*3.6.5. Prevent chronic condition*

Besides having immune modulatory activities, the effective biomolecules secreted by MSCs can prevent wounds from reaching a chronic state by their angiogenic and antiapoptotic characteristics [105]. For instance, transplantation of hMSCs intramyocardially has the ability to improve cardiac function via enhancing myogenesis and angiogenesis in the ischemic myocardium [136]. Also, MSCs enable a wound to progress to healing beyond the inflamma‐ tion stage and not regress into a chronic state [11].

### *3.6.6. Attenuation of scar formation*

The tissues in the scar have many disadvantages including their undesirable visual appearance and lack of structures that are present in the native skin such as hair follicles, sebaceous glands and sensory nerve receptors [30]. In addition, scar tissue weakens the skin making it more susceptible for re‐injury [137]. MSCs have been shown to overcome these disadvantages via attenuating scar formation [138].

### *3.6.7. Neutralising the reactive oxygen species (ROS)*

Although IL‐10 participates in preventing the invasion of neutrophils into the site of tissue injury and the enhancement of collagen deposition, the penetrations of some populations result in the release of ROS, which are oxygen molecules have an unpaired electron making them extremely reactive such as superoxide, hydrogen peroxide and alkyl peroxides [139]. Many tissues are susceptible to attack by ROS contributing to dangerous diseases including heart disease and cancer. Also, prolonged persistence of ROS induces fibrogenesis and the accumu‐ lation of fibrotic tissues [30]. To counter act such effects, MSCs significantly upregulate the expression of nitric oxide synthase [140] which alters the ROS balance preventing the formation of fibrotic tissues [141].

### *3.6.8. Producing antifibrotic factors*

MSCs release growth factors and cytokines characterised by their antifibrotic activities such as HGF and IL‐10 [142]. HGF has been shown to downregulate the expression of collagen type I and type III by fibroblasts therefore attenuating fibrosis and scar formation [143]. Moreover, HGF impacts on the keratinocyte behaviour by promoting their migration, proliferation and expression of VEGF‐A, thereby generating a well‐granulated tissue with a high degree of vascularisation and re‐epithelialisation [30].

### *3.6.9. Enhancing dermal fibroblast function*

In response to the wounding process, fibroblasts present at the injury site produce additional quantities of ECM to restore the integrity of the skin leading to scarred tissue [144]. Also, many endothelial cells undergo epithelial‐to‐mesenchymal transition (EMT) under the effect of TGF‐ β1 and become wound‐healing myofibroblasts [138]. Both of these actions affect the function of dermal fibroblasts. Therefore, MSCs present in the wound site enhance dermal fibroblast function by producing HGF and PGE2 which both play a role in inhibiting EMT [145] and secrete biomolecules promoting the function of dermal fibroblast in wound healing [90]. MSCs therefore enable the cells present in the wound site to release ECM similar to those produced by neighbouring dermal cells [30].

### *3.6.10. Promoting angiogenesis and vascular stability*

It has been well documented that BM‐MSCs play a major role in angiogenesis and microvas‐ cularisation via promoting proliferation, migration and differentiation of microvascular endothelial cells by producing basic FGF and VEGF‐A [146].

### **3.7. Requirements for the healing process**

### *3.7.1. Infection fighting*

Open wounds are at risk of infections by bacteria and other microorganisms causing serious conditions such tetanus and gangrene and giving rise to chronic wounds, bone necrosis, long‐ term disabilities and death. Unfortunately, in some wound types, the necrotic tissues exudate secretions which act as a medium for bacterial growth inside the wound and protect the bacteria from the host's immune defence [147]. The use of disinfectant is useless, because they damage the injured tissues and arrest wound contraction. Also, they are easily suppressed by the inorganic substances present in the wound including blood components and other tissue secretions [148]. Therefore, appropriate care is required to protect open wounds and reduce the possibility of bacterial infection. Included in such cares are the treatments of wounds with topical antibiotics to kill the invading bacteria and moisturise the wounded area [149, 150], accelerate the healing process [151] and modulate inflammation [152]. Failure of antibiotic treatment leads to non‐healing wounds in which bacteria thrive on dead tissue giving rise to uncontrolled infection leading to additional complicated treatments including draining and removal of dead tissues from the injured site or even amputation in the case of diabetic ulcers [147, 150].

