action of chemokines, while the level of N2O decreases, causing vasoconstriction and tissue non-perfusion phenomenon and aggravating tissue damage. Senescent cells are less responsive to ischemia-reperfusion injury, which may be one of the reasons why older patients are more likely to develop difficult-to-heal wounds.
1.2.2.3 Bacterial load, infection, and necrotic tissue retention Bacterial load, infection, and necrotic tissue retention are mutually dependent. Traumatic exudate and necrotic tissue not only serve as a good culture medium for bacteria, but also constitute a barrier for bacteria to evade the host immune response and increase the chance of infection. They also release proteases and toxins that degrade growth factors and attack adjacent normal tissue around the wound, forming a physical barrier that prevents the movement and re-epithelialization of cells involved in wound repair. In addition, necrotic material (mainly fibrin, denatured collagen and elastin) left behind by incomplete initial debridement can also retard wound healing by forming a fibrin network that retains growth factors. Both bacterial load and infection can increase inflammatory toxins and protein hydrolases, prolonging the inflammatory response and increasing necrotic tissue. It is important to note that bacterial load and infection are different. Bacterial load refers to proliferating bacteria that are numerous enough to impair wound repair and do not necessarily lead to infection. Infection causes a decrease in synthetic hormones, an increase in catabolic hormones, an infected hypermetabolic state, and sepsis that will make wound healing more difficult.
1.2.2.4 Diabetes mellitus is often associated with delayed vascularization, neuropathy, and infection of the trabecular surface, making it difficult to heal. It has been widely accepted that diabetic patients have delayed vasculogenesis leading to difficult wound healing. Possible mechanisms include dysregulation of NO levels and decreased levels of various growth factors that stimulate angiogenesis, such as vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). A recent study by Maruyama et al. in a mouse model suggests that the activity and number of macrophages and their effect on lymphatic vessel formation also play a crucial role in diabetic wound healing. Neuropathy dulls sensation in the lower extremities and thus makes patients more likely to suffer from repeated injuries and secondary infections. Lu Shuliang and other Chinese scholars have demonstrated that, in the absence of exogenous injury, the collagenous tissue of diabetic skin has been pathologically altered and cells have been stimulated by certain signals that activate p53 expression and induce apoptosis, with Bcl-2 family proteins being involved in the apoptotic process.
This stimulation may originate from changes in the environment in which the cells are located. Increased sugar content, accumulation of reactive metabolic intermediates, and increased reactive oxygen radicals in local tissues of diabetic skin are the more recognized pathological changes, while altered cell proliferation and apoptosis due to metabolic disorders are involved in the occurrence and development of diabetic nephropathy, neuropathy, and retinopathy trauma refractory to healing. The imbalance of pre-traumatic cell proliferation and apoptosis in diabetic skin tissues makes an abnormal starting point for the healing process, which is the basis for the delay of subsequent wound healing. In addition, the late stage glycosylation of diabetic skin has been shown to be a major factor in the prevention and treatment of diabetic skin. In addition, the influence of late glycosylation end-products on the formation of difficult-to-heal wounds in diabetic patients is of great interest. Possible mechanisms include the persistence of inflammatory response by advanced glycosylation end products (AGE), reduced collagen deposition by fibroblasts, and reduced growth factor activity.
1.2.2.5 Cellular senescence Cellular senescence includes not only cells that age normally in the organism, but also cells that senesce during continued exposure to exudate from chronic refractory wounds. It has been documented that fibroblasts in several types of refractory wounds including decubitus ulcers and varicose veins ulcers exhibit senescent features. Senescent cells are not only less responsive to normal wound healing stimuli, but also occupy limited wound space. During normal wound healing, these limited spaces are occupied by normal cells that respond well to healing stimuli.
1.2.2.6 Bacterial biofilms Recently, there has been greater interest in the role of bacterial biofilms (Biofilms) in the formation of chronic hard-to-heal wounds and as a possible new mechanism for hard-to-heal or non-healing wounds. The so-called bacterial biofilm is actually a membranous structure formed by some bacteria attached to and embedded in the wound surface, with extracellular matrix and so on. It is composed of bacteria and their products, extracellular matrix, necrotic tissue, etc. Since it is a membranous structure composed of multiple components present at the cellular level, it is often determined by fluorescein staining, etc. in studies. According to studies, the formation and role of bacterial biofilm is not evident in acute wounds, and the presence of this biofilm can be detected in only 6% of wounds, so bacteria are not a major factor in delaying wound healing. However, when a wound changes from acute to chronic, this biofilm can be detected in more than 60% of wounds, and when the bacterial population reaches a certain level, the bacterial biofilm may play a decisive role.
During the transition from acute to chronic, bacteria adhere to the wound surface and multiply to form clones, which later encapsulate themselves in a multilayered matrix of necrotic tissue and extracellular matrix, forming a protective layer that resembles a membrane-like structure, which is also clinically observed in the form of redness, swelling, heat, pain, and low oxygen partial pressure, so that the bacteria can resist various therapeutic measures. . In fact the establishment of this biofilm allows these bacteria to escape the killing effect of antibiotics on them. One hypothesis is that the biofilm formed on the infected surface of P. aeruginosa produces a resistance factor that allows the bacteria to escape the phagocytosis of the bacteria by neutrophils. Staphylococcus aureus has also been shown to have a similar effect.
2. Advances in the treatment of difficult-to-heal wounds
Traditional wound treatment techniques include standard debridement dressings, scabbing, wound decompression, and treatment of the underlying lesion. Since the formation of difficult-to-heal wounds is often multifactorial, it is hoped that targeted and comprehensive treatments can be used to improve the efficacy of treatment. With the in-depth understanding of the mechanism of difficult-to-heal wounds and the process of wound healing, as well as the application and maturation of various effective growth factor delivery techniques, new medical materials and new technologies for wound treatment such as negative pressure wound treatment, targeted treatment is expected to become a reality.
2.1 Application of skin substitutes There are three types of skin substitutes in common use: autologous or allogeneic epidermal sheets cultured in vitro, dermal scaffolds constructed by tissue engineering using natural biological materials (homogeneous or allogeneic dermis) or synthetic polymers, and hybrid skin substitutes that are both, which mainly function in two aspects, i.e. covering the wound and promoting wound healing. A major development is the hybrid skin substitute, and recently Gibbs et al. reported good results using epidermal cell cultures obtained from patients’ own biopsies as skin substitutes, using cell-free dermis as a scaffold for the treatment of chronic ulcers of the lower extremities. Mixed skin substitutes can lead to the death of some of the grown or seeded autologous cells due to the lack of blood supply because their vascularization process lags behind the cell growth entry.
One of the solutions is to apply various growth factors in the matrix of the substitute to promote the vascularization process. The practice used in the past of directly admixing various growth factors into the matrix does not allow the majority of the admixed growth factors to be degraded or bound at the lesion site without exerting the desired effect due to the inability to release growth factors in a relatively stable manner. Markowicz et al. showed that the collagen scaffold could also be modified to promote dermal regeneration and vascularization through animal studies. regeneration and vascularization. It is also possible to genetically modify various cells in the skin substitute to release various desired growth factors in a sustained and sufficient amount (described below).
With the development of tissue engineering technology, the development of different types of artificial skin by tissue engineering technology has been increasingly reported, mainly artificial skin sheets, artificial dermal substitutes and artificial composite whole skin. However, artificial skin sheets (i.e., epidermal skin sheets) are thin, brittle and contractile, poorly resistant to infection, poorly resistant to membranes after transplantation, prone to blistering, and not suitable for clinical manipulation. Artificial dermis mainly acts as a scaffold for full skin defect trauma, as a temporary substitute to cover the trauma, and then perform autologous dermis transplantation after the trauma is closed, its main products are Integra and Dermagraft series, generally divided into two layers, the inner layer adopts collagen fiber and chondroitin sulfate to form a porous scaffold or nylon mesh planted with fibroblasts to form a porous scaffold; DermagraftTM uses a biodegradable polyhydroxyacetic acid polymer as the dermal scaffold.
However, the ideal skin replacement product should be able to repair both the missing dermal and epidermal layers, the so-called whole skin composite, which includes at least two cellular components, epidermal cells in the superficial layer and fibroblasts in the dermis. Apligraft, which is approved for clinical use by the FDA, is a skin substitute containing human living cells. Apligraft is composed of living cells and structural proteins in two layers, mimicking the epidermis and dermis, and has been applied to diabetic ulcers and ulcers caused by venous blood flow disorders, significantly shortening the healing time of ulcers.