### *3.7.2. Prevention of ischemia and hypoxia*

I and type III by fibroblasts therefore attenuating fibrosis and scar formation [143]. Moreover, HGF impacts on the keratinocyte behaviour by promoting their migration, proliferation and expression of VEGF‐A, thereby generating a well‐granulated tissue with a high degree of

In response to the wounding process, fibroblasts present at the injury site produce additional quantities of ECM to restore the integrity of the skin leading to scarred tissue [144]. Also, many endothelial cells undergo epithelial‐to‐mesenchymal transition (EMT) under the effect of TGF‐ β1 and become wound‐healing myofibroblasts [138]. Both of these actions affect the function of dermal fibroblasts. Therefore, MSCs present in the wound site enhance dermal fibroblast function by producing HGF and PGE2 which both play a role in inhibiting EMT [145] and secrete biomolecules promoting the function of dermal fibroblast in wound healing [90]. MSCs therefore enable the cells present in the wound site to release ECM similar to those produced

It has been well documented that BM‐MSCs play a major role in angiogenesis and microvas‐ cularisation via promoting proliferation, migration and differentiation of microvascular

Open wounds are at risk of infections by bacteria and other microorganisms causing serious conditions such tetanus and gangrene and giving rise to chronic wounds, bone necrosis, long‐ term disabilities and death. Unfortunately, in some wound types, the necrotic tissues exudate secretions which act as a medium for bacterial growth inside the wound and protect the bacteria from the host's immune defence [147]. The use of disinfectant is useless, because they damage the injured tissues and arrest wound contraction. Also, they are easily suppressed by the inorganic substances present in the wound including blood components and other tissue secretions [148]. Therefore, appropriate care is required to protect open wounds and reduce the possibility of bacterial infection. Included in such cares are the treatments of wounds with topical antibiotics to kill the invading bacteria and moisturise the wounded area [149, 150], accelerate the healing process [151] and modulate inflammation [152]. Failure of antibiotic treatment leads to non‐healing wounds in which bacteria thrive on dead tissue giving rise to uncontrolled infection leading to additional complicated treatments including draining and removal of dead tissues from the injured site or even amputation in the case of diabetic ulcers

vascularisation and re‐epithelialisation [30].

*3.6.9. Enhancing dermal fibroblast function*

116 Wound Healing - New insights into Ancient Challenges

by neighbouring dermal cells [30].

*3.6.10. Promoting angiogenesis and vascular stability*

**3.7. Requirements for the healing process**

*3.7.1. Infection fighting*

[147, 150].

endothelial cells by producing basic FGF and VEGF‐A [146].

Ischemia is defined as the failure of blood to reach the target tissue and characterised by insufficient nutrient and oxygen supply resulting in turn in hypoxia and the insufficient availability or very low oxygen concentration at the injury site [153]. Therefore, the prevention of such circumstances is critical to the acceleration of wound‐healing process [154]. Cold environment causes continuous constriction of blood vessels thereby reducing blood flow into the injury site and causing ischemia. Therefore, keeping tissue warm will dilate blood vessels facilitating blood flow and decrease the probability of ischemia initiation; however, hyper‐ thermia is not recommended as this increases the likelihood of post‐surgical infection [147]. In some cases of diabetic ulcers and venous ulcers, surgery is required to treat ischemia by revascularisation the veins and arteries to correct their function [155]. Another approaches to combat ischemia include pressure‐assisted treatment or negative pressure wound therapy (NPWT), involving the creation of a vacuum to drain and remove wound exudate and their bacterial component, reducing tissue swelling, enhancing cell proliferation at the wounded site and the production of extracellular matrix thereby improving the healing process [156, 157]. Hypoxemia could arise due to vascular disease that arrests oxygen transfer, high demand for oxygen by tissue metabolism at the injured site and the formation of reactive oxygen species (ROS) [153]. The best therapy for hypoxemia is increasing the oxygenation of injured tissue by hyperbaric oxygen therapy (HBOT) to compensate oxygen limitations [155]. Also, higher oxygen content results in bacterial death, the acceleration of growth factor production, the enhancement of fibroblast growth and the promotion of angiogenesis [155, 157]. Another method to treat hypoxemia is the use of antioxidants to reduce the presence of oxidant substances [32].

MSCs could potentially be used to treat ischemia and hypoxia because they release angiogenic and mitogenic factors such as VEGF, IGF‐1 and HGF which are well known to induce angio‐ genesis and myogenesis [158]. Intravenous (IV) administration of BM‐MSCs increased the activity of matrix metalloproteinase‐2 and decreased the activity of matrix metalloproteinase‐ 9 resulting in improved cardiac function in a rat model of diabetic cardiomyopathy [159]. Wang et al. [160] showed that MSCs secrete hypoxia‐regulated haem oxygenase‐1, frizzled‐related protein‐2, hypoxic Akt‐regulated stem cell factor, heat‐shock protein‐20, adrenomedullin (AM) and SDF which collectively contribute to regeneration, neovascularisation and remodelling. MSCs were also be used to treat diabetic limb ischemia in the ischemic hind limb of type II diabetic mice due to the secretion of proangiogenic factors including hypoxia‐inducible factor and VEGF, responsible for vasculogenesis, blood flow regulation [161] and improvement of arterial perfusion in type 1 diabetic patients with gangrene [162]. A pilot study carried on patients of critical limb ischemia, who did not respond to other therapies, showed that multiple intramuscular injections of MSCs induced formation of vascular networks across the closed arteries, resulting in successful improvement of limb ischemia in terms of pain reduction and claudication [163]. In another study using a pig model of stenotic kidney blood flow, admin‐ istration of MSCs improved the architecture of microvessels in term of size and density [164].

### *3.7.3. Regulation of growth factors and hormones*

As previously described, growth mediatory factors play pivotal roles in wound healing. Therefore, more quantities of these growth factors are required to progress the healing process, in particular in case of chronic wounds, so they should be continuously upregulated [31, 32]. Several methodologies have been proposed to maintain efficient concentrations of growth factors at the injury site. Direct application of such biomolecules, however, requires large quantities and repetitive application. Spreading autologous platelets over the injury site which later secrete growth factors such as EGF, IGF‐1, IGF‐2, TGF‐β and VEGF has been proposed [165] as has the utilisation of keratinocytes and fibroblasts on a collagen matrix to enhance further secretion of growth factors when applied on the wound [32, 166]. Another potential way to protect efficient concentrations of growth factors at the wound site is through the prevention of their breakdown by analytical enzymes and thereby preventing the formation of proteases such as elastase [31, 32]. Oestrogen and prostaglandins have also been showed to play a role in the healing process; maintenance of their concentration at efficient levels may thus prevent excess neutrophils from reaching the injury site and produce more elastase [41, 49, 50, 167].

### **3.8. Limitations of using MSCs for wound healing**

MSCs can be considered as a promising tool for treating non‐healing wounds; however, some aspects of MSC biology need to be intensively studied before use in clinical application. One of these is the problem of finding a source of MSC isolation with no invasive procedure for autologous treatment, for example isolation of MSCs from peripheral blood instead of bone marrow. Other questions include the following: What is the ideal number and timing of MSC administration/implantation? How long can MSCs survive at the injury site after implantation? Are multiple administrations or implantations required for successful healing? When do the implanted MSCs start releasing their soluble secretions after implantation/administration? And finally, are the secretions of MSCs controllable? Answers to these questions are important and essential for the therapeutic use of MSCs and a safer and a more effective treatment for non‐healing wounds.