2.2 Application of growth factors Growth factors can bind to other molecules in the extracellular space or be degraded by proteases to fail, so the method of spraying and applying growth factors directly to the wounds is less effective. In contrast, gene therapy has shown promise by genetically modifying the cells involved in wound healing to synthesize and release the required growth factors in a more stable manner. Gene therapy should take into account three aspects: firstly, how to introduce the desired target gene into the target cells without causing damage or minimal damage to the organism; secondly, sufficient transfection rate of the target cells and sufficient expression of the target gene in the target cells to produce a therapeutic effect; and finally, good regulation of the introduced gene so that its expression can be maintained for a certain time and concentration as needed for treatment. Virus-based, chemical and mechanical transgenic techniques are available, each with its own advantages and disadvantages, and can be applied in vitro or in vivo. In vivo transgenic techniques are more practical as they avoid the isolation and culture of target cells and require less time. The safer and more promising clinical applications are gene gun technology and micro-implantation technology.
2.3 Stem cell transplantation Currently, there are increasing reports on the clinical application of ESC (epidermal stem cells), bone marrow MSC (mesenchymal stem cells), and embryonic stem cells to repair wounds; MSC has achieved significant results in promoting the healing of refractory wounds such as diabetic ulcers and varicose ulcers in the lower extremities, and studies are being conducted to induce the differentiation of embryonic stem cells and construct fully functional tissue-engineered skin. Theoretically, any cell with the potential to differentiate into epidermal tissue can be transplanted for tissue repair. Experiments have shown that the healing rate of myogenic stem cells is significantly higher than that of the control group. The application of stem cells in the treatment of refractory wounds will certainly have a profound impact on the clinical treatment of wound repair, but there are still many problems in stem cell identification technology, isolation culture and regulation technology, and ethics (embryonic stem cells can lead to the destruction of living embryonic tissues), and further research is needed.
2.4 Application of oxygen therapy In 2003, Cordilb et al. first reported that local oxygen therapy could accelerate the healing of wounds, and then conducted related basic research, suggesting that local hypoxemia after vascular dissection is a key factor limiting wound healing and increasing the chance of infection. Treatment with hyperbaric oxygen therapy increases the partial pressure of oxygen in tissues, accelerates fibroblast proliferation, collagen release, granulation tissue production, and accelerates epithelial growth. It inhibits a variety of metabolic reactions of bacteria and has a direct bactericidal or bacteriostatic effect on certain bacteria, especially those with anaerobic bacterial infections on the wound surface. Allen et al. found that an increase in partial pressure of oxygen in the tissue at the wound from 15 mmHg to 100 mmHg increases the bactericidal activity by 3-4 times. Early use of hyperbaric oxygen therapy promotes local blood flow, reduces edema, improves ischemia and hypoxia in damaged tissues and enhances resistance to infection, thus accelerating repair and healing of difficult wounds and reducing the amount of residual scar tissue to less than that expected from conventionally treated wounds. This method not only improves the healing rate, shortens the course of the disease and reduces patient pain, but also has the characteristics of being safe, effective and non-invasive, and can directly and rapidly eliminate wound edema, improve local hypoxia and promote wound healing. Combined with other treatment methods, it can achieve twice the effect with half the effort.
2.5 Negative pressure trauma treatment technology Negative pressure trauma treatment technology refers to the trauma treatment by placing a drainage tube connected to a special vacuum negative pressure pump on the trauma surface and wrapping it with gauze or polyurethane sponge, and then closing the trauma surface with a transparent film, and using the negative pressure pump to create a negative pressure environment on the trauma surface. Compared to traditional passive drainage measures, negative pressure trauma therapy is a more proactive means of drainage, allowing for uniform pressure transfer to the wound and preventing tissue debris from blocking the drainage tube. Negative pressure trauma techniques promote wound healing through a variety of mechanisms, including aspiration of exudate from the wound, reduction of tissue edema, promotion of granulation tissue growth, and maintenance of wound wetness. Braakenburg et al. showed that the efficacy of negative pressure wound therapy is comparable to that of some modern wound coverings, reducing patient pain and medical care tasks, especially for diabetic difficult-to-heal wounds. Thus, negative pressure wound therapy is expected to be an efficient and cost-effective treatment for difficult to heal wounds.